MX2008001345A - Colon cancer related gene tom34. - Google Patents

Abstract

Objective methods for detecting and diagnosing colon cancer are described herein. In one embodiment, the diagnostic method involves determining the expression level of TOM34 that discriminates between colon cancer cells and normal cells. Finally, the present invention provides methods of screening for therapeutic agents useful in the treatment of colon cancer, methods of treating colon cancer and method for vaccinating a subject against colon cancer.

Description

GEN TOM34 RELATED TO COLON CANCER The present application claims the benefit of the US Provisional Application Series No. 60 / 703,265 filed July 27, 2005, the total contents of which are incorporated herein by reference. Field of the Invention The present invention relates to methods for detecting and diagnosing colon cancer, as well as methods for preventing and preventing colon cancer. Background of the Invention Colorectal cancer is one of the most common causes of death from cancer worldwide. Despite several advances in the diagnosis and treatment of colorectal cancers, many patients with advanced colorectal cancer result in mortality. To improve this prognosis, the development of sensitive and specific diagnostic biomarkers is desired for the detection of early stage carcinomas and the development of more effective and less dangerous therapeutic drugs. For this purpose, it is a requirement to better understand the molecular mechanisms of colorectal carcinogenesis. Recent molecular studies have revealed that colorectal carcinogenesis involves an accumulation of genetic alternations that include genetic changes in tumor suppressor genes and / or oncogenes, including APC, p53, beta-catenin and K-ras (Nishisho I, and associates Science 253: 665-669, 1991; Baker SJ, and associates, Science 244: 217-221, 1989; Morin PJ , and associates, Science 275: 1787-1790, 1997; Forrester K, and associates, Nature 327: 298-303, 1987). In addition to these types of changes, epigenetic events such as altered methylation (Jones PA &Laird PW, Nat Genet 21: 163-167, 1999) and impression loss (Cui H, and associates, Nat Med 4: 1276-1280 , 1998), and / or transcriptional control deregulated by genetic changes or other unknown mechanism (s) are involved in the genesis of colorectal tumors. Among the genes involved in carcinogenesis, inhibition of gene products essential for the proliferation and / or survival of cancer cells that will result in growth inhibition or cell death is expected. Accordingly, molecules that exert oncogenic activity and that are specifically expressed in cancer cells represent promising targets for the development of novel anti-cancer drugs. Human TOM34 was discovered from EST and cDNA databases, and was anticipated as a component of the mitochondrial protein import machinery, since the anticipated protein shares sequence homology in the region of a residue-62 motif with the Tom70 family of known yeast from mitochondrial receptors (Nuttall SD, and associates, DNA Cell Biol 16: 1067-1074, 1997). However, recent studies have described that TOM34 is included mainly in the cytosolic fraction and partially in the mitochondrial and membrane fraction after fractionation of tissues and cells (Chewawiwat N, and associates, J Biochem (Tokyo) 125: 721-727, 1999). Another study showed its subcellular localization in HeLa cell cytoplasm by immunohistochemical staining (Chun-Song Yand Henry Y., Archives of Biochemistry and Biophysics 400: 105-110, 2002; Abhijit M, and associates, Archives of Biochemistry and Biophysics 400: 97 -104, 2002). The two-hybrid yeast classification system showed that TOM34 interacts in vitro with the Valosin-containing protein (VCP), a member of the AAA family (ATPases associated with a variety of cellular activities) (Chun-Song Y, and associated , Archives of Biochemistry and Biophysics 400: 105-110, 2002), or 90-kDa heat shock protein (hsp90) (Young JC, et al., J Biol Chem 273: 18007-18010, 1998). However, the biological role of TOM34 remains unresolved. It has been shown that CD8 + cytotoxic T lymphocytes (CTLs) recognize epitope peptides derived from tumor associated antigens (TAAs) presented in the MHC class I molecule, and smooth the tumor cells. Since the discovery of the MAGE family as the first example of TAAs, many others, have been discovered using immunological methods (Boon T., Int J Cancer. 1993; 54 (2): 177-80., Boon T & van der Bruggen P, J Exp Med. 1996; 183 (3): 725-9, van der Bruggen P, and associates, Science. 1991, 254 (5038): 1643-7, Brichard V, and associates, J Exp Med. 1993; 178 (2): 489-95., Kawakami Y, and associates, J Exp Med. 1994; 180 (1): 347-52), and some of them have been known in the process of clinical development as immunotherapy targets. TAAs discovered so far, includes MAGE (van der Bruggen P, and associates, Science, 1991; 254 (5038): 1643-7), gp100 (Kawakami Y, and associates, J Exp Med. 1994; 180 (1): 347 -52), SART (Shichijo S, and associates, J Exp Med. 1998; 187 (3): 277-88), NY-ESO-1 (Chen YT, and associates, Proc Natl Acad Sci USA 1997; 94 (5 ): 1914-8). At the same time, genetic products, which have already been shown to be overexpressed to a specific extent by tumor cells, have been shown to be targets for cellular immune responses. These include p53 (Umano Y, and associates, Br J Cancer, 2001; 84 (8): 1052-7), HER2 / neu (Tanaka H, and associates, Br J Cancer., 2001; 84 (1): 94-9 ), CEA (Nukaya I, and associates, Int J Cancer, 1999; 80 (1): 92-7), and others. Although these are examples of the significant progress that has been made in basic and clinical research (Rosenberg SA, and associates, Nat Med. 1998; 4 (3): 321 -7., Mukherji B, and associates, Proc Natl Acad Sci USA 1995; 92 (17): 8078-82., Hu X, and associates, Cancer Res. 1996,56 (11): 2479-83), there is a very limited number of candidate TAAs in general for the treatment of adenocarcinomas including colon cancer. If TAAs are present that are abundantly expressed only in cancer cells but not in normal cells, they could be promising candidates for immunotherapeutic purposes. Brief Description of the Invention To verify the molecular role in colorectal carcinogenesis and find novel therapeutic targets for patients with colorectal carcinoma (CRC), we have carried out expression profiles using a cDNA microformation consisting of 23040 genes (Lin YM, and associated , Oncogene 21: 4120-4128, 2002). Among the genes that show activated expression in colorectal tumors, we focused on TOM34 (34-kDa-translocase of the outer mitochondrial membrane) because their expression levels were increased frequently in 16 of 20 CRC samples reviewed. Analysis of multiple tissue northern spotting revealed that this gene was abundantly expressed in the testes and ovaries, and weakly in the prostate, spleen and colon but not in any of the other 11 normal adult tissues reviewed. Immunohistochemical staining of TOM34 showed significant accumulation in CRC tissues compared to their corresponding non-cancerous mucous membranes. In the present invention, it was confirmed that TOM34 was frequently active in CRCs, and which is expressed in the ovarian testes but not in 15 other normal adult tissues reviewed. Since the suppression of this TOM34 by siRNA markedly reduced the growth of colon cancer, the gene product may be a potential therapeutic target for human tumors, as well as a useful diagnostic marker. In addition, it was considered that TOM34 should serve as a TTA (antigen associated with tumor) which can induce a significant cellular immune response against colon cancer. To revise this hypothesis, we stimulate PBMC harvested from healthy volunteers with peptides derived from TOM34 and peptide-specific CTL clones obtained in a successful manner which also recognize and kill the tumor cells that express the antigen. Specifically, the transfection of colon cancer HCT116 and RKO with TOM34-specific small interfering RNA (siRNA), effectively suppressed its expression, and dramatically inhibited cell growth. In addition, we review the peptides with sequences derived from (PBMC) with respect to their potential to serve as antigenic epitope peptides and we have explored a novel epitope peptide for effective cancer immunotherapy, which can induce a significant cellular immune response against human cancer. colon. Peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated using peptides that have partial sequences of TOM34. Said peptides were chosen so that they were anticipated to bind to HLA-A * 2402. We have successfully identified a specific antigen peptide whose sequence derived from TOM34 that can induce cytotoxic T-lymphocyte cell lines (CTL) shows specific cytotoxicity against cells that present the antigen in a form restricted by HLA-A24. The CTL clones were established from these cell lines. Further analysis of the CTL clones showed that they had a potent cytotoxic activity not only against the peptide-loaded target cells but also against the cells that express endogenously TOM34. In addition, the cold target inhibition assay indicated that CTL clones specifically recognized the antigen peptide in the MHC class I molecule complex. These results strongly suggest that the peptide is an epitope peptide restricted by HLA-A424. which can induce a potent and specific immune response against colon cancer cells that express TOM34. These findings suggest that TOM34 is involved in the growth of cancer cells, and may contribute to the development of novel anti-cancer drugs and / or diagnosis of CRCs. The present invention is based on the discovery of a genetic expression pattern of TOM34. The nucleotide sequence and amino acid sequence of TOM34 are set forth in SEQ ID NO: 60 and 61, respectively. These sequences are also available in the Genbank Access Number AB085681. Accordingly, the present invention provides a method for diagnosing and determining a predisposition to colon cancer in a subject, by determining a level of expression of TOM34 in a biological sample derived from a patient, such as a tissue sample. A normal cell is one obtained from colon tissue. An 'alteration, for example, an increase in the level of expression of a gene compared to a level of normal control of the gene, indicates that the subject suffers from or is at risk of developing colon cancer. When used within the context of the present invention, the term "predisposition to colon cancer" comprises a condition of a subject to be predisposed to, having a tendency to, prevalence, inclination or susceptibility to colon cancer. In addition, said term also comprises that a subject is at risk of acquiring colon cancer. Within the context of the present invention, the phrase "control level" refers to a level of protein expression detected in a control sample. A control level can be a simple expression pattern derived from a simple reference population or from a plurality of expression patterns.

For example, the control level can be a database of expression patterns from previously tested cells. A "normal control level" refers to a level of genetic expression detected in a normal, healthy individual or in a population of individuals known not to have colon cancer. A normal individual is one without clinical symptoms of colon cancer. An increase in the level of TOM34 expression detected in a test sample compared to a normal control level indicates that the subject (from whom the sample was obtained) suffers from or is at risk of developing CRC. According to the present invention, the level of genetic expression is considered "altered" when the gene expression has increased 10%, 25%, 50% compared to the control level. Alternatively, a level of expression is considered "increased" when the gene expression is increased by at least 0.1, at least 0.2, at least 1, at least 2, at least 5, or at least 10 or more times compared to a control level. Expression is determined by detecting hybridization, for example, a TOM34 probe for a genetic transcription of a tissue sample derived from the patient. Within the context of the present invention, the tissue sample derived from the patient is any tissue obtained from a test subject, for example, a patient known to have, or suspected of having colon cancer. For example, the tissue may contain an epithelial cell. More particularly, the tissue may be an epithelial cell of a colorectal carcinoma. The present invention further provides methods for identifying an agent that inhibits the expression or activity of TOM34, by contacting a test cell that expresses TOM34 with a test compound, and determining the level of expression of TOM34 or the activity of its gene product. The test cell can be an epithelial cell, such as an epithelial cell obtained from a colorectal carcinoma. A decrease in the expression level of TOM34 or the activity of its gene product compared to a level of control or activity of the gene or gene product, indicates that the test compound is an inhibitor of TOM34 and can be used to reduce a symptom of colon cancer. The present invention also provides a kit comprising a detection reagent that binds TOM34 nucleic acids or polypeptides. Therapeutic methods of the present invention include a method of treating or preventing colon cancer in a subject by including the step of administering to the subject an agonist or inhibitor of TOM34, which, for example, is an antisense composition or an antibody composition. The agonist or inhibitor can either act at the level of nucleic acid or protein to reduce in this way or inhibit the expression or activity of TOM34. Within the context of the present invention, the antisense composition reduces the expression of the specific target gene. For example, the antisense composition may contain a nucleotide which is complementary to the sequence TOM34. Alternatively, the method of the present invention may include the steps of administering a small interfering RNA (siRNA) composition to a subject. Within the context of the present invention, the siRNA composition reduces the expression of TOM34. In yet another method, the treatment or prevention of colon cancer in a subject can be carried out by administering a ribozyme composition to a subject. Within the context of the present invention, the specific ribozyme composition of nucleic acid reduces the expression of TOM34. Actually, the inhibition effect of siRNA for TOM34 was confirmed. For example, it has been clearly shown that the siRNA for TOM34 inhibits the cell proliferation of colon cancer cells in the example section. Therefore, the present invention, TOM34 is preferably a therapeutic target of colon cancer. The present invention also includes vaccines and vaccination methods. For example, a method for treating or preventing colon cancer in a subject may involve administering to the same a vaccine containing a polypeptide encoded by a TOM34 nucleic acid or an immunologically active fragment of said polypeptide. Within the context of the present invention, an immunologically active fragment is a polypeptide that is shorter in length than the naturally occurring full length protein, which still induces an immune response analogue to that induced by the full length protein. For example, an immunologically active fragment must be at least 8 residues in length and have the ability to stimulate an immune cell, such as a T cell or a B cell. The stimulation of an immune cell can be measured by detecting cell proliferation, making cytokines (for example IL-2), or production of an antibody. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art to which the present invention pertains. Although methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated in their entirety by reference to the present invention. Nothing found in the present invention should be constructed as an admission that the present invention is not entitled to precede said description by virtue of the foregoing invention. In case of conflict, the present invention specification, including the definitions, will be controlled. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. An advantage of the methods described herein is that the disease is identified before the detection of palpable clinical symptoms of colon cancer. Other features and advantages of the present invention will be appreciated from the detailed description set forth below, and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the level of expression of the TOM34 gene in various tissues. (A) Semi-quantitative RT-PCR analysis of TOM34 in colon cancer tissues and their corresponding non-cancerous mucous membranes. T, tumor tissue; N, normal tissue. The expression of GAPDH served as an internal control. (B) TOM34 multiple tissue northern blot analysis. The transcription of TOM34 is approximately 2.0-kb in size. Figure 2 illustrates the level of expression of the protein encoded by the TOM34 gene in various tissues. (A) Western blot analysis of TOM34 in colon cancer tissues and its corresponding non-cancerous mucous membranes. T, tumor tissue; N, normal tissue. (B) Expression of TOM34 in colon cancer cell lines. The expression of beta-actin served as an internal control. Figure 3 illustrates a result of immunohistochemical staining of TOM34 in colon cancer tissues. (A and B) Representative images of spotting in cancer and non-cancer cells. Magnification: X40, (C and D) images of normal mucous membranes, and cancerous tissues (normal C, D; tumor). Magnification: X200. Figure 4 illustrates the deletion effect of TOM34 siRNA in the expression of the TOM34 gene or cell growth of the CRC cell line. (A) Effect of siRNAs on the expression of TOM34 in HCT116 cells. The expression of TOM34 was analyzed by Western blot analysis. The expression of beta-actin served as an internal control. EGFP-specific siRNA (siEGFP) was prepared for a negative control. (B) Effect of TOM34-siRNAs on the growth of HCT116 cells. Viability of HCT116 cells in response to EGFP-siRNA or TOM34-siRNAs was measured by MTT assay in triplicate. The error bars, SD; Asterisk denotes a significant difference (P <0.001) determined through a Scheff F test. Figure 5 illustrates the cytotoxic effect of the CTL line induced by TOM34-299 of 10 mer peptide. The CTL lines elevated through peptide 10 mer TOM34-299 showed peptide-specific cytotoxicity. The CTL lines showed high cytotoxic activity against target cells (TISI) loaded with TOM34-299, while these same ones did not show significant cytotoxic activity against the same target cells (TISI) without loaded peptide. This demonstrates that CTL lines have peptide-specific cytotoxicity. Figure 6 illustrates the peptide-specific and potent cytotoxicity of elevated CTL lines by TOM34-299. The CTL lines showed antigen-specific cytotoxicity and potent detectable in the E / T ratio as low as 1.2. Figure 7 illustrates potent cytotoxicity of CTL clones. CTL clones with very potent cytotoxicity can be established so that they are specific for TOM34-299. The CTL clones established from CTL lines against TOM34-299 showed varied extermination activities, having some of which extremely potent activity. Figure 8 illustrates HLA specificity of elevated CTL clones by TOM34-299. CTL clones raised by TOM34-299 recognize and lyse tumor cells endogenously expressing TOM34 in the HLA restricted mode.

