# Patent

## Title
Cancer diagnostic kit and cancer diagnostic method

## Abstract

The present invention provides a kit and method for diagnosing a cancer using a polynucleotide including a nucleotide sequence of SEQ ID No: 1 or 3 or a partial sequence thereof, a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof, or an antibody binding specifically to a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof. This makes it possible to find a CT antigen useful for testing, diagnosis, or treatment of a digestive system cancer and to develop a novel cancer diagnostic technique using the CT antigen.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the national stage application pursuant to 35 U.S.C. §371 of PCT International Application No. PCT/JP2008/063002, filed Jul. 18, 2008, which claims priority to Japanese patent application no. 236048/2007, filed Sep. 12, 2007. The contents of these applications are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a novel cancer diagnostic technique. More specifically, the present invention relates to a kit and method for diagnosing a cancer both of which uses the expression of CCDC62 or the presence of an anti-CCDC62 antibody as an index.
BACKGROUND ART
In order to establish a testing method, diagnostic method, and therapeutic method effective to cancers, it is necessary to find out a cancer antigen that expresses in an objective cancer with high frequency and has high antigenicity. However, there are few cancer antigens useful for testing, diagnosis, or treatment of digestive system cancers, especially gastric cancer and colon cancer. Therefore, it is very important to develop useful cancer antigens for testing, diagnosis, or treatment of digestive system cancers.
In terms of accompanying side effects, the most useful antigen for testing, diagnosis, or treatment of human cancer is considered to be a cancer-testis antigen (hereinafter referred to as “CT antigen”) that expresses in various cancers, but are confined to testis among normal tissues. To date, about 40 kinds of CT antigens have been identified and reported. However, all of these CT antigens do not possess strong immunogenicity to cancer patients, and there are a limited number of CT antigens promising for diagnosis marker of cancers, test marker of cancers, or treatment of cancers.
Typical CT antigens that have been reported to date include MAGE (see Non Patent Literature 1), SSX (see Non Patent Literature 2), and NY-ESO-1 (see Non Patent Literature 3). MAGE and NY-ESO-1 have been applied in clinical settings in the world, particularly in Europe and the United States, and the clinical settings have achieved some positive results (for example, for clinical application of NY-ESO-1, see Non Patent Literature 4). Thus, it can be said that NY-ESO-1 is currently the most promising CT antigen for applications to treatment of cancer and the like.
Non Patent Literature 1
van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, van den Eynde B, Knuth A, Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254: 1643-1647, 1991.
Non Patent Literature 2
Gure A O, Tureci O, Sahin U, Tsang S, Scanlan M J, Jager E, Knuth A, Pfreundschuh M, Old L J, Chen Y T. SSX: a multigene family with several members transcribed in normal testis and human cancer. Int. J. Cancer 72: 965-971, 1997.
Non Patent Literature 3
Chen Y T, Scanlan, M J, Sahin U, Tureci O, Gure A O, Tsang S, Williamson B, Stockert E, Pfreundschuh M, Old L J. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc. Natl. Acad. Sci. USA 94: 1914-1918, 1997.
Non Patent Literature 4
Jager E, Gnjatic S, Nagata Y, Stockert E, Jager D, Karbach J, Neumann A, Rieckenberg J, Chen Y T, Ritter G, Hoffman E, Arand M, Old L J, Knuth A. Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. Proc. Natl. Acad. Sci. USA 97: 12198-12203, 2000.
Non Patent Literature 5
Stockert E, Jager E, Chen Y T, Scanlan M J, Gout I, Karbach J, Arand M, Knuth A, Old L J. A survey of the humoral immune response of cancer patients to a panel of human tumor antigens. J. Exp. Med. 187: 1349-1354, 1998.
Non Patent Literature 6
Kurashige T, Noguchi Y, Saika T, Ono T, Nagata Y, Jungbluth A A, Ritter G, Chen Y T, Stockert E, Tsushima T, Kumon H, Old L J, Nakayama E. NY-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation with tumor grade. Cancer Res. 61: 4671-4674, 2001.
Non Patent Literature 7
Nakada T, Noguchi Y, Sato S, Ono T, Saika T, Kurashige T, Gnjatic S, Ritter G, Chen Y T, Stockert E, Nasu Y, Tsushima T, Kumon H, Old L J, Nakayama E. NY-ESO-1 mRNA expression and immunogenicity in advanced prostate cancer. Cancer Immunity 3: 10, 2003.
Non Patent Literature 8
Sugita Y, Wada S, Fujita S, Nakata T, Sato S, Noguchi Y, Jungbluth A A, Yamaguchi M, Chen Y T, Stockert E, Gnjatic S, Williamson B, Scanlan M J, Ono T, Sakita I, Yasui M, Miyoshi Y, Tamaki Y, Matsuura N, Noguchi S, Old L J, Nakayama E, Monden M. NY-ESO-1 expression and immunogenicity in malignant and benign breast tumors. Cancer Res. 64: 2199-2204, 2004.
Non Patent Literature 9
Fujita S, Wada H, Jungbluth A A. Sato S, Nakata T, Noguchi Y, Doki Y, Yasui M, Sugita Y, Yasuda T, Yano M, Ono T, Chen Y T, Higashiyama M, Gnjatic S, Old L J, Nakayama E, Monden M. NY-ESO-1 expression and immunogenicity in esophageal cancer. Clin. Cancer Res. 10: 6551-6558, 2004.
Non Patent Literature 10
Nakamura, S, Nouso K, Noguchi Y, Higashi T, Ono T, Jungbluth A A, Chen Y T, Old L J, Nakayama E, Shiratori Y. Expression and immunogenicity of NY-ESO-1 in hepatocellular carcinoma. J. Gastroenterol. Hepatol. 21: 1281-1285, 2006.
SUMMARY OF INVENTION