The cytotoxic activities against colon cancer cell lines of DLD-1 and HT29 which are expressed endogenously TOM34, were tested using two CTL clones raised by TOM34-299 as effector cells. The SNU-C2A cells were used as the target which expresses endogenously TOM34 but does not express HLA-A24. CTL clones showed high cytotoxic activities against both DLD-1 and HT29 cells expressing TOM34 as well as HLA-A24. On the other hand, they showed non-significant cytotoxic activity against SNU-C2A cells, which express TOM34, but not HLA-A24. Figure 9 illustrates HLA specificity of elevated CTL clones by TOM34-299. The CTL clone raised by TOM34-299 specifically recognizes TOM34 in the form restricted by HLA-A24. It was carried out in cold objective inhibition test as described in the section on "materials and methods". HT29 cells labeled by Na251CrO were prepared as a hot target, while TISI cells with or without loaded TOM34-299 peptide were used without 51Cr labeling as cold targets. The E / T ratio was set at 20. The cytotoxic activity of the CTL clone against HT29 cells was inhibited by the addition of TISI cells only when loaded with the peptide. Figure 10 illustrates specificity of the activity Cytotoxicity of the elevated CTL clone by TOM34-299. The cytotoxic activity of the CTL clone elevated by TOM34-299 is specifically blocked by antibodies that recognize the HLA class I or CDd T cell surface antigens. The cytotoxic activity of the CTL clone was determined against HT29 cells in the presence of antibodies that recognize the T-cell surface antigens. CTL activity was clearly blocked by adding antibodies that recognize HLA class I or CDd and was marginally affected by the addition of antibodies to HLA class II or CD4, while not being inhibited at all by adding control antibody matched with isotype. Detailed Description of the Invention The words "a", "an" and "the", as used in the present invention, mean "at least one" unless specifically indicated otherwise. Throughout the present specification in the appended claims, unless the context requires otherwise, the word "comprises", and variations such as "comprising" and "comprising" will be understood as implying inclusion. of a member or step or group of members or steps manifested, but not the exclusion of any member or step or group of members or steps. The present invention is based in part on the discovery of the elevated expression of TOM34 in cells from colon tissues of a patient with CRC. The high gene expression was identified using an integral cDNA microform system. Using a microformation of cDNA containing 23,040 genes, the integration gene expression profiles of 20 patients were previously constructed. TOM34 expresses the high level in CRC patients. TOM34 identified in the present invention was used for diagnostic purposes as a CRC marker and as a genetic target, whose expression is altered to treat or alleviate a CRC symptom. When measuring the expression of TOM34 in a sample of cells, CRC is diagnosed. Similarly, by measuring the expression of TOM34 in response to various agents and treatment agents, CRC can be identified. The present invention involves determining (e.g., measuring) the expression of TOM34. Using sequence information provided by the entries to the GeneBank ™ databases for the TOM34 sequence, TOM34 is detected and measured using techniques known to those skilled in the art. For example, a sequence within the entries to the sequence databases corresponding to TOM34 is used to construct probes to detect the TOM34 RNA sequence, for example, in Northern blot hybridization analysis. As another example, the sequence can be used to construct primers, to specifically amplify TOM34, for example, in detection methods based on amplification, such as polymerase chain reaction based on reverse transcription. The level of expression of TOM34 in the population of test cells, for example, a tissue sample derived from a patient, is subsequently compared to the level of expression of TOM34 in a reference population. The population of the reference cell includes one or more cells for which the compared parameter is known, ie, CRC cells or non-CRC cells. Whether a pattern of gene expression in the population of the test cell compared to the reference cell population indicates or does not indicate CRC or a predisposition to it, depends on the composition of the reference cell population. For example, if the reference cell population is composed of non-CRC cells, a similar gene expression pattern in the population of the test cell and reference cell population indicates that the population of the test cell is non- CRC Conversely, if the reference cell population is made up of CRC cells, a similar gene expression profile between the population of test cells and the reference cell population indicates that the population of test cells includes CRC cells. A level of expression of TOM34 in a population of Test cells are considered altered expression levels if their expression level varies from the reference cell population by more than 1.0, 1.5, 2.0, 5.0, 10.0 or more times the level of expression of the corresponding TOM34 in the cell population of reference. The differential gene expression between a population of test cells and a population of reference cells is normalized to a control nucleic acid, eg, a maintenance gene. For example, a control nucleic acid is one which is known to not differ depending on the carcinogenic or non-carcinogenic state of the cell. The levels of control nucleic acid expression in the test and reference nucleic acid can be used to normalize signal levels in compared populations. Control genes include β-actin, glyceraldehyde 3-phosphate dehydrogenase or ribosomal P1 protein. The population of test cells is compared with multiple populations of reference cells. Each of the multiple reference populations may differ in the known parameter. Therefore, a population of test cells can be compared to a second population of reference cells known to contain, for example, CRC cells, as well as a second reference population known to contain, for example, non-CRC cells ( normal cells). The test cell is included in a type of tissue or cell sample of a subject that is known to contain, or is suspected to contain, CRC cells. The test cell is obtained from a body tissue or a body fluid, i.e., a biological fluid (such as blood or urine). For example, the test cell is purified from a tissue. Preferably, the population of test cells comprises an epithelial cell. The epithelial cell proceeds in a tissue known to be, or suspected of being, a CRC. The cells in the reference cell population are derived from a type of tissue similar to the test cell. Optionally, the reference cell population is a cell line, for example, a CRC cell line (positive control) or a normal non-CRC cell line (negative control). Alternatively, the population of control cells is derived from a database of molecular information derived from cells for which the parameter or condition tested is known. The subject is preferably a mammal. The mammal can be, for example, a human, a non-human primate, a mouse, rat, dog, cat, horse or cow. The expression of TOM34 described herein is determined as the level of protein or nucleic acid using methods known in the art. For example, Northern hybridization analysis using probes that specifically recognize the sequence can be used to determine expression genetics. Alternatively, expression is measured using PCR tests based on reverse transcription, for example, using primers specific for TOM34. Expression is also determined at the protein level, i.e., by measuring the polypeptide levels encoded by the gene products described herein, or biological activity thereof. Such methods are known in the art and include, for example, immunoassays based on antibodies to proteins encoded by TOM34. The biological activity of the protein encoded by the gene is also well known. CRC Diagnostics: Within the context of the present invention, CRC is diagnosed by measuring the level of expression of TOM34 from a cell test population (eg, a biological sample derived from a patient). Preferably, the population of test cells contains an epithelial cell, for example, a cell obtained from colon tissue. Genetic expression can also be measured from blood or other bodily fluids such as urine. Other biological samples can be used to measure protein levels. For example, the level of protein in blood or serum derived from a subject to be diagnosed can be measured by immunoassay or other conventional biological assay. The expression of TOM34 is determined in the test cell or biological sample and compared to the level of normal control expression associated with TOM34 tested. A normal control level is an expression profile of TOM34 normally found in a population known to not have CRC. An alteration (eg, an increase) in a level of expression in the TOM34 patient's thin tissue sample indicates that the subject is suffering from, or at risk of developing CRC. For example, an increase in the expression of TOM34 in the test population compared to the normal control level indicates that the subject is suffering or at risk of developing. The alteration of TOM34 in the test population compared to the normal control level indicates that the subject suffers from or is at risk of developing CRC. The level of expression of TOM34 in a biological sample can be estimated by quantifying a mRNA that corresponds to, or is encoded by, protein by TOM34. Quantitation methods for mRNA are known to those skilled in the art. For example, mRNA levels can be estimated by northern staining or RT-PCR. Since the nucleotide sequences of TOM34 are known, one skilled in the art can design the nucleotide sequences for probes or primers to quantitate TOM34. For example, the TOM34-specific primer group comprising the nucleotide sequence of SEQ ID NOs: 43 and 44, is the preferred primer.

Also the expression level of TOM34 can be analyzed based on the activity or amount of TOM34 protein. A method for determining the amount of TOM34 protein is shown below. For example, immunoassay methods are useful for the determination of proteins in biological materials. Any biological materials such as the biological sample can be used for the determination of the protein or its activity, provided that the TOM34 gene is expressed in the sample of a patient with colon cancer. For example, the colon tissue sample can be referred to as the biological sample. However, body fluids, such as blood and urine can also be analyzed. On the other hand, a suitable method can be selected for the determination of the activity of a protein encoded by the TOM34 gene according to the activity of a protein that will be analyzed. The level of expression of the TOM34 gene in a biological sample is estimated and compared to that of a normal sample (eg, a sample derived from a non-diseased subject). When said comparison shows that the level of expression of the genes is higher than that of the normal sample, it is judged that the subject will be affected with colon cancer. The level of expression of the TOM34 gene in biological samples from a normal subject and the subject to be diagnosed can be determined at the same time. As Alternatively, the normal ranges of expression levels can be determined through a statistical method based on the results obtained by analyzing the level of expression of the gene in samples previously collected from a control group. A result obtained by comparing the sample of a subject is compared with the normal range; When the result is not within the normal range, the subject is judged to be affected with, or at risk of developing colon cancer. In the present invention, a diagnostic agent for diagnosing colon cancer is also provided. The diagnostic agent of the present invention comprising a compound that binds to a polynucleotide or a TOM34 gene polypeptide, preferably, an oligonucleotide that hybridizes to the TOM34 gene polynucleotide, or an antibody that binds to the polypeptide encoded by the TOM34 gene, is You can use as said compound. In addition, also an aptamer, such as RNA, DNA or a peptide aptamer can be used as a compound. Preferably, the diagnostic agent comprises a detectable label such as a fluorescent, luminescent or bioluminescent label which is commonly known in the art. The method of the present invention for diagnosing colon cancer can be applied to evaluate the efficacy of colon cancer treatment in a subject. According to the method, a biological sample, such as a population of Test cells, is obtained from a subject undergoing treatment for colon cancer. The evaluation method can be carried out according to conventional methods to diagnose colon cancer. If desired, biological samples are obtained from the subject at various time points before, during or after treatment. The level of expression of the TOM34 gene in the biological sample is subsequently determined and compared to a level of derived control, for example, a population of reference cells which includes cells whose colon cancer status (i.e., cancer cell or non-cancerous cell) is known. The level of control is determined in a biological sample that has not been exposed to treatment. If the level of control is derived from a biological sample that does not contain a cancer cell, a similarity between the level of expression in the biological sample derived from the subject and the level of control indicates that the treatment is effective. A difference between the level of expression of the TOM34 gene in the biological sample derived from the subject and the level of control indicates a prognosis or less favorable clinical outcome. Identification of agents that inhibit TOM34 expression: An agent that inhibits the expression of TOM34 or the activity of its gene product can be identified by contacting a population of test cells expressing TOM34 with a test agent, and subsequently determining the level of TOM34. expression of TOM34 or the activity of its genetic product. A decrease in the expression level of TOM34 or in the level of activity of its gene product in the presence of the agent compared to the expression or activity level in the absence of the test agent, indicates that the agent is an inhibitor of TOM34. and it is useful in the inhibition of CRC. The test cell population can be any cell that expresses TOM34. For example, the population of test cells may contain an epithelial cell, such as a cell derived from colon tissue. In addition, the test cell can be an immortalized cell line derived from a carcinoma cell or colorectal cancer. Alternatively, the test cell may be a cell that has been transfected with TOM34 or which has been transfected with a regulatory sequence (eg, promoter sequence) from TOM34 operably linked to a reporter gene. Efficacy evaluation of CRC treatment in a subject: The TOM34 expressed differentially identified in the present invention also allows the course of CRC treatment to be monitored. In this method, a population of test cells is provided from a subject undergoing treatment for CRC. If desired, the population of test cells is obtained from the subject at various time points, before, during and / or after treatment. The expression of TOM34 in the cell population subsequently it is determined and compared to a population of reference cells which include cells whose CRC status is known. Within the context of the present invention, reference cells must not have been exposed to the treatment of interest. If the population of reference cells does not contain CRC cells, a similarity in the expression of TOM34 in the population of test cells and the population of reference cells, indicates that the treatment of interest is effective. However, a difference in TOM34 expression in the test population and a normal control reference cell population indicates a less favorable clinical outcome or prognosis. Similarly, if the population of reference cells contains CRC cells, a difference between the expression of TOM34 in the population of test cells and the population of reference cells indicates that the treatment of interest is effective, while a similarity in the expression of TOM34 in the test population and a population of cancer control reference cells indicates a less favorable clinical outcome or prognosis. In addition, the level of expression of TOM34 determined in a biological sample derived from a subject obtained after treatment (i.e., post-treatment levels) can be compared to the level of TOM34 expression determined in a biological sample derived from a subject obtained before the treatment (ie, pre-treatment levels). A decrease in the expression level of TOM34 in a post-treatment sample indicates that the treatment of interest is effective, while an increase or maintenance in the level of expression in the post-treatment sample indicates a less favorable clinical outcome or prognosis. As used in the present invention, the term "effective" indicates that the treatment leads to a reduction in the expression of an activated gene in pathological form, or decrease in size, prevalence or metastatic potential of CRC in a subject. When the treatment of interest is applied prophylactically, the term "effective" means that the treatment delays or prevents the formation of CRC or delays, prevents or alleviates the symptom of clinical CRC. The evaluation of colon tumors can be elaborated using standard clinical protocols. In addition, effectiveness can be determined in association with any known method for diagnosing or treating CRC. CRC can be diagnosed, for example, by identifying symptomatic abnormalities, for example, weight loss, abdominal pain, back pain, anorexia, nausea, vomiting and malaise, weakness and generalized predisposition. Selection of a therapeutic agent to treat CRC that is suitable for a particular individual: The difference in genetic matching of individuals it can give results differences in their relative capacities to metabolize several drugs. An agent that is metabolized in a subject to act as an anti-CRC agent can manifest itself, inducing a change in a pattern of gene expression in the subject's cells, of which it is characteristic of a carcinogenic state with a pattern characteristic of genetic expression of a non-carcinogenic state. Accordingly, the differentially expressed TOM34 described herein allows a putative therapeutic or prophylactic inhibitor of CRC to be tested in a population of test cells from a selected subject in order to determine whether the agent is an adequate inhibitor of CRC in the subject. To identify a CRC inhibitor that is suitable for a specific subject, a population of test cells from the subject are exposed to a therapeutic agent, and the expression of TOM34 is determined. Within the context of the method of the present invention, the test cell population contains a CRC cell that expresses TOM34. Preferably, the test cell is an epithelial cell. For example, a population of test cells can be incubated in the presence of a candidate agent and the gene expression pattern of the population of the test cell can be measured and compared with one or more reference profiles, for example, a expression profile non-CRC reference or a reference expression profile. A decrease in the expression of TOM34 in the population of test cells relative to a population of reference cells containing CRC, indicates that the agent has therapeutic potential. Within the context of the present invention, the test agent can be any compound or composition. Exemplary test agents include, but are not limited to, immunomodulatory agents. Classification assay for identifying therapeutic agents: Using the TOM34 gene, the proteins encoded by the gene or transcription regulation region of the gene, compounds that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene can be classified. These compounds are used as pharmaceuticals to treat or prevent CRC. Accordingly, the present invention provides a method for classifying a compound, to treat or prevent CRC using the TOM34 polypeptide. In one embodiment of this classification method comprises the steps of: a) contacting a test compound with a polypeptide encoded by a TOM34 polynucleotide; b) detecting the binding activity between the polypeptide and the test compound; Y c) selecting the test compound that binds to the polypeptide. The TOM34 polypeptide to be used for classification may be a recombinant polypeptide or a protein derived from nature or a partial peptide thereof. The polypeptide will be contacted with a test compound and can be, for example, a polypeptide purified as a soluble protein or a form linked to a carrier or a fusion protein fused to other polypeptides. As a method for classifying proteins, for example, that bind to the TOM34 polypeptide using the TOM34 polypeptide, many methods can be used that are well known to those skilled in the art. Such classification can be carried out, for example, by an immunoprecipitation method, specifically, in the following manner. The gene encoding the TOM34 polypeptide is expressed in host cells (for example animal) and thus by inserting the gene into an expression vector for external genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8. The promoter that will be used for the expression can be any promoter that can be commonly used and that includes, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol 3. Academic Press, London, 63 -141 (1981)), the EF-a promoter (Kim DW, and associates, Gene 91: 217-23 (1990)), the CAG promoter (Niwa and associates, Gene 108: 193-9 (1991)), the RSV LTR promoter (Cullen, Methods in Enzymology 152: 664-704 (1987)), the SRa promoter (Takebe et al., Mol Cell Biol 8: 466-72 (198d)) , the CMV immediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 64: 3365-9 (1987)), the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), the late adenovirus promoter (Kaufman et al., Mol Cell Biol 9: 946-58 (1989)), the HSV TK promoter, etc. The introduction of the gene into the host cells to express an external gene can be carried out according to any methods, for example, electroporation method (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol. Cell Biol 4: 1641-3 (1984)), the lipofectin method (Derijard, B Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)), etc. The polypeptide encoded by the TOM34 gene can be expressed as a fusion protein comprising a recognition site (epitope) of a monoclonal antibody introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C-terminus of the polypeptide. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (nineteen ninety five)). Vectors that express a fusion protein, for example, with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescence protein (GFP) etc., through the use of its multiple cloning sites, They are commercially available. A fusion protein prepared by introducing only small epitopes consisting of several up to a dozen amino acids, so that the property of the TOM34 polypeptide is not changed by fusion, is also reported. Epitopes, such as polyhistidine (His-tag), has added influenza, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), 10 T7 gene protein (T7-tag), glycoprotein of human herpes simplex virus (HSV-tag), E-tag (an epitope in the monoclonal phage), etc., the monoclonal antibodies that recognize them, such as the epitope-antibody system to classify proteins that bind to the TOM34 polypeptide (Experimental Medicine 13: 85-90 (1995)). In immunoprecipitation, an immune complex is formed by adding these antibodies to prepared cell lysate using a suitable detergent. The immune complex consists of TOM34 polypeptide, a polypeptide comprising the ability to bind to the polypeptide and an antibody. Immunoprecipitation can also be carried out using antibodies against the TOM34 polypeptide, in addition to the use of antibodies against the above epitopes, wherein the antibodies can be prepared as described above. An immune complex can be precipitated, for example through a Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody. If the polypeptide encoded by the TOM34 gene is prepared as a fusion protein with an epitope, such as GST, an immune complex can be formed in the same way as in the use of the antibody against the TOM34 polypeptide, using a substance that binds specifically to these epitopes, such as glutathione-sepharose 4B. The immunoprecipitation can be carried out following or according to, for example, the methods described in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)). SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed through the molecular weight of the protein using gels with a suitable concentration. Since the protein bound to the TOM34 polypeptide is difficult to detect through a common spotting method, such as Coomassie staining or silver staining, the detection sensitivity of the protein can be improved by culturing cells in a culture medium containing Isotope labeling proteins, 35S- methionine or 35S-cysteine in cells and detecting proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed. As a method for classifying proteins that bind to TOM34 polypeptide using the polypeptide, for example, spotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used. Specifically, a protein binding to the TOM34 polypeptide can be obtained by preparing a cDNA library from cells, tissues, organs (e.g., tissues such as testes or ovary) or cultured cells (e.g., CW2, DLDI, HCTI 16, HCT15, RKO, LS174T) are expected to express a protein linkage with the TOM34 polypeptide using a phage vector (eg ZAP), which expresses the protein in LB-agarose, fixing the protein expressed on a filter, reacting the polypeptide TOM34 purified or labeled with the above filter, and detecting the plates expressing proteins bound to the TOM34 polypeptide according to the label. The polypeptide of the present invention can be labeled using the biotin-avidin bond, or by using an antibody that specifically binds the TOM34 polypeptide, or a peptide or polypeptide (eg, GST) that is fused to the TOM34 polypeptide. You can also use methods to use radioisotopes or fluorescence. Alternatively, in another embodiment of the classification method of the present invention, a two-hybrid system utilizing cells ("MATCHMAKER Two-Hybrid system", "MATCHMAKER Mammalian Two-Hybrid Assay", "MATCHMAKER one- Hybrid "(Clontech);" HybpZAP Two-Hybrid Vector System "(Stratagene), the reference" Dalton and Treisman, Cell 68: 597-612 (1992) "," Fields and Sternglanz, Trends Genet 10: 286-92 (1994) ) "). In the two-hybrid system, the polypeptide of the present invention is fused to the SRF binding region or GAL4 binding region and expressed in yeast cells. A cDNA library is prepared from cells that are expected to express a protein that binds to the polypeptide of the present invention, such that the library, when expressed, is fused to the transcription activation region VP16 or GAL4. Subsequently, the cDNA library is introduced into the previous yeast cells and the cDNA derived from the library is isolated from the detected positive clones (when a protein binds to the polypeptide of the present invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by cDNA can be prepared by introducing the cDNA previously isolated for E. coli and expressing the protein.

As a reporter gene, for example, the Ade2 gene, lacZ gene, CAT gene, luciferase gene, etc. can be used. in addition to the HIS3 gene. A compound that binds to the polypeptide encoded by the TOM34 gene can also be classified using affinity chromatography. For example, the polypeptide of the present invention can be immobilized on an affinity column transporter, and a test compound, which contains a protein with the ability to bind to the polypeptide of the present invention, is applied to the column. A test compound in the present invention can be, for example, cell extracts, cell lysates, etc. After the test compound is loaded, the column is washed, and compounds linked to the polypeptide of the present invention can be prepared. When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an olive DNA is synthesized based on the sequence, and the cDNA libraries are classified using the oligo DNA as a probe to obtain a DNA that it encodes the protein.

A biosensor can be used using the surface plasmon resonance phenomenon as a means to detect or quantify the bound compound in the present invention. When said biosensor is used, the interaction between the polypeptide of the present invention and a compound of Test can be observed in real time as a surface plasmon resonance signal, using only a small amount of polypeptide and without labeling (eg BIAcore, Pharmacia). Accordingly, it is possible to evaluate the linkage between the polypeptide of the present invention and a test compound using a biosensor such as BIAcore. Methods for classifying the molecules that bind when the immobilized TOM34 polypeptide is exposed to synthetic chemical compounds or libraries of natural substances or a random phage display library, are well known to those of skill in the art methods for classification using high performance based on combination chemistry techniques (Wrighton and associates, Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9 (1996)) to isolate not only proteins but also chemical compounds that bind to the TOM34 protein (including agonists and antagonists). Alternatively, the present invention provides a method for classifying a compound for treating or preventing CRC using the polypeptide encoded by the TOM34 gene comprising the following steps: a) contacting a test compound with a polypeptide, encoded by a TOM34 polynucleotide; b) detect the biological activity of the passage polypeptide (to); and c) selecting the test compound that suppresses the biological activity of the polypeptide encoded by the TOM34 polynucleotide compared to the biological activity of the detected polypeptide in the absence of the test compound. Since the TOM34 protein has the activity of promoting cell proliferation of CRC cells, a compound that inhibits this activity of this protein can be classified, using this activity as an index. Any polypeptides can be used for classification, provided they understand the biological activity of the TOM34 protein. Said biological activity includes cell proliferation activity of the human TOM34 protein. For example, human TOM34 protein can be used and polypeptides functionally equivalent to these proteins can also be used. Said polypeptides can be expressed endogenously or exogenously through the cells. The compound isolated through this classification is a candidate for agonists or antagonists of the polypeptide encoded by the TOM34 gene. The term "agonist" refers to molecules that activate the function of the polypeptide by linking thereto. Similarly, the term "antagonist" is refers to molecules that inhibit the function of the polypeptide, binding to them. In addition, a compound isolated through this classification is a candidate for compounds that inhibit the in vivo interaction of the TOM34 polypeptide with molecules (including DNAs and proteins). When the biological activity that will be detected in the method of the present invention is cell proliferation, it can be detected, for example, by preparing cells expressing the TOM34 polypeptide, culturing the cells in the presence of a test compound, and determining the speed of cell proliferation, measuring the cell cycle etc., as well as measuring the colony formation activity as described in the Examples section. In additional embodiments, the present invention provides methods for classifying compounds to treat or prevent CRC. As described in detail above, by controlling the expression levels of TOM34, the generation and progress of CRC can be controlled. Therefore, compounds that can be used in the treatment or prevention of CRC can be identified through classifications that use TOM34 expression levels as indices. Within the context of the present invention, said classification may comprise, for example, the following steps: a) contacting a candidate compound with cells that express TOM34; and b) selecting the candidate compound that reduces the expression level of TOM34 compared to a control level in the absence of the candidate compound. Cells that express TOM34 include, for example, cell lines established from CRC; said cells can be used for the above classification of the present invention (for example CW2, DLD1, HCT116, RKO, LS174T). The level of expression can be estimated by methods known to those skilled in the art. In the classification method, a compound that reduces the expression level of TOM34 can be selected as candidate agents that will be used for the treatment or prevention of CRC. Alternatively, the classification method of the present invention may comprise the following steps: a) contacting a candidate compound with a cell in which a vector, comprising the transcription regulatory region of TOM34 and a reporter gene that is expressed under the control of the region of transcription regulation, has been introduced; b) measure the expression or activity of the reporter gene; and c) selecting the candidate compounds that reduce the expression or activity of the reporter gene. Suitable reporter genes and host cells are well known in the art. The reporter construction required for classification can be prepared using the transcription regulation region of a marker gene. When the transcription regulatory region of a marker gene has been known to those skilled in the art, a reporter construct can be prepared using the previous sequence information. When the transcriptional regulatory region of a marker gene remains unidentified, a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the marker gene. Examples of supports that can be used to bind proteins include insoluble polysaccharides, such as agarose, cellulose and dextran; and synthetic resins, such as polyacrylamide, polystyrene and silicone; commercially available beads and plates (eg multiple deposit plates, biosensor chip, etc.) prepared from the above materials. When accounts are used, they can be filled into a column. The binding of a protein to a support can be carried out according to routine methods, such as chemical bonding and physical adsorption. Alternatively, a protein can be linked to a support through antibodies that specifically recognize the protein. In addition, the binding of a protein to a support can also be conducted through medium of avidin and biotin. The link between proteins is carried out in a regulator, for example, but they are not limited to phosphate buffer and Tris buffer, as long as the regulator does not inhibit the link between the proteins. In the present invention, a biosensor can be used that uses a surface plasmon resonance phenomenon as a means to detect or quantify the bound protein. When said biosensor is used, the interaction between the proteins can be observed in real time as a surface plasmon resonance signal, using only a small amount of polypeptide and without labeling (for example BIAcore, Pharmacia). Alternatively, the TOM34 polypeptide can be labeled, and the tag of the linked protein can be used to detect or measure the bound protein. Specifically, after pre-labeling one of the proteins, the tagged protein is contacted with the other protein in the presence of a test compound, and subsequently the bound proteins are detected or measured according to the label after of the washing. Labeling substances such as radioisotope enzymes (eg, 3 H, 14 C, 32 P, 33 P, 35 S, 125 I, 131 I), (eg alkaline phosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase), fluorescent substances ( by example fluorescence isothiocyanate (FITC), rhodamine) and biotin / avidin can be used for the labeling of a protein in the method of the present invention. When the protein is labeled with a radioisotope, detection or measurement can be carried out by liquid scintillation. Alternatively, protein tags with enzyme can be detected or measured by adding a substrate of the enzyme to detect enzymatic change of the substrate, such as color generation, with an absorptiometer. In addition, in the case where a fluorescent substance is used as the label, the bound protein can be detected or measured using a fluorophotometer. In the case of using an antibody in the classification of the present invention, the antibody is preferably labeled with one of the aforementioned labeling substances, and is detected or measured based on the labeling substance. Alternatively, the antibody against the TOM34 polypeptide or actin can be used as a primary antibody to be detected with a secondary antibody that is labeled with a labeling substance. In addition, the antibody bound to the protein in the classification of the present invention can be detected or measured using a G protein or protein A column. Alternatively, in another embodiment of the classification method of the present invention, a two-hybrid system that uses cells ("MATCHMAKER Two-Hybrid system", "MATCHMAKER Mammalian Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)"). In the two-hybrid system, the TOM34 polypeptide of the present invention is fused to the SRF binding region or the GAL4 binding region and is expressed in yeast cells. As a reporter gene, for example, the Ade2 gene, the lacZ gene, the CAT gene, the luciferase gene etc., can be used in addition to the HIS3 gene. Any test compound, e.g., cell extracts, cell culture supernatant, microorganism fermentation products, marine organisms extracts, plant extracts, purified or crude proteins, peptides, compounds without peptides, synthetic micromolecular compounds and natural compounds can be use in the classification methods of the present invention. In the present invention, the test compound can be obtained using any of a number of methods in the methods of combination libraries known in the art, including (1) biological libraries, (2) solid phase libraries or parallel solution phase blotted in spatial form, (3) synthetic library methods that require deconvolution; (4) the "one count, one compound" library method; and (5) synthetic bioblin library methods that utilize affinity chromatography selection. Biological library methods using affinity chromatography selection are limited to peptide libraries, while four other methods are applicable to peptides, non-peptide oligomer or libraries of small molecule compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt and associates (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb and associates (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann and associates (1994) J. Med. Chem. 37: 2678-85; Cho et al. (1993) Science 261: 1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994) J. Med. Chem. 37: 1233-51). Compound libraries can be presented in solution (see Houghten's publication (1992) Bio / Techniques 13: 412-21), or in beads (see the publication Lam (1991) Nature 354: 82-4), pieces (Fodor (1993) Nature 364: 555-6), bacteria (US Patent Number 5,223,409), spores (US Patent Number 5,571,698; 5,403,484 and 5,223,409), plasmids (Culi and associates (1992) Proc. Natl. Acad. Sci. EUA 89: 1865-9) or phage (Scott and Smith (1990) Science 249: 386-90; Devlin (1990) Science 249: 404-6; CwMa and associates (1990) Proc. Natl. Acad. Sci. E.U.A. 87: 6378-82; Felici (1991) J. Mol. Biol. 222: 301-10; U.S. Patent Application Number 2002103360). A compound isolated through the classification methods of the present invention is a candidate for drugs that inhibit the activity of the TOM34 polypeptide, to treat or prevent diseases attributed, for example, to cell proliferation diseases, such as CRC. A compound in which a part of the structure of the compound obtained by the classification methods of the present invention is converted by addition, elimination and / or replacement, includes the compounds obtained by the classification methods of the present invention. Pharmaceutical compositions for treating or preventing CRC When administering an isolated compound through the method of the present invention as a pharmaceutical for humans and other mammals, such as mice, rats, Guinea pigs, rabbits, cats, dogs, sheep, pigs, cattle, monkeys, baboons and chimpanzees, the isolated compound can be administered directly or can be formulated in a dosage form using known pharmaceutical preparation methods. For example, according to the need, the drugs can be taken orally, such as sugar-coated tablets, capsules, elixirs and microcapsules, or in non-oral form, in the form of injections of solutions of sterile suspensions with water or any other pharmaceutically acceptable liquid. For example, the compounds can be mixed with vehicles or pharmaceutically acceptable media, specifically, sterilized water, physiological saline solution, plant oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, linkers, etc., in a dosage unit form required for the implementation of generally accepted drugs. The amount of active ingredient contained in said preparation, makes a suitable dose within the indicated range acquirable. Examples of additives that can be mixed into tablets and capsules include, but are not limited to, linkers, such as gelatin, corn starch, tragacanth gum and gum arabic. Excipients, such as crystalline cellulose, blowing agents, such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose or saccharin; and flavoring agents such as peppermint, Gaultheria adenothrix oil and cherry. When the unit dosage form is a capsule, a liquid carrier such as an oil may be additionally included in the above ingredients. Sterile compositions for injection can be formulated following normal drug implementations using vehicles, such as distilled water, suitable for injection. Physiological saline, glucose and other isotonic liquids, including adjuvants, such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, can be used as aqueous solutions for injection. These can be used together with suitable solubilizers, such as alcohol, for example, ethanol; polyalcohols, such as propylene glycol and polyethylene glycol; and nonionic surfactants, such as Polysorbate 80 (TM) and HCO-50. Sesame oil or soy bean oil can be used as an oleaginous liquid, in conjunction with benzyl benzoate or benzyl alcohol as a solubilizer, and can be formulated with a regulator, such as phosphate buffer and sodium acetate buffer; a pain killer, such as procaine hydrochloride; a stabilizer, such as benzyl alcohol and phenol; and / or an antioxidant. A prepared injection can be filled in a suitable vial. Methods known to those skilled in the art can be used to administer the pharmaceutical composition of the present invention to patients, for example as an intraarterial, intravenous or percutaneous injection or as an intranasal, transbronchial, intramuscular or oral administration. The dosage and method of administration vary according to the body weight and age of the patient and the method of administration; however, those skilled in the art can routinely select a suitable method of administration. If the compound can be encoded by DNA, the DNA can be inserted into a vector for gene therapy and the vector is administered to a patient to carry out the therapy. The dosage and method of administration vary according to the body weight, age and symptoms of the patient; however, one skilled in the art can select them appropriately. For example, although the dose of a compound that binds a protein of the present invention and regulates its activity depends on the symptoms, the dose will generally be from about 0.1 mg to about 100 mg per day, preferably from about 1.0 mg to about 50. mg per day and more preferably about 20 mg per day, when orally administered to a normal adult human (60 kg weight). When the compound is administered parenterally, in the form of an injection to a normal adult human (weight 60 kg) although there are some differences according to the patient, target organ, symptoms and method of administration, it is convenient to inject intravenously a dose of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg per day and more preferably about 0.1 mg. approximately 10 mg per day. In the case of other animals, the amount of adequate dose can be calculated routinely by converting to 30 kilos of body weight. Prognostic evaluation of a subject with CRC: The present invention also provides a method for evaluating the prognosis of a subject with CRC including the step of comparing the expression of TOM34 in a population of test cells with the expression of TOM34 in a population of Reference cells derived from patients through a spectrum of stage of the disease. By comparing the genetic expression of TOM34 in the population of test cells and the population (s) of reference cells, or by comparing the pattern of gene expression with time in populations of test cells derived from the subject, the prognosis can be evaluated of the subject. For example, an increase in the expression of TOM34, compared to a normal control indicates a less favorable prognosis. Conversely, a similarity in the expression of TOM34 compared to normal control indicates a more favorable prognosis for the subject. Apparatus: The present invention also includes a CRC-detection reagent, e.g., a nucleic acid that specifically binds or identifies TOM34 nucleic acids, such as oligonucleotide sequences that are complementary to a part of the TOM34 nucleic acid, or an antibody that binds to proteins encoded by TOM34 nucleic acid or an aptamer. The detection reagents can be packaged together in the form of a team. For example, the detection reagents can be packaged in separate containers, for example, a nucleic acid or antibody (either bound to a solid matrix or separately packed with reagents to bind them to the matrix), a control reagent (positive and / or negative) and / or a detectable label. The instructions (for example, written, tape, VCR, CD-ROM, etc.) to carry out the test can be included in the equipment. The assay format of the kit may be a Northern hybridization or a sandwich ELISA, both of which are known in the art. For example, a CRC detection reagent can be immobilized on a solid matrix, such as a porous strip, to form at least one CRC detection site. The measurement or detection of the region of the porous strip can include a plurality of sites, each containing a nucleic acid. A test strip may also contain sites for negative and / or positive controls. Alternatively, the control sites can be located on a separate strip of the test strip. Optionally, different detection sites may contain different amounts of immobilized nucleic acids, i.e., a greater amount in the first site of detection and lower amounts in subsequent sites. At the time of the addition of the test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of CRC present in the sample. The detection sites can be configured in any suitable detectable form and are usually in the form of a bar or point that spans the entire width of the test strip. Methods of CRC Inhibition: The present invention further provides a method for treating or alleviating a symptom of CRC in a subject, by decreasing the expression of TOM34 (or the activity of its gene product). Suitable therapeutic compounds can be administered prophylactically or therapeutically to a subject who suffers from or is at risk of (or is susceptible to) developing CRC. Such subjects can be identified using standard clinical methods or by detecting an aberrant expression level of TOM34 or aberrant activity of their gene product. Within the context of the present invention, suitable therapeutic agents include, for example, inhibitors of cell cycle regulation and cell proliferation. Alternatively, the therapeutic method of the present invention may include the step of decreasing the expression, function, or both, of the TOM34 gene products whose expression is increased in aberrant form ("activated" gene or "over-expressed") in colon cells. The expression can be inhibited in any of the various ways known in the art. For example, expression can be inhibited by administering to the subject a nucleic acid that inhibits or antagonizes the expression of the overexpressed gene, for example, an antisense oligonucleotide or a small interfering RNA that disrupts the expression of the overexpressed gene.