The inventors of the present invention tested patients with various epithelial tumors for their capabilities of producing antibodies to NY-ESO-1. The result of the test was as follows: 7% for bladder cancer (9 of 124 cases, see Non Patent Literature 6), 4.6% for prostatic cancer (10 of 218 cases, see Non Patent Literature 7), 1.6% for breast cancer (1 of 62 cases, see Non Patent Literature 8), 3.9% (2 of 51 cases, see Non Patent Literature 9), 2.2% for liver cancer (2 of 92 cases, see Non Patent Literature 10). This reveals that the capabilities of producing antibodies are not so high in all these cancers. Thus, antibodies against the CT antigens reported in the past exist in sera from patients with epithelial tumor with extremely low frequency.
Further, in some rare cases, the CT antigens reported in the past express in digestive system cancers such as gastric cancer and colon cancer. Moreover, the antibodies against the CT antigens are produced with extremely low frequency in patients with digestive system cancers. For example, it has been reported that the capabilities of producing antibodies to NY-ESO-1, MAGE, and SSX of patients with colon cancer are all 0% (see Non Patent Literature 5). Incidentally, the inventors of the present invention tested sera from 58 patients with colon cancer for their capabilities of producing antibodies to NY-ESO-1. The result of the test showed that the capabilities of producing antibodies to NY-ESO-1 were 0% (not published). Further, even in the preliminary analysis using sera from patients with gastric cancer by the inventors of the present invention, the presence of the antibody to NY-ESO-1 has not been proved yet.
Thus, it is not easy to find out a useful cancer antigen for diagnosis of epithelial tumors including digestive system cancers, such as gastric cancer and colon cancer. Therefore, there is a very strong demand for the development of a useful cancer antigen for diagnosis of digestive system cancers.
The present invention has been attained in view of the above problem, and an object of the present invention is to provide a novel cancer diagnostic technique using a useful cancer antigen (preferably CT antigen) for testing, diagnosis, or treatment of epithelial tumors including digestive system cancers.
In order to solve the above problem, the inventors of the present invention carried out SEREX (serological analysis of cancer antigens by recombinant cDNA expression cloning) using a cDNA library derived from normal testis and a serum from a patient with gastric cancer. Then, the inventors of the present invention accomplished the present invention by finding that CCDC62 in an antibody-positive clone is a CT antigen.
That is, a kit according to the present invention is a kit for diagnosing a cancer, comprising a polynucleotide including a nucleotide sequence of SEQ ID No: 1 or 3 or a partial sequence thereof.
A kit according to the present invention is a kit for diagnosing a cancer, comprising a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof.
A kit according to the present invention is a kit for diagnosing a cancer, comprising an antibody binding specifically to a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof.
A kit according to the present invention is preferably used for diagnosis of an epithelial tumor or a skin cancer, more preferably for diagnosis of at least one cancer selected from the group consisting of a gastric cancer, a colon cancer, a breast cancer, a head and neck cancer, a lung cancer, a renal cancer, a prostatic cancer, and a malignant melanoma.
A method for diagnosing a cancer according to the present invention is characterized by comprising a polynucleotide measuring step of measuring a level of presence of a polynucleotide including a nucleotide sequence of SEQ ID No: 1 or 3 or a partial sequence thereof in a sample derived from a subject.
A method for diagnosing a cancer according to the present invention is characterized by comprising: a polypeptide measuring step of measuring a level of presence of a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof in a sample derived from a subject.
A method for diagnosing a cancer according to the present invention is characterized by comprising: an antibody measuring step of measuring a level of an antibody binding specifically to a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof in a sample derived from a subject.
A method for diagnosing a cancer according to the present invention is preferably used for diagnosis of an epithelial tumor or a skin cancer, more preferably for diagnosis of at least one cancer selected from the group consisting of a gastric cancer, a colon cancer, a breast cancer, a head and neck cancer, a lung cancer, a renal cancer, a prostatic cancer, and a malignant melanoma.
Additional objects, features, and strengths of the present invention will be made clear by the description below. Further, the advantages of the present invention will be evident from the following explanation in reference to the drawings.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a view schematically showing SEREX.
FIG. 2 is a view showing the result of analysis of CCDC62 mRNA expressions in human normal tissues, wherein (a) shows the result of RT-PCR analysis and (b) shows the result of real-time RT-PCR analysis.
FIG. 3 is a view showing the result of RT-PCR analysis of CCDC62-2 mRNA expressions in various cancer tissues, wherein (a) shows the result of analysis for lung cancer and (b) shows the result of analysis for colon cancer, and (c) shows the result of analysis for prostatic cancer.
FIG. 4 is a view showing the result of analysis of reactivity of sera from patients with lung cancer against various antigens by phage plaque assay.
FIG. 5 is a view showing the result of analysis of humoral immune responses of cancer patients against CCDC62-2 protein, wherein (a) shows the result of ELISA analysis and (b) shows the result of Western blotting analysis.