Of course, the herein described methods of treatment or relief apply, with due changes, to the use of a compound that decreases the expression of TOM34 (or the activity of its gene product) as described in the present invention. for the preparation of a pharmaceutical composition for treating or alleviating colon cancer in a subject. Said compound can be, for example, an antisense, siRNA, antibody, aptamers or a composition of ribozymes. Nucleic Acids Antisense and siRNA: As noted above, the antisense nucleic acids corresponding to the nucleotide sequence of TOM34 can be used to reduce the level of expression of the gene. The antisense nucleic acids correspond to TOM34 which are activated in CRC, and are useful for the treatment of CRC. Specifically, the antisense nucleic acids of the present invention can act by linking to the nucleotide sequence of 'TOM34, or mRNAs that they correspond to them, thereby inhibiting the transcription or translation of genes, promoting the degradation of mRNAs and / or inhibiting the expression of proteins encoded by TOM34, thus inhibiting the function of proteins. The term "antisense nucleic acids" as used in the present invention, comprises both nucleotides that are totally complementary to the target sequence and those that lack a match to one or more nucleotides, provided that the antisense nucleic acids can hybridize to specific to the target sequences. For example, the antisense nucleic acids of the present invention include polynucleotides having a homology of at least 70% or more, preferably at least 80% or more, more preferably at least 90% or more, even more preferably at least 95% or more. more or span at least 15 continuous nucleotides. Algorithms known in the art can be used to determine homology. The antisense nucleic acid of the present invention acts on cells that produce the proteins encoded by TOM34, binding to the DNA or mRNA that encodes the protein, inhibiting its transcription or translation, promoting the degradation of mRNA and inhibiting the expression of the protein, giving as result in this way the inhibition of protein function.

An antisense nucleic acid of the present invention can be prepared in an external preparation, such as a liniment or a cataplasm, by mixing it in additions as a suitable base material which is inactive against the nucleic acid. Also, as necessary, the antisense nucleic acids of the present invention can be formulated into tablets, powders, granules, capsules, liposome capsules, injections, solutions, nasal drops and freeze-drying agents by adding excipients, isotonic agents, solubilizers , stabilizers, preservatives, pain killers, etc. These can be prepared following known methods. The antisense nucleic acids of the present invention can be provided to the patient by direct application in the diseased site or by injection into a blood vessel, so that it reaches the diseased site. An antisense mounting means can also be used to increase the membrane's permeability and durability. Examples include, but are not limited to, liposomes, poly-L-lysine, lipids, cholesterol, lipofectin or derivatives thereof. The doses of the antisense nucleic acid derivative of the present invention can be adjusted appropriately according to the condition of the patient and used in desired amounts. For example, a range of 0.1 to 100 mg / kg, preferably from 0.1 to 50 mg / kg. The antisense nucleic acids of the present invention inhibit the expression of a protein thereof and are therefore useful for suppressing the biological activity of the protein of the present invention. In addition, expression inhibitors, comprising antisense nucleic acids of the present invention, are useful in that they can inhibit the biological activity of a protein of the present invention. The binding of the siRNA to a transcript corresponding to TOM34 in the target cell results in a reduction in protein production through the cell. The length of the oligonucleotide is at least 10 and can be as long as the transcription that occurs naturally. Preferably, the oligonucleotide is less than 75, 50, 25 nucleotides in length. More preferably, the oligonucleotide is 19-25 nucleotides in length. The antisense nucleic acids of the present invention include modified oligonucleotides. For example, thioated oligonucleotides may be useful for conferring nuclease resistance to an oligonucleotide. Likewise, a siRNA against TOM34 can be used to reduce the expression level of TOM34. In the present invention, the term "siRNA" refers to a double stranded RNA molecule that prevents translation of a target mRNA. Standard techniques for introducing siRNA into the cell can beUlE used, including those in which DNA is a template from which RNA is transcribed. Within the context of the present invention, the siRNA comprises a sense nucleic acid sequence and an antisense nucleic acid sequence against an activated marker gene, such as TOM34. The siRNA is constructed so that a single transcript has both sense and antisense sequences complementary to the target gene, e.g., a hairpin. A TOM34 siRNA hybridizes the target mRNA and in this way decreases or inhibits the production of the polypeptides encoded by TOM34, associating with the single-stranded mRNA transcription normally, interfering in this way with the translation and therefore, expression of the protein. Therefore, the siRNA molec of the present invention can be defined by their ability to hybridize specifically to TOM34 mRNA under stringent conditions. For the purposes of the present invention, the terms "hybridize" or "hybridize in specific form" are used to refer to the ability of two nucleic acid molec to hybridize under "stringent hybridization conditions". The phrase "stringent hybridization conditions" refers to conditions under which a nucleic acid moleccan anneal to its target sequence, usually in a complex mixture of nucleic acids, but not detectable for other sequences. Strict conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide for nucleic acid hybridization is found in the Tijssen publication, Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Probes, "Overview of Principles of Hybridization and the Strategy of Nucleic Acids Assays" (1993). Generally, stringent conditions are selected to have a temperature of about 5 to 10 ° C less than the thermal melting point (Tm) of the specific sequence at a pH of defined ionic strength. The Tm is the temperature (according to the defined ionic strength, pH and nucleic concentration) in which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (since the target sequences are in excess, in Tm 50% of the probes are occupied in equilibrium). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice the support, preferably 10 times the support hybridization. Exemplary stringent hybridization conditions may be the following: 50% formamide, 5x SSC and 1% SDS, incubating at a temperature of 42 ° C, or 5x SSC, incubating at a temperature of 65 ° C, with a 0.2 wash. x SSC and 0.1% SDS at a temperature of 50 ° C.

Within the context of the present invention, a siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides in length. More preferably, a siRNA is 19 to 25 nucleotides in length. The exemplary nucleic acid sequence for the production of siRNA TOM34 includes the nucleotide sequences of SEQ ID NO: 48 or 52 as the target sequence. In RNA or derivatives thereof, the base "t" must be replaced with "u" in the nucleotide sequences. Accordingly, for example, the present invention provides a double-stranded RNA moleccomprising the nucleotide sequence 5'-gaaagugucucuaucucca-3 '(SEQ ID NO: 48) or 5'-ggauggaaacugcagagac-3' (SEQ ID NO: 52). In order to increase the inhibition activity of siRNA, nucleotide "u" can be added at the 3 'end of the antisense strand of the target sequence. The number of "u" s that will be added is at least 2, generally from 2 to 10, preferably from 2 to 5. The aggregate "u" s forms a single strand at the 3 'end of the antisense strand of the siRNA. A TOM34 siRNA can be introduced directly into the cells in a form that has the ability to bind to the mRNA transcripts. In these embodiments, the siRNA molec of the present invention are usually modified as described above for antisense molec. Other modifications are also possible, example, siRNA conjugated to cholesterol have shown improved pharmacological properties (Song et al., Nature Med. 9: 347-351 (2003)). Alternatively, a DNA encoding the siRNA can be carried in a vector. Vectors can be produced, for example, by cloning the target sequence TOM34 into an expression vector having regulatory sequences linked in an operative manner flanking the sequence in a way that allows the expression (by transcription of the DNA molecule) of both strands (Lee, NS and associates, (2002) Nature Biotechnology 20: 500-505). An RNA molecule that is antisense to the mRNA of TOM34 is transcribed by a first promoter (e.g., a 3 'promoter sequence of the cloned DNA) and an RNA molecule that is the sense strand of the TOM34 mRNA is transcribed through a second promoter (e.g., a 5 'promoter sequence of the cloned DNA). The sense and antisense strands hybridize in vivo to generate siRNA constructs to silence TOM34. Alternatively, the two constructs can be used to create the sense and antisense strands in a siRNA construct. The cloned TOM34 can encode a construct having a secondary structure, for example, hairpins, wherein the simple transcript has both sense and antisense sequences complementary to the target gene. A loop sequence consisting of a sequence of Arbitrary nucleotides can be located between the sense and antisense sequence in order to form the hairpin loop structure. Therefore, the present invention also provides siRNA having the general formula 5 '- [A] - [B] - [A'] - 3 ', wherein [A] is a ribonucleotide sequence corresponding to a sequence that hybrid in a specific way to a mRNA or a TOM34 cDNA. In preferred embodiments, [A] is a ribonucleotide sequence corresponding to a sequence of TOM34, [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and [A '] is a ribonucleotide sequence consisting of the complementary sequence of [A]. The region [A] hybridizes to [A '], and subsequently a loop consisting of the region [B] is formed. The loop sequence may preferably be 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from the group consisting of the following sequences (http://www.ambion.com/tec lib / tb / tb 506.html). In addition, the loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque, J.-M., and associates, (2002) Nature 418: 435-438). CCC, CCACC or CCACACC: Jacque, J. M. and associates, (2002) Nature, Vol. 418: 435-438. UUCG: Lee, N.S., and associates. (2002) Nature Biotechnology 20: 500-505. Fruscoloni, P., and associates, (2003) Proc. Natl. Acad. Sci. USA 100 (4): 1639-1644. UUCAAGAGA: Dykxhoorn, D.M., and associates, (2002) Nature Reviews Molecular Cell Biology 4: 457-467. Accordingly, the loop sequence can be selected from the group consisting of CCC, UUCG, CCACC, CCACACC and UUCAAGAGA. Preferably the loop sequence is UUCAAGAGA ("ttcaagaga" in DNA). The exemplary hairpin siRNA suitable for use within the context of the present invention includes: for TOM34-siRNA-D (siD, for the target sequence of SEQ ID NO: 48) 5'-gaaaguguucucuacucca- [B] -uggaguagagaacacuuuc-3 '(SEQ ID NO: 49) and for TOM34-siRNA-E (siE, for the target sequence of SEQ ID NO: 52) 5'-ggauggaaacugcagagac- [B] -gucucugcaguuuccaucc-3 '(SEQ ID NO: 53) The nucleotide sequence of suitable siRNAs can be designed using a computer program of siRNA design available on the Ambion website (http://www.ambion.com/techlib/miscAsiARN finder.html). The computer program selects nucleotide sequences for the siRNA synthesis based on the following protocol. Site Selection Obiective siRNA: 1. Beginning with the AUG start codon of the object transcript, the downstream of the AA dinucleotide sequences is scanned. The emergence of each AA is recorded in the 19 nucleotides adjacent to the 3 'end with potential siRNA target sites. Tuschl, and associated, does not recommend against the siRNA design for the 5 'and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) and these may be richer in regulatory protein binding sites . The UTR binding proteins and / or translation initiation complexes can interfere with the binding of the endonuclease complex siRNA. 2. Compare the potential target sites with the human genome database and eliminate from consideration any target sequences that have significant homology to other coding sequences. The homology search can be carried using BLAST, which can be found on the NCBI server at www.ncbi.nlm.nih.gov/BLAST/. 3. Select target rating sequences for synthesis. In Ambion, several target sequences can be preferentially selected along the length of the gene for evaluation. The regulatory sequences flanking the TOM34 gene sequences can be identical or different, so that their expression can be modulated independently, or a temporary or spatial form. siRNA is transcribed intracellularly by cloning TOM34 templates, respectively, into a vector containing, for example, a polymerase transcription unit III of small nuclear RNA RNA (snRNA) U6 or the human H1 RNA promoter. To introduce the vector into the cell, a transfection enhancing agent can be used. FuGENE (Rochediagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen) and Nucleofector (Wako mash Chemical) are useful as the transfection enhancing agent. The antisense or siRNA oligonucleotide of the present invention inhibits the expression of a polypeptide thereof and is therefore useful for suppressing the biological activity of a polypeptide of the present invention. Also, expression inhibitors comprising the antisense oligonucleotide or siRNA of the present invention are useful at the point where they can inhibit the biological activity of the polypeptide of the present invention. Accordingly, a composition comprising an antisense or siRNA oligonucleotide of the present invention is useful for treating a CRC. Antibodies: Alternatively, the function of the TOM34 gene product which is overexpressed can be inhibited in CRC by administering a compound that binds to or inhibits the function of the gene products. For example, the compounds of a antibody that binds to the gene product of TOM34. The present invention relates to the use of antibodies, particularly antibodies against a protein encoded by TOM34, or a fragment of said antibody. As used in the present invention, the term "antibody" refers to an immunoglobulin molecule having a specific structure, which interacts (e.g., binds) only with the antigen that has been used to synthesize the antibody (e.g. , the gene product of an activated marker) or with an antigen closely related to it. In addition, an antibody can be a fragment of an antibody or a modified antibody, provided that it binds to the protein encoded by TOM34. For example, the antibody fragment can be Fab, F (ab ') 2, Fv, or single chain Fv (scFv), where the Fv fragments of the H and L chains are linked by a suitable linker (Huston JS and associates , Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). More specifically, an antibody fragment can be generated by treating an antibody with an enzyme, such as a papain or pepsin. Alternatively, a gene encoding the antibody fragment can be constructed, inserted into an expression vector, and expressed in a suitable host cell (see for example, Co MS and associate publications, J. Immunol., 152: 2968- 2976 (1994): Better M. and Horwitz AH Methods Enzymol 178: 476-496 (1989), Pluckthun A. and Skerra A. Methods Enzymol. 178: 497-515 (1989); Lamoyi E. Methods Enzymol. 121: 652-663 (1986); Rousseaux J. and associates, Methods Enzymol. 121: 663-669 (1986); Bird R. E. and Walker B. W. Trends Biotechnol. 9: 132-137 (1991)). An antibody can be modified by conjugation with a variety of molecules, such as polynetin I (PEG). The present invention provides modified antibodies. The modified antibody can be obtained by chemical modification of an antibody. Said methods of modification are conventional in this field. Alternatively, an antibody can comprise a chimeric antibody having a variable region derived from a non-human antibody and a constant region derived from a human antibody, or a humanized antibody, comprising a region of complementarity determination (CDR) derived from a non-human antibody, a framework region (FR) and a constant region derived from a human antibody. Said antibodies can be prepared using known technologies. Humanization can be carried out by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (see for example, Verhoeyen and associated Science 239: 1534-1536 (1988)). Accordingly, said humanized antibodies are chimeric antibodies, wherein less than one intact human variable domain has been replaced by the corresponding sequence from non-human species. Fully human antibodies comprising human variable regions in addition to human and constant arm regions can also be used. Such antibodies can be produced using various techniques known in the art. For example, in vitro methods involve the use of recombinant libraries or human antibody fragments deployed in bacteriophage (eg, Hoogenboom &Winter, J. Mol. Biol. 227: 381-8 (1991). Human antibodies can be made by introducing the human immunoglobulin loci into transgenic animals, for example, mice in which the endogenous immunoglobulin genes have been partially or completely deactivated.This method is described, for example, in US Patents Nos. 6,150,584; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016. Targeted cancer therapies in specific molecular alterations that occur in cancer cells have been validated through clinical development and regulatory approval of anti-cancer drugs such as trastuzumab (Herceptin) for the treatment of advanced breast cancer, imatinib mesylate (Gleevec) for chronic myeloid leukemia, gefitinib (Ire ssa) for non-small cell lung cancer (NSCLC), and rituximab (anti-CD20 mAb) for B-cell lymphoma and mantle cell lymphoma (Ciardiello F. and associates, Clin Cancer Res. 2001 October; 7 (10): 2958-70. Slamon DJ. and associates, N Engl J Med. 2001 March 15; 344 (11): 783-92; Rehwald U. and associates, Blood. 2003 January 15; 101 (2): 420-424; Fang G. and associates, (2000). Blood, 96, 2246-2253). These drugs are clinically effective and better tolerated than traditional anti-cancer agents because they target only transformed cells. Therefore, these drugs not only improve the survival and quality of life of cancer patients, but also validate the concept of molecularly directed cancer therapy. In addition, targeted drugs can increase the efficacy of standard chemotherapy when used in combination with them (Gianni L. (2002), Oncology, 63 Suppl 1, 47-56, Klejman A. and associates, (2002). , 21, 5868-5876). Accordingly, future cancer treatments will likely involve a combination of conventional drugs with target-specific agents for different characteristics of tumor cells, such as angiogenesis and invasiveness. These ex vivo or in vitro modulation methods can be carried out (e.g., by culturing the cell with the agent), or alternatively, in vivo (e.g., by administering the agent to a subject). The methods involve administering a protein or combination of proteins or a nucleic acid molecule or combination of nucleic acid molecules as therapy to counteract the aberrant expression of genes expressed in differential form or aberrant activity of their genetic products. Diseases and disorders that are characterized by increased levels of expression (relative to a subject that does not suffer from the disease or disorder) or biological activities of genes- and gene products, respectively, can be treated with antagonistic therapeutics (i.e. , reduce or inhibit) the activity of the gene or genes over-expressed. Therapeutics that antagonize with activity can be administered in a therapeutic or prophylactic manner. Accordingly, therapeutics that can be used within the context of the present invention include, for example, (i) a polypeptide of the overexpressed gene or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to the over-expressed gene or gene products; (iii) nucleic acids encoding the overexpressed gene; (iv) antisense nucleic acids or nucleic acids that are "dysfunctional" (ie, due to a heterologous insertion within the nucleic acids of the overexpressed gene); (v) small interference RNA (siRNA); or (vi) modulators (i.e., inhibitors, antagonists that alter the interaction between an overexpressed polypeptide and < its link part). Dysfunctional antisense molecules are used to "eliminate" the endogenous function of a polypeptide by recombination homologous (see for example, the publication of Capecchi, Science 244: 1288-1292 1989). The increased levels can be easily detected by quantifying the peptide and / or RNA, obtaining a tissue sample from the patient (for example, from biopsy tissue) and analyzing it in vitro with respect to RNA or peptide levels, structure and / or activity of expressed peptides (or mRNAs of a gene whose expression is altered). Methods that are well known within the art, include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS), immunocytochemistry, etc. .) and / or hybridization assays for detecting the expression of mRNAs (eg, Northern, spotted, in situ hybridization, etc.). Prophylactic administration occurs before the manifestation of palpable clinical symptoms of the disease, so that a disease or disorder can be avoided, or alternatively, delayed in its progress. The therapeutic methods of the present invention may include the step of contacting a cell with an agent that modulates one or more of the activities of the gene products of the genes expressed differentially. Examples of agents that modulate protein activity include, but are not they are limited to nucleic acids, proteins, naturally occurring cognate ligands of said proteins, peptides, peptide mimetics and other small molecules. Vaccination against CRC: In the present invention, the peptide derived from TOM34 proved to be TAA epitopes restricted by HLA-A24, which is an HLA allele common in Japanese and Caucasian populations. Candidates of HLA-A24 binding peptides derived from TOM34 were identified using the information with respect to their binding affinities to HLA-A24. After in vitro stimulation of the T cells by dendritic cells (DCs) loaded with these peptides, CTL was established in a successful manner using TOM34-299 (KLRQEVKQNL (SEQ ID No. 7)). These CTLs demonstrated potent cytotoxic activity against colorectal carcinoma cells. In addition, clones and CTL lines derived from these cells also showed specific cytotoxicity against HLA-A24 positive colorectal carcinoma cell lines that endogenously over-express TOM34. The cytotoxic activities of these clones and CTL lines were not demonstrated against cell lines lacking the expression of either HLA-A24 or a target TAA. The specific cytotoxic activities of these clones and CTL lines were significantly inhibited by the cold target. These results show that TOM34 is useful as TAAs of CRC and that TOM34 are epitope peptides of TAA restricted by HLA-A24. Since the TOM34 antigen is over-expressed in most CRCs and is associated with tumor cell proliferation, they are good targets that will be used for CRC immunotherapy. Accordingly, the present invention further provides methods for treating or preventing CRC, wherein the methods comprise the steps of administering an immunogenic peptide or less than about 40 amino acids, often less than about 20 amino acids, usually less than about 15 amino acids comprising the amino acid sequence of SEQ ID NO: 7. Alternatively, the immunogenic peptide may comprise a derivative of the sequence of SEQ ID NO: 7, wherein 1, 2, 3 or several amino acids are modified, substituted or added. In preferred embodiments, the immunogenic peptide is a non-peptide or a decapeptide. Alternatively, the present invention provides a method for inducing antitumor immunity for CRC, and wherein the method comprises the steps of administering an immunogenic peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 7 or a derivative thereof. as described above. In the present invention, the peptide or derivative thereof can be administered to a subject in vivo or ex vivo. In addition, the present invention also provides the use of a decapeptide comprising the amino acid sequence of SEQ ID NO: 7 or a derivative thereof to make an immunogenic composition for treating or preventing CRC. The homology analyzes of TOM34 showed that they have no significant homology with the peptides derived from any known human gene products. This decreases the possibility of unknown or unwanted immune responses with immunotherapy against these molecules. With respect to HLA antigens, the use of type A-24 that is highly expressed among Japanese, is favorable for effective results, and the use of subtypes such as A-2402 is even more preferred. Normally, in the clinical aspects, the type of HLA antigen of the patient that requires treatment is investigated in advance, which allows the appropriate selection of peptides that have high levels of binding affinity with this antigen, or that have inducibility of cytotoxic T cell. (CTL) by presentation of antigens. In addition, in order to obtain peptides showing high binding affinity and CTL inducibility, substitution or addition of 1, 2, 3, 4 or several amino acids based on the amino acid sequence of the partial peptide TOM34 can be carried out. It occurs naturally. Here, the term "several" means 5 or less, or preferably 3 or less. In addition, in addition to the peptides that are naturally deployed, since the regularity of the sequences of Peptides displayed by binding to HLA antigens is already known (Kubo, and associates, J. Immunol., 152, 3913, 1994; Rammensee, and associates, Immunogénetics., 41: 178-228, 1995; Kondo, and associates, J. Immunol. 155: 4307-12, 1995), modifications based on such regularity can be carried out on the immunogenic peptides of the present invention. For example, peptides showing high-level HLA-24 binding affinity have their second amino acid of the N-terminus substituted with phenylalanine, tyrosine, methionine or tryptophan, and peptides whose amino acid in C-term is substituted with phenylalanine, leucine, triptofan or methionine, can also be used favorably. However, when the peptide sequence is identical to a part of the amino acid sequence an endogenous or exogenous protein having different functions, side effects such as autoimmune disorders or allergic symptoms against specific substances can be induced, therefore, preferably, they avoid situations in which the sequence matches the amino acid sequence of another protein, carrying out a homology investigation using available databases. Furthermore, it is clear from the homology investigations that even when there are no peptides in which 1 or 2 amino acids are different, there is no danger that the modifications of the amino acid sequences before mentioned, in order to increase the binding affinity with HLA antigens and / or increase CTL inducibility, these problems arise. Although peptides that have high binding affinity to HLA antigens as described above are expected to be highly effective as cancer vaccines, the candidate peptides, which are selected according to the presence of the high binding affinity as a indicator, should be checked for the actual presence of CTL inducibility. Confirmation of CTL inducibility is achieved by inducing cells presenting antigens having human MHC antigens (e.g., B-lymphocytes, macrophages and dendritic cells), or more specifically dendritic cells derived from human peripheral blood mononuclear leukocytes, and after stimulation with the peptides, mixed with CD-8 positive cells, and subsequently measuring the cytotoxic activity against target cells. In the form of the reaction system, transgenic animals that have been produced to express a human HLA antigen can be used (for example, those described in the publication by BenMohamed L. et al., Hum. Immunol., 2000 August; 61 (8) : 764-79). For example, the target cells can be radiolabelled with 5 Cr, and therefore, a cytotoxic activity can be calculated from the radioactivity released from the target cells. As an alternative, you can check by measuring the IFN-? produced and released by CTL in the presence of cells presenting antigens carrying immobilized peptides, and visualizing the zone of inhibition in the medium using anti-IFN-α monoclonal antibodies. As a result of the CTL inducibility review of peptides as described above, those with high affinity binding to an HLA antigen do not necessarily have high inducibility. In addition, the decapeptide comprising the amino acid sequences indicated by KLRQEVKQNL (SEQ ID NO: 7), showed particularly high CTL inducibility. As noted above, the present invention provides peptides having cytotoxic T-cell inducibility, and comprising the amino acid sequence of SEQ ID NO: 7 wherein 1, 2, 3, 4 or some amino acids are substituted or aggregated. The amino acid sequences comprising 9 or 10 amino acids indicated in SEQ ID NO: 7, in which 1, 2, 3, 4 or several amino acids are substituted or aggregated, preferably do not coincide with the amino acid sequence of other proteins. In particular, the substitution of amino acids to phenylalanine (F), tyrosine, (Y), methionine (M) or tryptophan (W) in the second amino acid of the N-term, and to phenylalanine (F), leucine (L), isoleucine ( I), tryptophan (W) or methionine (M) at the C-terminal amino acid, and the addition of amino acids of 1 or 2 amino acids N-term and / or C-term are favorable examples. Specifically, the present invention provides a decapeptide comprising the amino acid sequence KLRQEVKQNL (SEQ ID NO: 7), or K- [L, F, Y, M or W] -RQEVKQN- [L, F, L, I , W or M]. The peptides of the present invention can be prepared using well known techniques. For example, the peptides can be prepared in synthetic form, by recombinant DNA technology or chemical synthesis. The peptide of the present invention can be synthesized individually, or in the form of longer polypeptides comprising two or more peptides. The peptides are preferably isolated, i.e., they are substantially free of other naturally occurring host cell proteins and fragments thereof. The peptides may contain modifications such as glycosylation, side chain oxidation or phosphorylation; provided that the modifications do not destroy the biological activity of the peptides as described in the present invention. Other modifications include incorporation of D-amino acids or other amino acid mimetics that can be used, for example, to increase the serum half-life of the peptides. The peptides of the present invention can be prepared in a combination, which comprises two or more peptides of the present invention, to be used as a cancer vaccine that can induce CTL in vivo. The peptides are in a cocktail or can be conjugated to each other using standard techniques. For example, the peptides can be expressed as a simple polypeptide sequence. The peptides in the combination may be the same or different. In administering the peptides of the present invention, the peptides are presented in a high density of the HLA antigens that present cells, then CTL are induced that react in a specific manner to the complex formed between the unfolding peptide of the HLA antigen. Alternatively, the antigen presenting the cells having the peptides of the present invention immobilized on their cell surface, is obtained by removing dendritic cells from the subjects, these are stimulated through the peptides of the present invention, CTL is induced in the subjects administering these cells again to the subjects, and as a result, the aggressiveness towards the target cells can be increased. More specifically, the present invention provides drugs for treating tumors or preventing proliferation, metastasis of said tumors, which comprise 1 or more of the peptides of the present invention. The peptides of the present invention can be used to treat CRC. The peptides of the present invention can be administered directly as a pharmaceutical composition that has been formulated through formulation methods conventional In such cases, in addition to the peptides of the present invention, vehicles, excipients, and those ordinarily used for drugs, as appropriate without particular limitations, may be included. The immunogenic compositions of the present invention can be used for treatment and prevention of CRC. The immunogenic compositions for treatment and / or prevention of CRC, which comprise the peptides of the present invention as the active ingredients, may comprise an adjuvant so that cellular immunity is established effectively, or can be administered with other active ingredients. such as antitumor agents, and can be administered by granule formulation. An adjuvant that can be applied includes those described in the literature (Johnson, Clin Microbiol. Rev., 7: 277-289, 1994). Adjuvant examples include, aluminum phosphate, aluminum hydroxide or alum. In addition, liposome formulations, granular formulations in which the drug binds to beads with a few μm in diameter, and formulations in which the lipid binds to the peptide, can be used conveniently. The method of administration may be oral, intradermal, subcutaneous, intravenous, etc., and systemic administration or local administration in the vicinity of the targeted tumor is possible. The dose of the peptides of the present invention is it can adjust in an appropriate manner according to the disease to be treated, the age of the patient, the weight, method of administration, etc., and is ordinarily 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, more preferably 0.1 mg to 10 mg, and is preferably administered once from a few days to a few months. Those skilled in the art can appropriately select the correct dose. Alternatively, the present invention provides intracellular vesicles called exosomes, which present complexes formed between the peptides of the present invention and the HLA antigens on their surface. Exosomes can be prepared, for example, using the methods described in detail in the Japanese Published Translation of International Publications Nos. Hei 11-510507 and 2000-512161, and is preferably prepared using cells that present antigens obtained from subjects to whom the treatment and / or prevention is directed to them. The exosomes of the present invention can be inoculated as cancer vaccines, similar to the peptides of the present invention. The type of HLA antigen used must coincide with that of the subject that requires treatment and / or prevention. For example, for Japanese, HLA-A24, particularly HLA-A2402 is often appropriate. In some embodiments, the vaccine compositions of The present invention comprises a component which primes cytotoxic T lymphocytes. Lipids have been identified as agents with the ability to prime CTL in vivo against viral antigens. For example, palmitic acid residues can be adhered to the e and a-amino groups of a lysine residue and subsequently linked to an immunogenic peptide of the present invention. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated in a liposome, or emulsified in an adjuvant. As another example of lipid primer of CTL responses, E. coli lipoproteins such as tripalmitoyl-S-glycerylcysteiniseryl-serine (P3CSS) can be used to prime CTL when they are covalently adhered to a suitable peptide (see for example, the publication of Deres and associates, Nature 342: 561-4, 1989). The immunogenic compositions of the present invention may also comprise nucleic acids encoding the immunogenic peptides described herein. See, for example, the publication by Wolff and associates (1990) Science 247: 1465-1468; US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647 and publication WO 98/04720. Examples of DNA-based delivery technologies include "discovered DNA", facilitated administration (transmitted by bupivicaine, polymers, peptides), cationic lipid complexes and administration transmitted by particles ("genetic history") or transmitted by pressure (see for example, US Patent No. 5,922,687). The immunogenic peptides of the present invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccines or pox "fowlpox" viruses. This method involves the use of vaccine viruses, for example, as a vector to express nucleotide sequences encoding the peptide. At the time of introduction into a host, the recombinant vaccine virus expresses the immunogenic peptide, and therefore elicits an immune response. Vaccine vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in the publication of Stover, et al. (1991) Nature 351: 456-460. A wide variety of other vectors useful for therapeutic administration or immunization, eg, adeno- and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, may be appreciated. See, for example, the publications of Shata and associates (2000) Mol. Med. Today 6: 66-71; Shedlock, and associates (2000) J. Leukoc. Biol. 68: 793-806; and Hipp, and associates (2000) In vivo 14: 571- The present invention also provides methods for inducing cells that present antigens using the peptides of the present invention. Cells presenting antigens can be induced by inducing dendritic cells of peripheral blood monocytes and subsequently contacting them (stimulation) with the peptides of the present invention in vitro, ex vivo or in vivo. When the peptides of the present invention are administered to the subjects, cells presenting antigens having the peptides of the present invention immobilized thereto are induced in the body of the subject. Alternatively, after the peptides of the present invention are immobilized to antigen presenting cells, the cells can be administered to the subject in the form of a vaccine. For example, ex vivo administration may comprise the steps of: a: collecting cells that present antigens from the subject; and b: contacting the cells that present antigens from step a, with the peptide. Cells presenting antigens obtained through step b can be administered to the subjects in the form of a vaccine. The present invention also provides a method for inducing cells that present antigens that have a high level of cytotoxic T cell inducibility, wherein the method comprises the step of transferring genes comprising polynucleotides encoding the peptides of the present invention to cells presenting antigens in vitro. The introduced genes can be in the form of DNAs or RNAs. For the introduction method, without particular limitations, various methods carried out in a conventional manner in this field can be used, such as lipofection, electroporation and calcium phosphate methods. More specifically, it can be carried out as described in the publications of Reeves, et al., Cancer Res., 56: 5672, 1996; Butterfield, et al., J. Immunol., 161: 5607, 1998; Boczkowski, et al., J. Exp. Med., 184: 465, 1996; Published Japanese Translation of International Publication No. 2000-509281. When transferring the gene into cells that present antigens, the gene goes through transcription, translation and as such in the cell, and subsequently the protein obtained is processed by MHC Class I or Class II, and proceeds through a presentation path to present partial peptides. In addition, the present invention provides methods for inducing CTL using the peptides of the present invention. When the peptides of the present invention are administered to a subject, CTL is induced in the body of the subject, and the resistance of the immune system to direct the CRC cells in the tumor tissues is increased. Alternatively, they can be used for an ex vivo therapeutic method, wherein the cells presenting antigens derived from the subject, and the CD8 positive cells or peripheral blood mononuclear leukocytes are contacted (stimulated) with the peptides of the present invention in vitro, and after induce CTL, the cells are returned to the subject. For example, the method may comprise the steps of: a: collecting cells that present antigens from the subject; b: contact cells that present antigens from step a, with the peptide; c: mix the cells presenting antigens from step b with CD8 + T cells, and culture them together to induce cytotoxic T-cells; and d: collecting CD8 + T cells from the culture as a whole from step c. CD8 + T cells having cytotoxic activity obtained through step d, can be administered to the subject in the form of a vaccine. Accordingly, the present invention relates to CD8 + cells that have the ability to recognize the peptides described herein, which are presented through MHC molecules, for example, HLA-24. In addition, the present invention also relates to cells, for example, APCs comprising the MHC-class I-type HLA-24 molecule having the peptide described herein. In addition, the present invention provides isolated cytotoxic T cells that are induced using the peptides of the present invention. The cytotoxic T cells, which have been induced by stimulation from cells presenting antigens presenting the peptides of the present invention, are preferably derived from subjects who are targeted for treatment and / or prevention, and can be administered by themselves. or in combination with other drugs including the peptides of the present invention or exosomes for the purposes of antitumor effects. The cytotoxic T cells obtained act in a specific manner against target cells presenting the peptides of the present invention, or preferably the same peptides used for induction. The target cells can be cells that express endogenously TOM34, or cells that are transfected with TOM34 genes, and cells that present the peptides of the present invention to the cell surface due to stimulation through these peptides, can also become objects of attack.