DESCRIPTION OF EMBODIMENTS
The inventors of the present invention carried out SEREX for a cDNA library derived from normal testis using a serum from a specific patient with gastric cancer. As a result of SEREX, we found out that a positive clone was a clone expressing CCDC62 and that anti-CCDC62 antibody existed in the serum from the patient with gastric cancer. That is, the inventors of the present invention found out that CCDC62 was a cancer antigen in a patient with gastric cancer. Further, the inventors of the present invention carried out analysis of CCDC62 expression and confirmed that CCDC62 was strongly expressed only in testis among human normal tissues and also was expressed in cancer tissues and cancer cell lines. On the basis of these findings, the inventors of the present invention accomplished the present invention using the CT antigen, CCDC62.
SEREX (serological analysis of cancer antigens by recombinant cDNA expression cloning) is a method for identifying an antigen gene recognized by an antibody that exists in a patient serum from a cDNA expression library derived from cancer tissue (Sahin U, Tureci O, Schmitt H, Cochlovius B, Johannes T, Schmits R, Stenner F, Luo G, Schobert I, Pfreundschuh M. Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci USA. 1995 Dec. 5; 92(25):11810-3). SEREX is known as an excellent method for screening a cancer antigen. It should be noted that the present invention, for the first time, provided the accomplishment using the cDNA library derived from normal testis, not using a cDNA library derived from autologous cancer tissue.
As above, the sequence of human CCDC62 is known, but the relevance of human CCDC62 to a disease has not been reported at all. The inventors of the present invention first found out the expression of CCDC62 in cancers and the presence of the anti-CCDC62 antibody in a serum from a cancer patient.
SEQ ID No: 1 represents a nucleotide sequence of transcript variant 1 of the human CCDC62 gene. SEQ ID No: 2 represents an amino acid sequence of a protein coded by transcript variant 1 of the human CCDC62 gene. SEQ ID No: 3 represents a nucleotide sequence of transcript variant 2 of the human CCDC62 gene. SEQ ID No: 4 represents an amino acid sequence of a protein coded by transcript variant 2 of the human CCDC62 gene. As for the nucleotide sequence of the human CCDC62 gene, transcript variant 1 and transcript variant 2 are registered respectively as NM—032573 and NM 201435 in the GenBank.
[1. A Method for Diagnosing a Cancer]
The present invention provides a method for diagnosing a cancer. In one embodiment, the cancer diagnostic method according to the present invention is a method for diagnosing a cancer by measuring the expression level of CCDC62 in a sample derived from a subject and then comparing the resultant expression level with a control level (e.g. normal level). More specifically, the cancer diagnostic method according to the present embodiment comprises a polynucleotide measuring step or a polypeptide measuring step. The expression level of CCDC62 can be determined by measuring the amount of transcripts (mRNA) or the amount of translation products (protein) by means of a technique known in the art.
In another embodiment, the cancer diagnostic method according to the present invention is a method for diagnosing cancer by measuring the level of the anti-CCDC62 antibody in a sample derived from a subject and then comparing the resultant level with a control level (e.g. normal level). More specifically, the cancer diagnostic method according to the present embodiment comprises an antibody measuring step. The level of the anti-CCDC62 antibody can be determined by measuring the level of the anti-CCDC62 antibody in a serum derived from the subject.
A subject objective to the cancer diagnostic method of the present invention, which is not particularly limited and includes a wide range of animals in general, is preferably a human. If the subject is a human, the subject can be not only a cancer patient and a patient suspected of having cancer but also a healthy person.
The sample derived from the subject used in the cancer diagnostic method of the present invention is not particularly limited as long as it is obtained from the subject (i.e. a sample separated from the subject). Examples of the sample include blood (serum, plasma, hemocyte, etc.), urine, feces, expectoration, ascites, peritoneal lavage fluid, biopsy tissue, and a surgically resected specimen.
The normal level means the expression level of CCDC62 mRNA, the expression level of CCDC62 protein, or the level of the anti-CCDC62 antibody in a normal healthy individual (healthy person). The normal level is preferably a measurement value obtained by using a normal cell, a normal tissue, or a normal body fluid as a sample that is of the same kind as the sample derived from the subject to be compared with (i.e. the sample used in the polynucleotide measuring step, the polypeptide measuring step, or the antibody measuring step of the present invention). Further, it is more preferable that the normal level is a mean value for the group consisting of normal healthy individuals.
As used herein, “diagnosis” encompasses not only “determination” but also “testing”, “detection”, and “prediction”. That is, the diagnosis of a cancer means determination or prediction of whether a subject has a cancer, determination or prediction of a stage of cancer progression, and determination or prediction of a cancer treatment result, more preferably inspection/detection/prediction of whether a subject has a cancer, inspection/detection/prediction of a stage of cancer progression, and inspection/detection/prediction of a cancer treatment result.
The type of a cancer to be diagnosed is not particularly limited, but the cancer is preferably an epithelial tumor or a skin cancer. Examples of the epithelial tumor include a gastric cancer, a colon cancer, a breast cancer, a head and neck cancer, a lung cancer, a liver cancer, a renal cancer, an epithelial ovarian cancer, a prostatic cancer, and the like. Examples of the skin cancer include a malignant melanoma.
(1) Cancer Diagnostic Method Using mRNA Level as Index
In one embodiment, the cancer diagnostic method according to the present invention may be a method for diagnosing a cancer using a sample derived from a subject, the method including a polynucleotide measuring step of measuring the level of the presence of a polynucleotide including a nucleotide sequence of SEQ ID No: 1 or 3 or a partial sequence thereof in the sample derived from the subject. Note that the polynucleotide includes both DNA and RNA.
In the present embodiment, mRNA level of CCDC62 may be measured in the polynucleotide measuring step.
A method of measuring mRNA level, which is not particularly limited as long as it enables the measurement of a specific mRNA level, is selected for use as appropriate from among the known methods. Examples of the method of measuring mRNA level include a method using primers or a probe containing a polynucleotide consisting of nucleotide sequences of mRNAs or cDNAs of CCDC62 or a portion of complementary sequences of nucleotide sequences, wherein the polynucleotide is site-specifically bound to (hybridized with) any of mRNAs or cDNAs of CCDC62. The primers or probe may be subjected to various modifications for the measurement/detection of mRNA, as long as it forms site-specific base pairs with mRNA of CCDC62 or its corresponding cDNA.
The polynucleotide used as the above-described primers is not limited as long as it is designed on the basis of the nucleotide sequence of SEQ ID No: 1 or 3 or its complementary sequence. Examples of such a polynucleotide include a polynucleotide consisting of a nucleotide sequence of any of SEQ ID Nos: 5 through 8. These polynucleotides are the primers which were actually used by the inventors of the present invention in carrying out RT-PCR, and the polynucleotides are proved to specifically amplify cDNAs of CCDC62. The polynucleotide used as the above-described probe is not limited as long as it is designed on the basis of the nucleotide sequence of SEQ ID No: 1 or 3. Note that it is well known in the art that a polynucleotide available as primers that specifically amplify a mRNA (cDNA) of interest is usable as a probe for specifically detecting the mRNA (cDNA).