The present invention also provides cells that present antigens comprising the presentation of complexes formed between HLA antigens and the peptides of the present invention. The cells that present antigens that are obtained by contacting the peptides of the present invention, or the nucleotides encoding the peptides of the present invention, are derived preferably from subjects that are targets of treatment and / or prevention, can be administered as vaccines per se same or in combination with others drugs including the peptides of the present invention, exosomes or cytotoxic T cells. In the present invention, the phrase "vaccine" (also referred to as an immunogenic composition) refers to a substance that has the function of inducing anti-tumor immunity or immunity to suppress CRC at the time of inoculation in animals. In accordance with the present invention, polypeptides comprising the amino acid sequence of SEQ ID NO: 7, are suggested to be epitope peptides restricted by HLA-A24 that can induce a potent and specific immune response against CRC cells expressing TOM34. . Therefore, the present invention also comprises the method of inducing anti-tumor immunity using polypeptides comprising the amino acid sequence of SEQ ID NO: 7. In general, anti-tumor immunity includes immune responses such as the following: induction of cytotoxic lymphocytes against tumors comprising cells expressing TOM34, induction of antibodies recognizing tumors comprising cells expressing TOM34, and induction of antitumor cytokine production. Accordingly, when a certain protein induces any of these immune responses at the time of inoculation in an animal, it is decided that the protein has an effect of inducing anti-tumor immunity. The induction of Anti-tumor immunity through a protein can be detected by observing in vivo or in vitro the response of the immune system in the host against the protein. For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. An external substance that enters the living body is presented to T cells and B cells by the action of antigen-presenting cells (APCs). The T cells that respond to the antigen presented by APC in a specific form of the antigen, differentiate into cytotoxic T cells (or cytotoxic T lymphocytes; CTLs) due to stimulation through the antigen, and subsequently proliferate (this is referred to as T cells). Accordingly, CTL induction through a certain peptide can be evaluated by presenting the peptide to a T cell by APC, and detecting the induction of CTL. In addition, APCs have the effect of activating CD4 + T cells, CD8 + T cells, macrophages, eosinophils and NK cells. Since CD4 + T cells are also important in antitumor immunity, the action that induces anti-tumor immunity of the peptide can be evaluated using the activation effect of these cells as indicators. A method for evaluating the CTL induction action using dendritic cells (DCs) in the form of APC is well known in the art. DC is -a representative APC that has the strongest CTL induction action among APCs. In In this method, the test polypeptide is contacted initially with DC and subsequently this DC is contacted with T cells. The detection of T cells having the cytotoxic effect against the cells of interest after contact with DC, shows that the test polypeptide has an activity to induce cytotoxic T cells. CTL activity against tumors can be detected, for example, by using lysis of 51Cr-labeled tumor cells as the indicator. Alternatively, the method for evaluating the degree of tumor damage using 3H-thymidine uptake activity or release by LDH (lactose dehydrogenase) as the indicator, is also well known. In addition to DC, peripheral blood mononuclear cells (PBMCs) can also be used as APC. The CTL induction is reported to be increased by PBMC culture in the presence of GM-CSF and I L-4. Similarly, CTL has been shown to be induced by PBMC culture in the presence of magnetic penetration hormone (KLH) and I L-7 hemocyanin. Test polypeptides confirmed to possess CTL-inducing activity through these methods are polypeptides that have a DC activation effect and subsequent CTL induction activity. Accordingly, polypeptides that induce CTL against tumor cells are useful as vaccines against CRC. In addition, the APC that acquired the ability to induce CTL against CRC by contacting the polypeptides are useful as vaccines against CRC. In addition, CTL that acquired cytotoxicity due to the presentation of the polypeptide antigens by APC can also be used as vaccines against CRC. Such therapeutic methods for CRC using anti-tumor immunity due to APC and CTL are referred to as cellular immunotherapy. Generally, when a polypeptide is used for cellular immunotherapy, the efficiency of induction of a CTL is known to increase by combining a plurality of polypeptides having different structures and contacting them with DC. Therefore, when DC is stimulated with protein fragments, it is convenient to use a mixture of multiple types of fragments. Alternatively, the induction of anti-tumor immunity through a polypeptide can be confirmed by observing the induction of antibody production against tumors. For example, when antibodies against a polypeptide are induced in a laboratory animal immunized with the polypeptide, and when they grow, the proliferation or metastasis of tumor cells is suppressed by these antibodies, the polypeptide can be determined as having the ability to induce anti-tumor immunity. Anti-tumor immunity is induced by administering the vaccine of the present invention, and induction of immunity Anti-tumor enables the treatment and prevention of CRC. Therapy against or prevention of CRC generation includes, any steps such as inhibition of CRC cell growth, involution of CRC cells and suppression of CRC cell emergence. The decrease in mortality of individuals who have CRC, decrease in CRC markers in the blood, relief of detectable symptoms that accompany CRC and the like, is also included in the therapy or prevention of CRC. Said therapeutic and preventive effects are preferably statistically significant. For example, under observation, at a level of significance of 5% or less, where the therapeutic or preventive effect of a CRC vaccine is compared to a control without vaccine administration, for example, Student's t-test, Mann-Whitney test-U or ANOVA can be used -for statistical analysis.

The above-mentioned protein, which has immunological activity, or a polynucleotide or vector encoding the protein can be combined with an adjuvant. An adjuvant refers to a compound that improves the immune response against the protein when administered together (or in succession) with the protein having immunological activity. Examples of adjuvants include cholera toxin, salmonella toxin, alum and the like, but are not limited thereto. In addition, the vaccine of the present invention can be combined in a suitable manner with a pharmaceutically acceptable carrier. The examples of such vehicles are sterile water, physiological saline solution, phosphate buffer, culture fluid and the like. In addition, the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants, etc. The vaccine is administered systemically or locally. The administration of vaccines can be carried out by simple or reinforced administration by multiple administration. When APC or CTL is used as the vaccine of the present invention, CRC can be treated or prevented, for example, by the ex vivo method. More specifically, the PBMCs are collected from the subject, who receives treatment or prevention, the cells are contacted with the polypeptide ex vivo, and after the induction of APC or CTL, the cells can be administered to the subject. APC can also be induced by introducing a vector encoding the polypeptide in PBMCs ex vivo. One can clone APC or CTL induced in vitro before administration. By cloning and growing cells that have high activity to damage target cells, cellular immunotherapy can be carried out more effectively. In addition, APC and CTL isolated in this form can be used for cellular immunotherapy not only against individuals from whom the cells are derived, but also against similar types of diseases in other individuals.

Pharmaceutical compositions for inhibiting CRC: Within the context of the present invention, suitable pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) or for administration by inhalation or insufflation. Preferably, the administration is intravenous. The formulations are optionally packaged in separate dosage units. Pharmaceutical formulations suitable for oral administration include capsules, lozenges or tablets, each containing a predetermined amount of active ingredient. Suitable formulations also include powders, granules, solutions, suspensions and emulsions. The active ingredient is optionally administered as bolus or paste remedies. Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, fillers, lubricants, disintegrating and / or wetting agents. A tablet can be made by compression or molding, optionally with one or more formulation ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in the free-flowing form, such as a powder or granules, optionally mixed with a linker, lubricant, inert diluent, lubrication, active surface and / or dispersion. The molded tablets can be made by molding in a suitable machine a mixture of a pulverized compound moistened with an inert liquid diluent. The tablets can be coated according to methods known in the art. The oral fluid preparations may be in the form, for example, of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicles before use. Said liquid preparations may contain conventional additives, such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) and / or preservatives. The tablets may optionally be formulated to provide slow or controlled release of the active ingredient in the present invention. A pack of tablets may contain a tablet to be taken once a month. Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions, optionally containing antioxidants, regulators, bacteriostats and solutes which render the formulation isotonic with the blood of the projected receptor; as well as sterile aqueous and non-aqueous suspensions including suspending agents and / or thickening agents. The formulations can be presented in a unit dose or in multiple dose containers, for example, as sealed vials and flasks, and can be stored in a freeze-dried (lyophilized) condition, which requires only the addition of a sterile liquid carrier, for example , saline, water for injection, immediately before use. Alternatively, the formulations may be presented for continuous infusion. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above. Formulations suitable for rectal administration include suppositories with standard carriers such as cocoa butter or polyethylene glycol. Formulations suitable for topical administration in the mouth, for example, in buccal or sublingual form, include dragees, which contain the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and lozenges, which comprise the active ingredient in a base such as gelatin and glycerin or sucrose and acacia. For intranasal administration, the compounds of the present invention can be used as a liquid spray, a dispersible powder or in the form of drops. The drops can be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents and / or suspending agents. For administration by inhalation, the compounds they can be conveniently supplied from an insufflator, nebulizer, pressurized packages or other convenient means for the supply of an aerosol spray. The pressurized packages may comprise a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide and some other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to supply a measured quantity. Alternatively, for administration by inhalation or insufflation, the compounds may take the form of a dry powder composition, for example, a mixture of the powder of the compound and a suitable powder base, such as lactose or starch. The powder composition can be presented in a unit dosage form, for example, as capsules, cartridges, gelatin or bubble packets, of which the powder can be administered with the aid of an inhaler or insufflators. Other formulations include implantable devices and adhesive patches that release a therapeutic people. When desired, the formulations described above, adapted to provide sustained release of the active ingredient, can be employed. The pharmaceutical compositions may also contain other active ingredients, such as antimicrobial, immunosuppressive and / or preservative agents.