Examples of the known method of measuring mRNA using the primers or probe containing the polynucleotide site-specifically bound to the foregoing mRNA or cDNA include RT-PCR, real-time RT-PCR, competitive PCR, in situ hybridization, in situ PCR, DNA array method and the like.
For example, the above-described RT-PCR is a method of synthesizing cDNA, using reverse transcriptase, from total RNA or mRNA prepared from a sample, and then amplifying a region of interest by PCR using the syntherized cDNA as a template. Real-time RT-PCR is a method of, during amplification of a region of interest by PCR using cDNA as a template, monitoring in real time and analyzing the generation process of a resulting product of the amplification using a reagent for real-time monitoring. Examples of the reagent for real-time monitoring include SYBR (registered trademark: Molecular Probes Inc.) Green, TaqMan (registered trademark: Applied Biosystems Inc.) probe and the like.
For example, in the above-described DNA array method, cDNA or its fragment of CCDC62 is immobilized on a support and incubated with mRNA or cDNA prepared from a sample. At this time, the mRNA or cDNA may be labeled with a fluorescent dye or the like so that hybridization between the DNA immobilized on the support and the mRNA or cDNA prepared from the sample can be detected, and the mRNA level in the sample can be measured.
In the comparing step, the mRNA level of CCDC62 in the sample, which level has been measured in the polynucleotide measuring step, may be compared with the normal level. As described previously, the normal level is preferably measured by the same method using a normal cell, tissue, or body fluid, as a sample, that is of the same kind as the subject-derived sample to be compared (i.e. sample used in the polynucleotide measuring step of the present embodiment). The normal level may be obtained, concurrently with the polynucleotide measuring step, by measurement conducted for a sample derived from a normal and healthy individual, or may be data stored as background data. If mRNA level in the sample derived from the subject is higher than the normal level, it is possible to determine that the subject has a cancer. The level higher than the normal level is preferably a level twice higher than the normal level, more preferably a level three times higher than the normal level.
(2) Cancer Diagnostic Method Using Protein Level as Index
In one embodiment, the cancer diagnostic method according to the present invention may be a method for diagnosing a cancer using a sample derived from a subject, including a polypeptide measuring step of measuring the level of the presence of a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof in the sample derived from the subject.
In the present embodiment, the level of CCDC62 protein may be measured in the polypeptide measuring step.
A method of measuring protein expression level, which is not particularly limited as long as it enables the measurement of a specific protein level, is selected for use as appropriate from among the known methods. Examples of the method of measuring protein expression level include a method using an antibody binding specifically to CCDC62 protein. The antibody may be a polyclonal antibody or a monoclonal antibody. Further, the antibody may be a complete antibody molecule or an antibody fragment capable of specifically binding (e.g. Fab fragment or F(ab′)2 fragment).
Examples of the known method of measuring a protein level using an antibody include radioimmunoassay (RIA), ELISA (enzyme linked immunoassay), Western blotting, immunoprecipitation method, immunohistochemical method, and antibody array method. Among these methods, ELISA is preferable in terms of high sensitivity and convenience.
In the comparing step, the level of the CCDC62 protein in the sample, which level has been measured in the polypeptide measuring step, may be compared with the normal level. As described previously, the normal level is preferably measured by the same method using a normal cell, tissue, or body fluid as a sample that is of the same kind as the subject-derived sample to be compared (i.e. sample used in the polypeptide measuring step of the present embodiment). The normal level may be obtained, concurrently with the polypeptide measuring step, by measurement conducted for a sample derived from a normal and healthy individual, or may be data stored as background data. If the protein level in the sample derived from the subject is higher than the normal level, it is possible to determine that the subject has cancer. The level higher than the normal level is preferably a level twice higher than the normal level, more preferably a level three times higher than the normal level.
(3) Cancer Diagnostic Method Using Antibody Level as Index
In one embodiment, the cancer diagnostic method according to the present invention may be a method for diagnosing a cancer using a sample derived from a subject, including an antibody measuring step of measuring the level of the presence of an antibody binding specifically to a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof in the sample derived from the subject.
In the present embodiment, the level of anti-CCDC62 antibody may be measured in the antibody measuring step. Note that a sample used in measuring the level of the anti-CCDC62 antibody is preferably a serum, but is not particularly limited as long as it enables the measurement of the antibody level.
An antibody level measuring method is not particularly limited as long as it enables the measurement of a level of antibody titer against a specific antigen and the measurement using an antibody against an antibody of interest, and is selected for use as appropriate from among the known methods. Examples of the antibody level measuring method include ELISA using CCDC62 protein as an antigen and a protein array. The antigen protein used in measuring the antibody titer can be obtained by purification of a biological sample, but is preferably obtained as a recombinant protein. The recombinant protein can be obtained by introducing an expression vector including a CCDC62 gene into a host for expression and then purifying from the host.
In the comparing step, the level of the anti-CCDC62 antibody in the sample, which level has been measured in the antibody measuring step, may be compared with the normal level. The normal level may be obtained, concurrently with the antibody measuring step, by measurement conducted for a sample (preferably a serum) derived from a normal and healthy individual, or may be data stored as background data. If the level of the anti-CCDC62 antibody in the sample derived from the subject is higher than the normal level, it is possible to determine that the subject has a cancer. The level higher than the normal level is preferably a level twice higher than the normal level, more preferably a level three times higher than the normal level.
[2. Kit]
The present invention provides a kit used for diagnosis of cancers. The kit according to the present invention may include a reagent or an instrument required for implementation of the diagnostic method described previously. As used herein, the term “kit” refers to a packaging that includes a container (e.g. bottle, plate, tube, dish, etc.) storing a particular material therein. Preferably, the kit includes instructions for use of each reagent or instrument. As used herein concerning the kit, the term “includes (including)” refers to the inclusion of a reagent or an instrument in any one of individual containers that constitutes the kit. The “instructions” may be printed on a sheet of paper or other medium, or may be recorded in an electronic medium such as a magnetic tape, a computer-readable disk or tape, or a CD-ROM. The kit according to the present invention may further include a reagent or an instrument required for application to diagnosis of cancers.
In one embodiment, a kit according to the present invention can be a kit applicable to a cancer diagnostic method using the above-described mRNA level as an index. A kit according to the present embodiment may be a kit for diagnosing a cancer using a sample derived from a subject, the kit including a polynucleotide comprising a nucleotide sequence of SEQ ID No: 1 or 3 or a partial sequence thereof. Examples of the polynucleotide comprising a nucleotide sequence of SEQ ID No: 1 or 3 or a partial sequence thereof include primers and a probe both used for measurement of mRNA of CCDC62. The kit according to the present embodiment can be used suitably for measurement of a CCDC62 mRNA expression level.
A kit according to the present embodiment may be a RT-PCR kit for measurement of mRNA of CCDC62 or a real-time RT-PCR kit for measurement of mRNA of CCDC62. Further, a kit according to the present embodiment can be a kit by which RT-PCR or real-time RT-PCR can be carried out by user selection. In this case, a kit according to the present embodiment may include a primer pair for amplifying mRNA of CCDC62 by RT-PCR. Kit components other than the primer pair are not particularly limited. For example, the kit preferably includes a reagent for preparing RNA from tissues or cells, a reverse transcriptase, a buffer solution used for reverse transcription reaction, a heat-resistant DNA polymerase, a reagent for PCR, a reagent for real-time PCR, a tube for PCR, and a plate for PCR.
In another embodiment, a kit according to the present invention can be a kit applicable to a cancer diagnostic method using the above-described protein level as an index. A kit according to the present embodiment may be a kit for diagnosing a cancer using a sample derived from a subject, the kit including an antibody binding specifically to a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof. The kit according to the present embodiment can be used suitably for measurement of CCDC62 protein level.
In yet another embodiment, a kit according to the present invention can be a kit applicable to a cancer diagnostic method using the above-described antibody level as an index. A kit according to the present embodiment may be a kit for diagnosing a cancer using a sample derived from a subject, the kit including a polypeptide consisting of an amino acid sequence of SEQ ID No: 2 or 4 or a partial sequence thereof. In this case, a kit according to the present embodiment can be used for measurement of a level of antibody titer of the anti-CCDC62 antibody. Further, a kit according to the present embodiment may include an antibody against the anti-CCDC62 antibody. A technique of preparing such an antibody is well known in the art.
A kit according to the present embodiment can be a kit for ELISA. Such a kit preferably includes either (i) an ELISA plate having an antibody binding specifically to the CCDC62 protein immobilized (solid-phased) thereon or (ii) an ELISA plate having the CCDC62 protein immobilized (solid-phased) thereon. Further, if a kit according to the present embodiment is a kit including both of the ELISA plates (i) and (ii), such a kit can be a kit capable of measuring both the CCDC62 protein level and the anti-CCDC62 antibody level.
Kit components other than the ELISA plate are not particularly limited. For example, a kit according to the present embodiment preferably includes a labeled secondary antibody, a coloring reagent, and a washing buffer solution.
Specific embodiments implemented in the description of the embodiments and the following examples only show technical features of the present invention and are not intended to limit the scope of the invention. Variations can be effected by a person skilled in the art within the spirit of the present invention and the scope of the following claims.
Further, all of scientific literatures and patent literatures listed herein are herein incorporated by reference.
EXAMPLES
The following will describe the present invention in more detail by way of Examples. It should be noted however that the present invention is not limited in any way by the following Examples. Unless otherwise specified, methods required for general genetic recombination, such as extraction, cleavage, and connection of nucleic acids, transformation of Escherichia coli, and determination of nucleotide sequence of a gene, are carried out according to a manual attached to a commercially-available reagent, equipment, and others for use in each operation and an experimental manual (e.g. “Molecular Cloning, a Laboratory Manual, 3rd Ed (Sambrook et al. (2001), Cold Spring Harbor Laboratory Press)”).
Example 1
SEREX Analysis of Antigen Recognized by Antibody in Gastric Cancer Patient Serum
As schematically shown in FIG. 1, SEREX is a method of searching for a cancer antigen using a serum from a cancer patient in a cDNA expression library prepared by direct extraction of mRNAs from a cancer tissue isolated from the cancer patient. In Example 1, a cancer-testis antigen useful for diagnosis of gastric cancer was isolated by SEREX using a cDNA library derived from normal testis and a serum from a patient with gastric cancer.
(1) Gastric Cancer Patient Serum
A serum was obtained from a patient with gastric adenocarcinoma whose primary lesion and liver metastasis both showed a tendency to reduce. The obtained serum was confirmed to strongly react with a protein fraction obtained from normal testis, before selected. The serum was diluted 10-fold with TBS (Tris-Buffered Saline) and then subjected to absorption of anti-bacterial antibodies (nonspecific antibodies against Escherichia coli and a phage) using an affinity column of E. coli Y1090/Y1089 (BioDynamics Lab Inc., Tokyo).
(2) Preparation of cDNA Expression Library
Using Quick Prep Micro mRNA purification kit (Stratagene, La Jolla, Calif., USA), mRNAs were purified from normal testis Total RNA (BD Biosciences Clontech, Palo Alto, Calif., USA). From 5 μg of mRNAs, cDNAs were synthesized. The cDNAs were then subcloned into γZAP Express vectors (Stratagene). The recombinant γZAP Express vectors were packaged into phages to prepare a cDNA expression library.
(3) Immunoscreening of Normal Testis cDNA Library
About 4,000 phages were planted on a 150 mm-plate and incubated for 6 to 8 hours. IPTG-induced protein was transferred into a 135 mm-diameter nitrocellulose membrane (Schleicher 86 Schuell, Dassel, Germany). Thereafter, the resultant membrane was reacted for 15 hours with the serum from the patient with gastric cancer (diluted 200-fold with TBS) absorbing the anti-bacterial antibodies, and then subjected to detection using a peroxidase-labeled anti-human IgG antibody (Jackson ImmunoResearch, West Grove, Pa., USA). At this time, in order to remove IgG clones derived from antibody production cells, the membrane was processed with a secondary antibody alone. Antibody-positive clones were isolated therefrom and then subjected to secondary screening and third screening using a 82 mm-diameter membrane and a 47 mm-diameter membrane, respectively, to obtain a single clone.
(4) Determination of Insert cDNA Nucleotide Sequence
The antibody-positive clone was converted into pBK-CMV plasmid by in vivo excision. Thereafter, a nucleotide sequence of insert cDNA was analyzed (ABI PRISM R310 Genetic Analyzer; PE Applied Biosystems, Foster City, Calif, USA), and homology search was carried out according to a gene database (www.ncbi.nih.gov/BLAST/Blast.cgi).
(5) Result
As a result of screening about 200,000 clones, fifty-five antibody-positive clones shown in Table 1 were isolated.