It should be understood that in addition to the ingredients mentioned in particular above, the formulations of the present invention may include other agents conventional in the art with respect to the type of formulation in question. For example, the formulations suitable for oral administration. they may include flavoring agents. Preferred unit dose formulations contain an effective dose, as will be mentioned below, or a suitable fraction thereof, of the active ingredient. For each of the aforementioned conditions, the compositions, for example, polypeptides and organic compounds, may be administered orally or by injection in a dose ranging from about 0.1 to about 250 mg / kg per day. The dose range for adult humans is generally from about 5 mg to about 17.5 g / day, preferably about 5 mg to about 10 g / day, and most preferably from about 100 mg to about 3 g / day. Tablets or other unit dosage presentation forms provided in separate units may conveniently contain an amount that is effective at said dose or as a multiple of the same, eg, units containing about 5 mg to about 500. mg, normally about 100 mg up to approximately 500 mg. The dose used will depend on the number of factors, including the age and sex of the subject, the precise disorder that is being treated and its severity. Likewise, the administration route may vary depending on the condition and its severity. In any case, suitable and optimal doses can be routinely calculated by those skilled in the art, taking into consideration the aforementioned factors. The aspects of the present invention will be described in the following examples, which are not intended to limit scope of the present invention described in the appended claims. The examples that follow illustrate the identification and characterization of genes expressed differentially in CRC cells. EXAMPLES Cell Lines and Clinical Materials The human colon cancer cell lines HCT116 and RKO were obtained from the American Type Culture Collection (Rockville, MD). All these cells were cultured as monolayers in appropriate media, Mochis 5A (Invitrogen, Carlsbad, CA), for HCT116 and RPMl 1640 (Sigma-Aldrich Corporation, St. Louis, MO) for RKO, supplemented with 10% fetal bovine serum. (Cansera International Inc., Ontario, Canada) and 1% antibiotic / antifungal solution (Sigma-Rich). The cells were maintained at a temperature of 37 ° C in an atmosphere of humidified air with 5% CO 2. The carcinogenic tissues and the corresponding non-cancerous mucous membranes were cut from 12 patients during surgery, after consent was obtained. The TISI (HLA-A24 / 24) and EHM (HLA-A3 / 3) cells, the human B-lymphoblastoid cell lines, were generous gifts from Takara Shuzo Co., Ltd. (Otsu, Japan). The TISI cells were used for peptide-transmitted cytotoxicity assays. The cell lines of colorectal carcinoma DLD-1 (HLA-A24 / 02), HT29 (HLA-A24 / 01) and SNU-C2A (HLA-A31 / 26), were purchased from ATCC. A cell line of chronic myelogenous leukemia cell K562 was purchased from ATCC. Semi-quantitative RT-PCR Total RNA was extracted from cultured cells or clinical cultures using the TRIZOL reagent (Invitrogen) according to the manufacturer's protocol. RNA extracted with DNasel was treated (Roche Diagnostics, Mannheim, Germany), and reverse transcribed into single-stranded cDNAs using oligo (dT) 12.18 primer and Superscript II reverse transcriptase (Invitrogen). We prepared adequate dilutions of each single-stranded cDNA for subsequent PCR amplification, monitoring the GAPDH gene as a quantitative control. The primer sequences used are 5'-ACAACAGCCTCAAGATCATCAG-3 '(SEQ ID NO: 41) and 5'- GGTCCACCACTGACACGTTG-3 '(SEQ ID NO; 42) for GAPDH; 5'-TGGTATAAACCTAAGGCCCTGAT-3 '(SEQ ID NO: 43) and 5'-TAAACAGCTTAGGTGCCTCTCTG-3' (SEQ ID NO: 44) for TOM34. All amplification reactions were followed by initial denaturation at a temperature of 94 ° C for 2 minutes, followed by 18 (for GAPDH) or 29 cycles (for TOM34) of amplification at a temperature of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds in a GeneAmp 9700 PCR system (PE Applied Biosystems, Foster, CA). Northern blotting Human multiple tissue stained (BD Bioscience, Palo Alto, CA) was hybridized with a 32-labeled PCR product of TOM34. The probe was prepared by RT-PCR using a set of primers, 5'-GAACGTGAAGGCATTCTACAGA-3 '(SEQ ID NO: 45) and 5'-TAAACAGCTTAGGTGCCTCTCTG-3' (SEQ ID NO: 44), and an oligonucleotide labeling. Subsequent randomization with 32P-dCTP with a Mega Label team (Amersham Biosciences, Buckinghamshire, United Kingdom). Prehybridization, hybridization and washes were carried out according to the supplier's recommendations. The stains were autoradiografeados with classifications of intensification at a temperature of -80 ° C for 10 days. Preparation of Polyclonal Antibody against TOM34 All the coding region. of TOM34 was amplified using a set of primers, 5'-CATAAGCTTGCATGGCCCCCAAATTCCCA-3 '(SEQ ID NO: 58) and 5'-GTTAAGCTTTTAGTGTAGGTTCT-3', (SEQ ID NO: 59) and subsequently cloned into a suitable pET28 vector cloning site ( Novagen, Madison, Wl) to generate plasmids expressing His-tagged TOM34 protein. The recombinant protein was expressed in Escherichia coli strain, BL21-CodonPlus (DE3) -RIL (Stratagene, LaJolla, CA), and purified using a Superflow Metal Affinity Resin HEPA resin (BD Bioscience) according to the manufacturer's protocol. The protein was inoculated in rabbits, and the immunized sera were purified on affinity columns according to the standard method. A Western blot analysis was performed using proteins extracted from the cells using a 0.1% RIPA type regulator containing 50 mM Tris-HCl (pH7.5), 250 mM NaCl, 0.1% SDS, and 0.5% NP40 with a Group of Protease Cocktail III (CALBIOCHEM, La Jolla, CA). Immunohistochemistry Immunohistochemical staining using affinity purified polyclonal antibody against human TOM34 was carried out. They were subjected to a peroxidase immunoassay system SAB-PO (Nichirei, Tokyo, Japan), tissue sections embedded in paraffin, then the antigens were recovered from dewaxed tissues and hydrated by pre-treatment of the slices in 0.01M citrate buffer (pH6.0) at a temperature of 108 ° C for 10 minutes by autoclaving. Construction of siRNAs expressing psiU6BX for TOM34 The siRNAs expressing plasmids were prepared by cloning double-stranded oligonucleotides at a Bbsl site in the vector psiU6BX as previously described (publication WO2004 / 076623). The sequences of the paired oligonucleotides are; 5'- CACCGAAAGTGTTCTCTACTCCATTCAAGAGATGGAGTAGAGAA CACTTTC-3 '(SEQ ID NO: 46) and 5'- AAAAGAAAGTGTTCTCTACTCCATCTCTTGAATGGAGTAGAGAAC ACTTTC-3' (SEQ ID NO: 47) for siD; 5'- CACCGGATGGAAACTGCAGAGACTTCAAGAGAGTCTCTGCAGTT TCCATCC-3 '(SEQ ID NO; 50) and 5'-AAAAGGATGGAAACTGCAGAGACTCTCTTGAAGTCTCTGCAGTT TCCATCC-3 '(SEQ ID NO; 51) for yesE; After phosphorylating with T4-polynucleotide kinase, the paired oligonucleotides were boiled for five minutes, and then hardened to produce double-stranded oligonucleotides by cooling them slowly. HE prepared a control plasmid, psiU6BX-EGFP using an oligonucleotide consisting of the following sequences. Each nucleotide sequence, target sequences of siRNA and SEQ ID NOs are shown in table 1. 5'- CACCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTG CTGCTTC-3 '(SEQ ID NO: 54) and 5'-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTG CTGCTTC-3' (SEQ ID NO: 55). To check the effect of TOM34-siRNA, western blot analysis using anti-TOM34 antibody was carried out five days after transfection with the plasmids. Table 1. Sequence of specific double-stranded oligonucleotide inserted into siRNA expression vector and target sequences of each siRNA.

Colony Formation Assay Silvered HCT116 cells on a 10 cm dish (4x105 cells / dishes) were temporarily transfected with plasmids expressing TOM34-siRNAs using the FuGENEß reagent (Roche Diagnostics), and kept in medium containing 10% fetal bovine serum with 800 μg / ml Geneticin for two weeks. The surviving cells were fixed with 100% methanol and stained with a Giemsa solution. In another experiment, viable cells were measured with a cell counting device (DOJINDO, Kumamoto, Japan). Statistical Analysis Statistical significance was analyzed by ANOVA with a Scheffes F test, using software commercially available (Statview, SAS Institute, Cary, NC). Peptide derived from TOM34 The peptides of nonamer and 10-mer derived from the peptide sequence TOM34 that can bind to the HLA-A24 molecule, were anticipated by the link anticipation software (http://bimas.dcrt.nih.gov / cgi-bin / molbio / ken parker comboform) (Parker KC, and associates, J Immunol., 1994; 152 (1): 163-75). These peptides were synthesized through Mimotopes, San Diego, CA, according to the standard solid phase synthesis method and purified by reverse phase HPLC. The purity (> 90%) and identity of the peptides was determined by analytical HPLC and mass spectrometric analysis, respectively. The peptides were dissolved in dimethylsulfoxide (DMSO) at 20 mg / ml and stored at a temperature of -80 ° C. CTL induction in vitro Dendritic cells derived from monocytes (DCs) were used as antigen presenting cells (APCs) to induce CTLs against peptides presented in HLA. In vitro DCs were generated as described in many publications (Nukaya I, and associates, Int J Cancer 1999, 80 (1): 92-7, Tsai V, and associates, J Immunol. 1997; 158 (4): 1796 -802). In synthesis, peripheral blood mononuclear cells (PBMCs) were isolated from normal volunteers ((HLA-A24) by means of Ficoll- solution.

Paque (Pharmacia) and separated by adhesion to a plastic tissue culture flask (Becton Dickinson) to enrich them in this way for the monocyte fraction. The monocyte-enriched population was cultured in the presence of 1,000 U / ml of GM-CSF (provided by Kirin Brewery Company) and 1,000 U / ml of I L-4 (Genzyme) in AIM-V (Invitrogen) containing autologous serum deactivated by 2% heat (AS). After 7 days in the culture, DCs generated by cytokines were loaded with 20 μg / ml of binding peptides by HLA-A24 in the presence of 3 μg / ml of β2-microglobulin for 4 hours at a temperature of 20 ° C. in AIM-V. These DCs loaded with peptide were subsequently irradiated (5,500 rad) and mixed in a ratio of 1:20 with autologous CD8 + T cells, obtained by positive selection with Dynabeads M-450 CDd (Dynal) and Detachabead (Dynal). This mixture was subdivided and the aliquots were incubated in plates of 48 tanks (Corning); each contained 1.5 x 104 charged with DCs peptide and 3 x 105 CD8 + T cells in 0.5 ml of AIM-V / 2% AS containing 10 ng / ml of I L-7 (Genzyme). Three days later, these cultures were supplemented with IL-2 (CHIRON) to a final concentration of 20 lU / ml. On day 7 and 14, the T cells were again stimulated in addition with the DCs loaded with autologous peptides. The DCs were prepared each time in the same manner as described above. Cytotoxicity was tested against cells TISI loaded with peptide after the third round of peptide stimulation on day 21. CTL Expansion Procedure CTLs were expanded in culture using the method similar to that described in the Riddell Publication, and associates (Walter and associates, N Engl J Med 333: 1038-1044, (1995), Riddel et al., Nature Med. 2: 216-223, (1996)). A total of 5 x 104 CTLs were resuspended in 25 ml of AM-V / 5% AS with irradiated PBMC 25 x 106 (3300 rad) and irradiated EHM cells 5 x 106 (8, 000 rad) in the presence of 40 ng / ml of anti-CD3 monoclonal antibody (Pharmingen). One day after the start of culture, 120 IU / ml of I L-2 was added thereto. The culture was fed fresh AIM-V / 5% AS containing 30 lU / ml of IL-2 on days 5, 8 and 11. Establishment of CTL clones The expanded CTL cultures were diluted in the cells were distributed in the tanks of 96-well round bottom microtiter plates (Nalge Nunc International) adjusting the number of cells to be 0.3, 1, and 3 CTLs / deposit. Subsequently, CTLs were cultured with 7 x 10 4 cells / deposition of allogenic PBMCs, 1 x 10 4 cells / EHM deposit, 30 ng / ml of anti-CD3 antibody, and 125 U / ml of IL-2 in total of 150 μl / AIM-V deposit containing 5% AS. 50 μl / deposit of I L-2 was added to the medium 10 days later, so that I L-2 was converted to 125 U / ml in the final concentration. The cytotoxic activity of CTLs was tested at day 14, and the CTL clones were expanded using the same method as described above. Cytotoxicity assay Target cells were labeled with 100 μCi of Na251CrO (Perkin Elmer Life Sciences) for 1 hour at a temperature of 37 ° C in a CO2 incubator. Target cells loaded with peptide were prepared by incubating the cells with 20 μg / ml of the peptide for 16 hours at a temperature of 37 ° C before labeling. The labeled target cells were rinsed and mixed with effector cells in a final volume of 0.2 ml in round bottom micro-titration plates. The plates were centrifuged (4 minutes at 800 x g) to increase cell-to-cell contact and placed in a CO2 incubator at a temperature of 37 ° C. After 4 hours of incubation, 0.1 ml of the supernatant from each deposit was collected and the radioactivity was determined with a gamma counter. In case of evaluation of the cytotoxicity of target cells expressing endogenously TOM34, cytolytic activity is provided in the presence of a 30-fold excess of the non-labeled K562 cells to eliminate any non-specific lysis due to NK-type effectors. The specificity of antigens was confirmed through the cold target inhibition assay, which used unlabeled TISI cells that were loaded with the antigen peptide (20 μg / ml during 16 hours at a temperature of 37 ° C) to complete the recognition of 51Cr tumor cells labeled with HT29. The percentage of specific cytotoxicity was determined by calculating the percentage of specific 51Cr release through the following formula:. { (cpm of the release of the test sample - cpm of the spontaneous release) / (cpm of the maximum release - cpm of the spontaneous release)} x 100. Spontaneous release was determined by incubating the target cells alone, in the absence of effector cells, and maximum release was obtained by incubating the targets with HCl. All measurements were carried out by supplication, and IQS standard errors of the averages were consistently less than 10% of the average value. Results High expression of TOM34 in CRCs In our previous study, we identified a number of genes whose expression levels were frequently altered in colon tumors by analyzing the width-genome expression profile of 11 CRCs and nine colon adenomas using a micro -CADN formation consisting of 23040 genes. Among the genes whose expression levels were frequently activated in the 11 carcinomas, we focused on the present invention in a gene with a local identification number of D3124, which corresponded TOM34 (Access GeneBanck No: AB085681, SEQ ID NO; 60, 61) (34-kDa Translocase of the External Mitochondrial Membrane). Subsequent semi-quantitative RT-PCR analysis revealed its improved expression in 16 of 20 revised CRC clinical samples (Figure 1A). In addition, TOM34 were also significantly activated in the eight carcinomas or hepatocellular carcinomas, five of 19 lung cancers, 3 of 9 bladder cancers, seven of 27 acute myeloid leukemias, and nine of 49 soft tissue sarcomas in our microformation data. of cDNA. To investigate their expression in normal human tissues, we carried out multiple tissue Northern staining using TOM34 cDNA as a probe. As a result, the analysis revealed a transcript of approximately 2.0 kb in size, which was abundantly expressed in the testes and ovaries, and weakly in the prostate, spleen and colon, but was not expressed in any of the other 11 tissues reviewed. (figure 1B). TOM34 protein accumulated in colorectal cancer cell lines in CRC tissues We prepared a TOM34 polyclonal antibody and checked expression of TOM34 protein in eight CRC cell lines and 12 CRC tissues by immuno-histochemical staining. Immunoblot analysis detected a 34-kDa band of TOM34 that was abundantly expressed in all the revised CRC cell lines. On the other hand, low expression levels were shown in NIH3T3 and COS7, two lines of non-cancerous cells (Figure 2B). Western blot analysis using 16 CRCs and corresponding noncancerous mucosal tissues demonstrated their enhanced expression in 15 or 16 tumors compared to normal mucosa (Fig. 2A). To review the subcellular localization of TOM34, we carried out immuno-histochemical staining using HCT116 and RKO cells with anti-TOM34 antibody, which showed cytoplasmic localization of the protein. In addition, we carried out immuno-histochemical staining using 12 colon cancer tissues embedded in paraffin. In 11 of the 12 tumors, TOM34 was strongly stained in the cytoplasm of cancer cells, although it was scarcely stained in noncancerous mucosa (Figure 3). Effect of TOM34-siRNAs on growth of colon cancer cells To elucidate the role of TOM34 in cancer cells, we prepared plasmids expressing siRNA for TOM34 and its effect on the growth of cancer cells was reviewed. We prepared two forms of plasmids expressing siRNAs designed to suppress TOM34 (psiU6BX-TOM34-siD, and -siE) and a control plasmid (psiU6BX-siEGFP). We transfected HCT116 cells with psiU6BX-TOM34-siD, psiU6BX-TOM34-siE, or psiU6BX-siEGFP. Western blot analysis of transfected cell extracts revealed that psiUTBX-TOM34-siD and psiU6BX-TOM34-siE suppressed in the form significant expression of TOM34 in transfected cells compared to psiU6BX-siEGFP (Figure 4A). We transfected with the neomycin resistance gene of joint expression with plasmids in HCT116 cells, and cultured them with an adequate concentration of Geneticin for 13 days. In accordance with the reduced expression TOM34, both psiU6BX-TOM34-siD and psiU6BX-TOM34-siE markedly retarded the growth of transfected cells compared to psiU6BX-siEGFP (Figure 4B). These data indicated that the expression of TOM34 is associated with the growth of cancer cells. Anticipation of peptides that bind to HLA-A24 derived from TOM34 Tables 2 and 3 show the anticipated peptides for the antigen in a high-order binding affinity order. Twenty 10-mer peptides (Table 2) and twenty 9-mer peptides (Table 3) were selected for the antigen and reviewed as indicated below. Table 2. Anticipation of HLA-Class I Epitope Candidates (HLA-A24: 10 mer) Table 3. Anticipation of HLA-Class I Epitope Candidates (HLA-A24: 9 mer) Stimulation of T cells using the anticipated peptides The cytotoxic T cells that recognize the peptides derived from TOM34 were generated in the manner described in the section "Materials and Methods". Resulting CTLs with detectable cytotoxic activity were expanded, and said CTL lines were established to show higher cytotoxic activity against the target loaded with peptide compared to the target without peptide pulsing.