TABLE 1







SEREX-identified antigen genes











No. of




Gene
clones
Identity/similarities
Expression













OY-ST-1
10
cytochrome b5 reductase 2 (CYB5R2), transcript variant 2
Ubiquitous


OY-ST-2
8
no homology
Testis


OY-ST-3
2
G kinase anchoring protein 1 (GKAP1)
Testis


OY-ST-4
3
pleckstrin and Sec7 domain containing 3
Ubiquitous


OY-ST-5
4
palladin, cytoskeletal associated protein
Ubiquitous


OY-ST-6
1
synaptonemal complex protein 1, SYCP1 (SCP-1)
Testis


OY-ST-7
1
ankyrin repeat domain 13B
Ubiquitous


OY-ST-8
1
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9)
pseudogene




pseudogene 3 (ATP5GP3) on chromosome 14


OY-ST-9
1
lymphotoxin beta receptor (TNFR superfamily, member 3)
Ubiquitous


OY-ST-10
1
leucine-rich repeats and calponin homology (CH) domain containing 4
Ubiquitous


OY-ST-11
1
heat shock transcription factor 2
Ubiquitous


OY-ST-12
1
MAX gene associated, transcript variant 8 (MGA)
Ubiquitous


OY-ST-13
4
peroxisomal D3, D2-enoyl-CoA isomerase, transcript variant 1
no data


OY-ST-14
7
actin related protein 2⁄3 complex, subunit 2 34 kDa (ARPC2)
Ubiquitous


OY-ST-15
1
suppressor of Ty 5 homolog (S. cerevisiae) (SUPT5H)
Ubiquitous


OY-ST-16
1
polymerase (RNA) III (DNA directed) polypeptide H (22.9 KD) (POLR3H)
Ubiquitous


OY-ST-17
1
centrosomal protein 290 kDa) (CEP290)
Ubiquitous


OY-ST-18
2
coiled-coil domain containing 62 (CCDC62)
Testis


OY-ST-19
1
dihydrouridine synthase 1-like (S. cerevisiae)
Ubiquitous


OY-ST-20
1
leiomodin 1 (smooth muscle) (LMOD1)
Ubiquitous


OY-ST-21
1
activating signal cointegrator 1 complex subunit 2 (ASCC2)
Ubiquitous


OY-ST-22
1
chromosome 1 open reading frame 57 (Clorf57)
Ubiquitous


OY-ST-23
1
F-box and leucine-rich repest protein 5 (FBXL5), transcript variant 1
Ubiquitous