CTL lines stimulated by 10-mer peptide TOM34-299 (KLRQEVKQNL (SEQ ID NO: 7)) showed potent cytotoxic activity against the peptide loaded target without showing any significant cytotoxic activity against targets not loaded with the peptide (Figure 5). The CTL lines showed antigen-specific cytotoxicity and potent detectable in the E / T ratio as low as 1.2 (figures 6). Establishment of CTL clones The clones of CTLs were established by limiting the dilution of cultures of CTL lines followed by expansion as described in the section on "Materials and Methods". CTL clones specific for TOM34-299 obtained in this way, obtained clones with very high level cytolytic activities with activities of the general clones ranging from 20% to 70%) of lysis in the proportion of E / T of 1.2 and 40% a > 90% in the E / T ratio of 10 (figure 7). Cytotoxic activity against colon cancer cell lines expressing endogenously TOM34 as targets The CTL clones that emerged against the peptide TOM34-299 were reviewed for their ability to recognize and kill tumor cells expressing endogenously TOM34. Figure 8 shows the result with the two clones CTL clones raised against TOM34-299. Both CTL clones showed potent cytotoxic activity against lines of colon cancer cell DLD-1 and HT29, which expresses TOM34 as well as HLA-A24. On the other hand, neither of the two CTL clones showed any relevant cytotoxic activity against a colon cancer cell line SNU-C2A, which expresses TOM34 but not HLA-A24 (figure 8). Cold Target Inhibition Assay The cold target inhibition assay was carried out to confirm the specificity of the CTL lines. HT29 cells labeled by 51 Cr were used as a hot target, while TISI cells with or without loaded peptide were used without 51 Cr labeling as cold targets. Specific cell lysis against the target HT29 cells was significantly inhibited when the cold target cells loaded with peptide were added in the assay in various proportions but were not inhibited at all by the addition of the cold target cells without loaded peptide. The result is shown as a percentage of the specific lysis in the Efector / Hot Target ratio of 20 (Figure 9). Homology analysis of antigen peptides The CTL clones established against TOM34-299 showed very potent cytotoxic activity. This may mean that the sequence of this peptide is homologous with those derived from other molecules that are known to sensitize the human immune system. To exclude this possibility, it was carried out a homology analysis with the peptide sequences as a query using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) (Altschul SF, and associates, Nucleic Acids Res. 1997; 25 (17): 3389-402; Altschul SF, and associates, J Mol Biol. 1990; 215 (3): 403-10) and revealed that there was no sequence with significant homology. This indicates that the sequence of TOM34-299 is unique and there is a small possibility, for our better knowledge, of raising the unprojected immune response to any unrelated molecule. Blocking CTL activity by antibodies binding to T-cell surface antigens To see if the observed extermination activity is transmitted by the cytotoxic T-cells, the effects of the antibodies to kill the activity were investigated, using antibodies that neutralize the T cell-surface antigen functions related to CTL activity. CTL activity was clearly blocked by adding antibodies that recognize HLA Class I or CDd, and to a lesser degree by antibodies to HLA Class II or CD4, as shown in Figure 10. These results show that the cytotoxic activity of the cell clone T observed against colorectal carcinoma cells HT29 is the HLA-restricted activity of CD8-positive T lymphocytes. Description The present invention has shown that TOM34 is frequently activated in CRCs and that it is abundantly expressed in normal testes and ovaries and expressed weakly in the prostate, spleen and colon, although it is rarely detected in other normal adult tissues. reviewed. One of the most promising therapeutic methods is cancer immunotherapy, which includes T cell-mediated antitumor vaccination. The recognition of peptide antigens represented by the major histocompatibility complex (MHC) molecules, target cell of CD8 + cytotoxic T cells that display antigens. Since tumor cells originate from non-cancerous autologous cells, most tumor cells escape immune surveillance. It is of great importance to identify antigens expressed in specific form in cancer cells to apply cancer immuno-therapy. The antigens are not expressed in the testes due to the lack of MHC class I molecules (Jassim A, and associates, Eur J Immunol 19: 1215-1220, 1989). Therefore, genes expressed specifically in cancer and testes should be targeted for immunotherapy. Since TOM34 was not abundantly expressed in normal organs, except for testes and ovaries, and is active in most colon cancers, TOM34 may serve a therapeutic purpose for CRC as a novel testis-cancer antigen.

Tom34 was initially considered to function as a component of the translocase for the import of proteins into the mitochondria, because this protein showed sequence homology with the yeast Tom protein (Nuttall SD, and associated, DNA Cell Biol 16: 1067- 1074, 1997). Although Tom34 was anticipated to be located on an outer mitochondrial membrane, a recent study revealed that it was included in the cytosol of Hela cells (Chun-Song Y &Henry W, Archives of Biochemistry and Biophysics 400: 105-110, 2002; Abhijit M , and associates, Archives de Biochemistry and Biophysics 400: 97-104, 2002). Consistent with these data, we found that the subcellular localization of TOM34 was cytoplasmic in CRC cells by in-situ-histochemical staining using a polyclonal anti-Tom34 antibody. In another report, the classification using a yeast two-hybrid system identified Valosin-containing protein (VCP) and hsp90 as an interaction protein with TOM34. VCP, a member of the AAA family (ATPases associated with a variety of cellular activities) also known as p97, forms a complex with the ubiquitinated l? Ba and 26S proteasome subunits, suggesting that VCP may be involved in the translocation of the factor Transcription NFKB for the nucleus degrading l? Ba (Dai RM, and associates, J Biol Chem 273: 3562-3573, 1998). HSP90 is a molecular chaperone whose interaction is required for the stability and function of a large number of molecules Therefore, TOM34 can be stabilized by binding with HSP90. Since HSP90 is associated with oncogenic molecules such as Rafl, AKT and SMYD3, a number of HSP90 inhibitors have been developed for the treatment of human cancer. These inhibitors are assumed to cause several adverse effects, hindering HSP90, normal functional, because HSP90 is expressed ubiquitously in most normal tissues. In conclusion, TOM34 was activated in CRCs but was expressed sparingly in normal organs except the testicles and ovaries. Therefore, it is likely that the inhibition of TOM34 does not lead to severe adverse effects in normal cells. In addition, the suppression of TOM34 resulted in the inhibition of cell growth of cancer cells, suggesting that TOM34 plays an essential role in its growth. These data implied that TOM34 is a promising target for immunotherapy and developed anticancer drugs for human colorectal cancer. In addition, the identification of new TAAs, which induce powerful and specific anti-tumor immune responses, guarantees the further development of the clinical application of the peptide vaccination strategy in several types of cancer (Boon T &van der Bruggen P, J Exp Med. 1996; 183 (3): 725-9, van der Bruggen P, and associates, Science, 1991; 254 (5038): 1643-7, Brichard V, and associates, J Exp Med., 1993; 178 (2): 489-95, Kawakami Y, and associates, J Exp Med. 1994; 1d0 (1): 347-52., Shichijo S, and associates, J Exp Med. 1998; 187 (3): 277- dd, Chen YT, and associates, Proc Natl Acad Sci USA 1997; 94 (5): 1914-8, Harris CC, J Natl Cancer Inst. 1996; 88 (20): 1442-55, Butterfield LH, and associates, Cancer Res. 1999; 59 (13): 3134-42, Vissers JL, and associates, Cancer Res. 1999; 59 (21): 5554-9, van der Burg SH, and associates, J Immunol. 1996; 156 (9): 3308-14, Tanaka F, and associates, Cancer Res. 1997; 57 (20): 4465-d, Fujie T, and associates, Int J Cancer. 1999; d0 (2): 169-72., Kikuchi M, and associates, Int J Cancer. 1999; 81 (3): 459-66, Oiso M, and associates, Int J Cancer. 1999; 81 (3): 387-94). We analyzed the peptides derived from TOM34 as TAA epitopes restricted by HLA-A24, one of the frequent HLA alleles in Japanese, as well as in the population of Caucasians (Date Y, and associates, Tissue Antigens, 1996; 47: 93-101, Kondo A , and associates, J Immunol, 1995; 155: 4307-12, Kubo RT, and associates, J Immunol, 1994,152: 3913-24). In the present invention, we selected candidate epitope peptides restricted with HLA-A24 derived from TOM34 using the information with respect to their binding affinities in advance with HLA-A24. After in vitro stimulation of T cells by DCs loaded with these candidate peptides, the CTLs were successfully established using a peptide TOM34-299 (KLRQEVKQNL) (SEQ ID NO: 7) which showed potent cytotoxic activity against TISI cells loaded with peptides. In addition, the CTL clones derived from these CTLs showed cytotoxicity 'also specific against colorectal carcinoma cell lines that overexpress TOM34 endogenously and HLA-A24 positive. The cytotoxic activities of these CTL clones did not unfold against cell lines that lack expression of either HLA-A24 or target TAA and are significantly inhibited through the cold target. These results strongly suggest that TOM34 is a novel TAA for colon cancer and that TOM34-299 is a specific epitope peptide of this TAA restricted by HLA-A24. Since this antigen is over-expressed in most cases of colon cancer and is associated with tumor cell proliferation, it seems to be an objective model to be used in immunotherapy against colon cancers. The homology analysis of TOM34-299 showed that it has no significant homology with the peptides of any known human gene products. INDUSTRIAL APPLICABILITY The genetic expression analysis of colon cancer described in the present invention, obtained through a combination of laser-capture dissection and wide-genome cDNA microformation, has identified specific genes as targets for cancer prevention and therapy . Based on the Expression of a subgroup of these differentially expressed genes, the present invention provides molecular diagnostic markers for identifying and detecting CRC. The methods described here are also useful in the identification of additional molecular targets for CRC prevention, diagnosis and treatment. The data reported here adds a comprehensive understanding of CRC, facilitates the development of novel diagnostic strategies and provides clues to the identification of molecular targets for therapeutic drugs of preventive agents. This information contributes to a deeper understanding of colon tumorigenesis, and provides indicators to develop novel strategies for the diagnosis, treatment and finally prevention of CRC. For example, agents that block the expression of TOM34, prevents its activity has therapeutic utility as anti-cancer agents, particularly anti-cancer agents for CRC treatment. Examples of such agents include anti-sense oligonucleotides, small interfering RNAs, and ribozymes against the TOM34 gene, and antibodies that recognize TOM34. As an alternative, the identification of new TAAs, which induce preferential and specific anti-tumor immune responses, guarantee the further development of clinical application of peptide vaccination strategies in CRC. In addition, although the present invention has been described in detail and with reference to the specific modalities thereof, it will be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the present invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications to the present invention can be made without departing from the spirit and scope thereof. Therefore, the present invention is intended to be defined not by the foregoing description, but by the claims that follow and their equivalents.

Claims (33)

1. CLAIMS 1. A method for diagnosing colon cancer or a predisposition to develop colon cancer in a subject, wherein the method comprises determining the level of expression of TOM34 in a biological sample derived from a patient, wherein the increase in the level Expression of the sample, compared to a normal level of control of the gene, indicates that the subject suffers from or is at risk of developing colon cancer.
2. 2. The method as described in the claim 1, characterized in that the level of expression of the sample is at least 10% higher than the normal control level.
3. The method as described in claim 1, characterized in that the level of gene expression is determined through a method selected from the group consisting of: a) detect mRNA of TOM34, b) detect a protein encoded by TOM34 , and c) detect a biological activity of TOM34.
4. 4. The method as described in the claim 1, characterized in that the biological sample derived from the patient comprises an epithelial cell.
5. The method as described in claim 1, characterized in that the biological sample derived from the patient comprises a colon cancer cell.
6. 6. The method as described in claim 1, characterized in that the biological sample derived from the patient comprises an epithelial cell from a colon cancer cell.
7. 7. A method for classifying a compound for treating or preventing colon cancer, wherein the method comprises the steps of: a) contacting the test compound with a polypeptide encoded by a TOM34 polynucleotide; b) detecting the binding activity between the polypeptide and the test compound; and c) selecting the test compound that binds to the polypeptide.
8. 8. A method for classifying a compound for treating or preventing colon cancer, wherein the method comprises the steps of: a) contacting a candidate compound with a cell expressing TOM34; and b) selecting the candidate compound that reduces the expression of the TOM34 level compared to the expression level of TOM34 detected in the absence of the test compound.
9. 9. The method as described in claim d, characterized in that the cell comprises a colon cancer cell.
10. 10. A method for classifying a compound for treating or preventing colon cancer, wherein the method comprises the steps of: a) contacting the test compound with a polypeptide encoded by a TOM34 nucleotide; b) detecting the biological activity of the polypeptide of step (a); and c) selecting the test compound which suppresses the biological activity of the polypeptide encoded by the nucleotide of TOM34 as compared to the biological activity of the polypeptide detected in the absence of the test compound.
11. 11. A method for classifying a compound for treating or preventing colon cancer, wherein the method comprises the steps of: a) contacting a candidate compound with a cell into which a vector has been introduced, comprising the transcription regulatory region of TOM34 and a reporter gene that is expressed under the control of the transcription regulatory region; b) measure the expression or activity of the reporter gene; and c) selecting the candidate compound that reduces the expression or activity of the reporter gene.
12. 12. A kit comprising a detection reagent that binds to (a) TOM34, or (b) polypeptides encoded by TOM34.
13. 13. A method for treating or recovering colon cancer in a subject, wherein the method comprises administering to the subject an anti-sense composition, wherein the antisense composition comprises a nucleotide sequence complementary to a TOM34 coding sequence.
14. 14. A method for treating or preventing colon cancer in a subject, wherein the method comprises administering to the subject a siRNA composition, wherein the siRNA composition reduces the expression of TOM34.
15. 15. The method as described in the claim 14, characterized in that the siRNA comprises the sense strand comprising a nucleotide sequence of SEQ ID NO: 4d or SEQ ID NO: 52.
16. 16. The method as described in claim 15, characterized in that the siRNA has the formula general 5'- [A] - [B] - [A '] - 3 \ where [A] is a ribonucleotide sequence corresponding to a sequence of SEQ ID NO: 48 or SEQ ID NO: 52, [B] is a ribonucleotide loop sequence consisting of 3 to 23 nucleotides, and [A '] is a ribonucleotide sequence consisting of the complementary sequence of [A].
17. 17. A method for treating or preventing colon cancer in a subject, wherein the method comprises the step of administering to the subject a pharmaceutically effective amount of an antibody, or an immunologically active fragment of the same, that binds to a protein of TOM34.
18. 18. A method for treating or preventing colon cancer in a subject, wherein the method comprises administering to the subject a vaccine comprising (a) a polypeptide encoded by a TOM34 nucleic acid, (b) an immunologically active fragment of the polypeptide, or (c) a polynucleotide encoding the polypeptide or the immunologically active fragment of (b).
19. The method as described in claim 1d, characterized in that the immunologically active fragment is either or both of (i) a decapeptide comprising the amino acid sequence of SEQ ID NO: 7, and (ii) a peptide having cytotoxic T cell inducibility, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 7, wherein 1, 2, or several amino acids are substituted or added.
20. A method for inducing an anti-tumor immunity, wherein the method comprises the step of contacting a cell presenting antigens with a polypeptide, a polynucleotide encoding the polypeptide or a vector comprising the polynucleotide, wherein the polypeptide is encoded by TOM34 or an immunologically active fragment of the polypeptide.
21. The method as described in claim 20, characterized in that the immunologically active fragment is either or both of (i) a decapeptide comprising the amino acid sequences of SEQ ID NO: 7, and (ii) a peptide having cytotoxic T cell inducibility, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 7, wherein 1, 2, or several amino acids are substituted or aggregated.
22. 22. The method as described in the claim 20, characterized in that the method further comprises the step of administering the cells presenting antigens to a subject.
23. 23. A method for treating or preventing colon cancer in a subject, characterized in that the method comprises the step of administering a compound obtained by a method according to any of claims 7 to 11.
24. 24. A composition for treating or preventing colon cancer, characterized in that the composition comprises a pharmaceutically effective amount of an antisense polynucleotide or siRNA against a TOM34 polynucleotide.
25. 25. The composition as described in claim 24, characterized in that the siRNA comprises the sense strand comprising a nucleotide sequence of SEQ ID NO: 48 or SEQ ID NO: 52.
26. 26. The composition as described in Claim 25, characterized in that the siRNA has the general formula 5 '- [A] - [B] - [A'] - 3 ', where [A] is a ribonucleotide sequence corresponding to a sequence of SEQ ID NO : 48 or SEQ ID NO: 56, [B] is a loop sequence of ribonucleotide consisting of 3 to 23 nucleotides, and [A1] is a ribonucleotide sequence consisting of the complementary sequence of [A].
27. 27. A composition for treating or preventing colon cancer, characterized in that it comprises a pharmaceutically effective amount of an antibody or fragment thereof that binds to a protein encoded by TOM34.
28. 28. A composition for treating or preventing colon cancer, characterized in that it comprises as an active, a pharmaceutically effective ingredient of a compound selected through a method as described in any of claims 7 to 11, and a pharmaceutically acceptable carrier.
29. 29. A decapeptide characterized in that it comprises the amino acid sequence of SEQ ID NO: 7.
30. 30. A peptide having a cytotoxic T cell inducibility, characterized in that the peptide comprises the amino acid sequence of SEQ ID NO: 7, wherein , 2, or several amino acids are substituted or added.
31. 31. The peptide as described in the claim 30, characterized in that the second amino acid of the N-terminus is phenylalanine, tyrosine, methionine or tryptophan.
32. 32. The peptide as described in claim 30, characterized in that the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan or methionine.
33. 33. A pharmaceutical composition for treating or preventing colon cancer, characterized in that it comprises a pharmaceutically effective amount of the peptide as described in claim 29 to 30, in the form of an active ingredient.