As the result of the homology search, 23 kinds of genes (OY-ST-1 through OY-ST-23) were identified. Then, normal tissue expressions of these genes were searched according to the database. The result of the search showed that among these 23 kinds of genes 4 kinds of genes were specific to testis (Table 1). Regarding the other 19 kinds of genes, they were expressed in a broad area of normal tissue, or the data on their expressions was not obtained.
As genes expressing specifically in testis, 2 clones of OY-ST-3 (G kinase anchoring protein 1 (GKAP1)) were obtained. Regarding the invention concerning GKAP1, a patent application was filed by the same inventors of the present invention and is now pending (not published yet). Further, 1 clone of OY-ST-6 (synaptonemal complex protein 1, SYCP1 (SCP-1)) was obtained. SCP-1 (OY-ST-6), which is one of the typical cancer-testis antigens (CT antigens) discovered shortly after the development of SEREX, is known to have a weak immunogenicity (Tureci, et al., Proc. Natl. Acad. Sci. USA, 1998).
2 clones of OY-ST-18 were isolated, and the homology search showed that these 2 clones were CCDC 62 (coiled-coil domain containing 62), and that they were transcript variant 1 (CCDC62-1, NM—032573: SEQ ID No: 1) and transcript variant 2 (CCDC62-2, NM—201435: SEQ ID No: 3), respectively. It should be noted that it has not been reported that CCDC62 is related to diseases including human cancers.
Example 2
Analysis of CCDC62 Expression
C62-1-S (sense: 5′-TCCCCGGCAAGTGAGCTAAT-3′, SEQ ID No: 5) and C62-1-AS (antisense: 5′-ATACATCCCCATTCCCGAGG-3′, SEQ ID No: 6) as primers specific for CCDC62 (variant 1) and C62-2-S (sense: 5′-AAGTCAGAGGTCCCAGAAGA-3′, SEQ ID No: 7) and C62-2-AS (antisense: 5′-CTATGCAGGGGTTCTTTCTC-3′, SEQ ID No: 8) as primers specific for CCDC62 (variant 2) were synthesized by a DNA synthesizer. By RT-PCR, cDNA derived from human normal tissues (MTC panel, BD Biosciences Clontech) and cDNAs derived from various cancers were amplified to analyze the expression of genes.
The cancer tissues as used were the donated ones that were obtained by a surgery or a biopsy. Note that all the samples derived from cancer patients or normal healthy individuals in Examples 1 and 2 were obtained by donation only from donors who had given informed consents after they had been directly given oral explanation and explanation in writing by their doctors in charge. The samples derived from cancer patients or normal healthy individuals were held anonymously and used in this study with their personal information strictly protected. The details of this study, an explanatory document for informed consent, and a certificate of informed consent were authorized by Okayama University, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Institutional Review Board of Human Genome and Gene Analysis Research.
Cancer cell lines as used were 11 kinds of lung cancer cell lines, 8 kinds of malignant melanoma cell lines, and 3 kinds of prostatic cancer cell lines. The malignant melanoma cell lines, which were established in the Sloane-Kettering Cancer Center in the U.S., were obtained therefrom by the inventors of the present invention. The other cell lines are commonly used for cancer research and usually available from a known cell preservation organization or the like.
From the cancer tissues and the cancer cell lines, respective total RNAs were extracted (RNeasy; Qiagen, Hilden, Germany). From the total RNAs, cDNAs were synthesized with MMLV reverse transcriptase and oligo(DT)15 primers (Ready-To-Go You-Prime First-Strand Beads; Amersham Biosciences, Buckinghamshire, UK). RT-PCR for CCDC62 was carried out under the following conditions: 35 cycles of denaturation at 94° C. for 1 minute; annealing at 60° C. for 1 minute; and extension reaction at 72° C. for 1.5 minutes. The PCR products were analyzed by agarose gel electrophoresis. At the same time, the amplification of G3PDH gene from the syntherized cDNAs was confirmed.
Regarding CCDC62-1 mRNA and CCDC62-2 mRNA, their expressions in 14 kinds of normal tissues were analyzed using CCDC62-1-specific primers or CCDC62-2-specific primers. The result of the analysis confirmed strong expressions in testis only ((a) of FIG. 2). The expression of CCDC62 genes in human normal tissues was quantitatively analyzed by real-time RT-PCR. Using random primers, cDNAs were synthesized from 2 μg of total RNAs (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, Foster City, Calif.). Using CCDC62-specific TagMan probe (TaqMan Gene Expression Assays, Applied Biosystems), the synthesized cDNAs were quantitatively analyzed with ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The TagMan probe (AssayID: Hs00261486) as used is a probe specific to a common sequence between 2 kinds of CCDC62 splice variants (CCDC62-1 and CCDC62-2). As an endogenous control, G3PDH was used (TaqMan Pre-Developed Assay Reagents, Applied Biosystems). The expression of CCDC62 was shown as compared with the expression of CCDC62 in normal testis, which was used as a calibrator. A strong CCDC62 expression in testis was confirmed. The level of such expression was equivalent to the level of expression of NY-ESO-1, which is a typical CT antigen, confirmed by the analysis carried out in the same procedure ((b) of FIG. 2).
The result of the analysis of CCDC62-1 mRNA and CCDC62-2 mRNA expressions in various cancer tissues showed no CCDC62-1 mRNA expressions. Table 2 shows the result of RT-PCR analysis of CCDC62-2 expression in various cancer tissues using specific primers.







TABLE 2







CCDC62-2 mRNA expressions


in cancer tissues and cancer cell lines (RT-PCR)










Positive/total












Subject
Tissues
Cell lines
















Breast cancer
1/4
(25%)
ND



Colon cancer
5/40
(12%)
ND



Esophageal cancer
7/33
(21%)
ND



Gastric cancer
8/117
(7%)
ND



Head and neck cancer
4/34
(12%)
ND



Liver cancer
0/5
(0%)
ND



Lung cancer
5/19
(26%)
4/11 (36%) 



Ovarian cancer
0/6
(0%)
ND



Prostate cancer
3/9
(33%)
1/3 (33%)



Renal cancer
1/4
(25%)
ND











Malignant melanoma
ND
3/8 (38%)










The CCDC62-2 mRNA expression was observed in an oral cavity cancer, an esophagus cancer, a gastric cancer, a breast cancer, a colon cancer, a lung cancer, a prostatic cancer, and a renal cancer (FIG. 3). Particularly, the CCDC62-2 mRNA expression in gastric cancer was confirmed in 8 of 117 cases (7%), the CCDC62-2 mRNA expression in lung cancer was confirmed in 5 of 19 cases (26%), and the CCDC62-2 mRNA expression in prostatic cancer was confirmed in 3 of 9 cases (33%). Further, the CCDC62-2 mRNA expression was also confirmed in cancer cell lines derived from lung cancer and prostatic cancer. Besides, high CCDC62-2 mRNA expression in malignant melanoma was confirmed in 3 of 8 cases (38%).
Example 3
Reactivity Against SEREX-Identified Antigens in Lung Cancer Patient Sera
Reactivity of sera from lung cancer patients against CCDC62-1, CCDC62-2, and one more kind of identified CT antigen, GKAP1 clone was analyzed by phage plaque assay (FIG. 4).







TABLE 3







Reactivity against various antigens


in sera from lung cancer patients










Antigen
Positive/total







OY-ST-2 (clone 1)
6/29



OY-ST-2 (clone 2)
2/29



CCDC62 (variant 1)
0/29



CCDC62 (variant 2)
4/29



GKAP1
2/29










Four out of 29 sera from patients with lung cancer were CCDC62-2 positive. Further, two out of 29 sera from patients with lung cancer were GKAP1-positive. Against CCDC62-1, none of the sera from patients with lung cancer reacted (Table 3). These antigens did not react with 7 healthy individual sera at all.
Example 4
Analysis of Humoral Immune Responses of Cancer Patients Against CCDC62-2 Protein
Into pGEX-6P-1 expression vector, cDNA encoding C terminus (amino acid residue 366-684) of CCDC62-2 protein was inserted. Escherichia coli BL-21 was transformed with the vector. GST fused-CCDC62-2 protein, which had been obtained by IPTG induction, was purified using GSTrap FF column (Amersham Biosciences).
Humoral immune responses of cancer patients against CCDC62-2 were analyzed by ELISA ((a) of FIG. 5). Specifically, GST-fused recombinant CCDC62-2 protein (1 μg/ml) was adsorbed on a 96-well plate (100 ng per well), subjected to blocking with 5% FCS/PBS for 1 hour, and then reacted with the serum (100 μl) for 2 hours. After the plate was washed, the CCDC62-2 protein on the plate was reacted with peroxidase-labeled anti-human IgG antibody (Jackson ImmunoResearch) for 1 hour. The reactant was made colored with a substrate (1,2-phenylenediamine dihydrochloride). Thereafter, absorbance was measured for 191 of sera from cancer patients and 41 of sera from healthy individuals. Antibody titer was evaluated for 400-fold diluted sera, and an OD value not less than 4 SD, which corresponds to an average OD value (490 nm) of healthy individuals, was determined to be positive. The result is shown in Table 4.







TABLE 4







Detection of CCDC62-2-specific IgG antibody


in sera from cancer patients (ELISA analysis)










Subject
Positive/total















Colon cancer
2/11
(18%)



Gastric cancer
6/104
(5.8%)



Lung cancer
5/76
(6.6%)



Healthy donor
0/41
(0%)










Out of 191 cancer patients, 13 patients (6.8%) were antibody-positive. Forty-one healthy individuals examined were all negative. Out of 104 gastric cancer patients, 6 patients (5.8%) were antibody-positive. Out of 76 lung cancer patients, 5 patients (6.6%) were antibody-positive. Out of 11 colon cancer patients, 2 patients (18%) were antibody-positive.
Sera from patients with gastric cancer and sera from patients with lung cancer were examined by Western blotting ((b) of FIG. 5(b)). Specifically, the recombinant CCDC62-2 protein was separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and then reacted with 100-fold diluted ELISA-positive and negative cancer patient sera. The protein after the reaction was detected using peroxidase-labeled anti-human IgG antibody (Jackson ImmunoResearch) to thereby analyze whether the antibodies in the sera were specific to the CCDC62-2 protein. The result of the analysis confirmed that the antibodies of the ELISA-positive patient sera were specific to the CCDC62-2 protein.
The present invention can provide a novel kit and method for diagnosing a cancer both using a CT antigen effective for diagnosis of digestive system cancer, such as gastric cancer and colon cancer. The CT antigen of interest is effectively used for not only diagnosis of digestive system cancer but also diagnosis of other epithelial tumors, such as breast cancer, head and neck cancer, lung cancer, renal cancer, and prostatic cancer, and diagnosis of skin cancer, such as malignant melanoma.
Specific embodiments or examples implemented in the description of the embodiments only show technical features of the present invention and are not intended to limit the scope of the invention. Variations can be effected within the spirit of the present invention and the scope of the following claims.
Industrial Applicability
The present invention can provide panels of a cancer antigen gene, a vector, a protein, an antibody, and cytotoxic T cell (CTL), and the like, for use in testing or diagnosis of cancer (determination of the presence or absence of cancer and a stage of cancer progression or prognostication of a cancer patient), prevention of cancer, treatment of cancer (e.g. vaccine against cancer), or the like. With these used alone or in combination or used as a kit, it is possible to evaluate a malignancy grade of cancer, a form of cancer tissue, treatment result, and prognostication. The use of CCDC62 enables a testing for cancer or a diagnosis of cancer with high accuracy. Moreover, in combination with CT antigens previously identified by the inventors of the present invention (e.g. OY-TES-1, RFX4, AKAP3, XAGE-1, and GKAP1), CCDC62 enables application to a wider variety of cancers.
The present invention is provided for use in diagnosis of cancer, and the present invention can be not only conductive to advances in medical sciences and medical care objective to cancers but also applicable to clinical test agent industry, reagent industry, medical equipment industry, and the like industries.

## Claims

1. A method for diagnosing an epithelial tumor or a melanoma, comprising:
mixing a sample derived from a subject and a polypeptide consisting of an amino acid sequence of position 366 to position 684 of SEQ ID No: 4, and
measuring a level of an antibody binding specifically to the polypeptide in the sample.

2. The method according to claim 1, the method being used for diagnosis of at least one cancer selected from the group consisting of a gastric cancer, a colon cancer, a breast cancer, a head and neck cancer, a lung cancer, a renal cancer, a prostatic cancer, and a malignant melanoma.

3. The method according to claim 1 , the antibody measuring step further comprising detecting an antibody which binds to the polypeptide.

4. The method according to claim 1, wherein the sample is sera.

5. The method according to claim 1, further comprising a step of comparing the level of the antibody binding specifically to the polypeltide obtained by the measuring with a control level.