JP2008505601A - Genes and polypeptides related to prostate cancer - Google Patents

Abstract

The present application provides a novel human gene CCDC4 whose expression is markedly elevated in prostate cancer. The gene and the polypeptide encoded by the gene can be used, for example, in the diagnosis of prostate cancer, as a target molecule for developing drugs against the disease, and to attenuate cell proliferation of prostate cancer.

Description

TECHNICAL FIELD The present invention relates to the field of biological science, and more particularly to the fields of cancer therapy and cancer diagnosis. In particular, the present invention relates to a novel polypeptide encoded by a novel gene B3537 (CCDC4) associated with prostate cancer. Furthermore, the present invention relates to a novel gene CCDC4. The genes and polypeptides of the invention can be used, for example, in the diagnosis of prostate cancer, as a target molecule for developing drugs against the disease, and to attenuate prostate cancer cell growth. This application claims priority from US Provisional Application No. 60 / 555,810, filed Mar. 23, 2004, the contents of which are hereby fully incorporated by reference.

Background Art Prostate cancer is one of the most common cancers in men in Western countries (Gronberg, Lancet 361: 859-64 (2003)). In developed countries, the incidence of prostate cancer is steadily increasing due to the spread of Western-style diets and an increasing elderly population. Early diagnosis by serum testing for prostate specific antigen (PSA) provides an opportunity for curative surgery and significantly improves the prognosis of prostate cancer. However, up to 30% of patients treated with radical prostatectomy relapse cancer (Han et al., J Urol 166: 416-9 (2001)). The most recurrent or most advanced cancers respond favorably to androgen deprivation therapy because prostate cancer growth is androgen-dependent at an early stage. However, most patients treated with this therapy eventually progress to androgen-independent disease, at which point they do not respond. The most significant clinical problem of prostate cancer is that androgen-independent prostate cancer does not respond to any other treatment (Gronberg, Lancet 361: 859-64 (2003)). Therefore, establishing a new treatment method other than androgen deprivation therapy for prostate cancer is an urgent issue for the management of prostate cancer.

  cDNA microarray technology provided a comprehensive profile of gene expression in normal and malignant cells, as well as the ability to compare gene expression in malignant and corresponding normal cells (Okabe et al., Cancer Res 61: 2129-37 (2001) Kitahara et al., Cancer Res 61: 3544-9 (2001); Lin et al., Oncogene 21: 4120-8 (2002); Hasegawa et al., Cancer Res 62: 7012-7 (2002)). This approach allows us to understand the complex nature of cancer cells and helps to elucidate the mechanisms of carcinogenesis. Identification of genes that are deregulated in tumors may lead to a more accurate and error-free diagnosis of individual cancers and the development of new therapeutic targets (Bienz and Clevers, Cell 103: 311-20 (2000) ). In order to elucidate the underlying mechanisms of tumors from a genome-wide perspective, and to explore target molecules for the development of diagnostics and novel therapeutics, we have cDNA containing 23040 genes. Microarrays have been used to analyze tumor cell expression profiles (Okabe et al., Cancer Res 61: 2129-37 (2001); Kitahara et al., Cancer Res 61: 3544-9 (2001); Lin et al., Oncogene 21: 4120- 8 (2002); Hasegawa et al., Cancer Res 62: 7012-7 (2002)).

  It is already easy to identify the molecular targets of antitumor drugs by studies designed to elucidate the mechanism of carcinogenesis. For example, a farnesyltransferase inhibitor (FTI), originally developed to inhibit growth signaling pathways involving Ras, whose activation depends on post-translational farnesylation, is a treatment for Ras-dependent tumors in animal models. (He et al., Cell 99: 335-45 (1999)). Clinical trials are being conducted in humans in combination with anti-cancer drugs and anti-HER-2 monoclonal antibody trastuzumab for the purpose of antagonizing proto-oncogene receptor HER2 / neu. Clinical efficacy and overall survival of breast cancer patients Improvement has been achieved (Lin et al., Cancer Res 61: 6345-9 (2001)). The tyrosine kinase inhibitor STI-571, which selectively inactivates the bcr-abl fusion protein, is used in chronic myeloid leukemia where constitutive activation of the bcr-abl tyrosine kinase plays a crucial role in leukocyte transformation. Developed for therapeutic purposes. These types of drugs are designed to suppress the carcinogenic activity of specific gene products (Fujita et al., Cancer Res 61: 7722-6 (2001)). For this reason, gene products that are frequently upregulated in cancer cells may serve as target candidates for developing new anticancer agents. Indeed, novel drugs that target dysregulated molecules that have the effect of causing cancer growth and progression have proven effective against certain types of cancer. Such agents include Herceptin for breast cancer, Glivec (STI571) for CML, and Iressa (ZD1839) for non-small cell lung cancer.

  Several molecules are known to be overexpressed in prostate cancer, which have been identified as prostate cancer therapeutic targets or markers (Xu et al., Cancer Res 60: 6568-72 (2000); Luo et al., Cancer Res 62: 2220-6 (2002)). However, most of them are also highly expressed in other major organs. Therefore, drugs targeting these molecules are considered to be toxic to cancer cells, but are also considered to have an adverse effect on normally proliferating cells of other organs.

  CD8 + cytotoxic T lymphocytes (CTLs) have been shown to recognize tumor peptides from tumor associated antigens (TAA) presented on MHC class I molecules and lyse tumor cells. Since the discovery of the MAGE family as the first example of TAA, many other TAAs have been discovered using immunological approaches (Boon, Int J Cancer 54: 177-80 (1993); Boon and van der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52 (1994)). Some of the discovered TAAs are currently in clinical development as targets for immunotherapy. TAAs discovered so far include MAGE (van der Bruggen et al., Science 254: 1643-7 (1991)), gp100 (Kawakami et al., J Exp Med 180: 347-52 (1994)), SART (Shichijo et al. J Exp Med 187: 277-88 (1998)) and NY-ESO-1 (Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997)). On the other hand, gene products that have been shown to be specifically overexpressed in tumor cells have been shown to be recognized as targets that induce cellular immune responses. Such gene products include p53 (Umano et al., Brit J Cancer 84: 1052-7 (2001)), HER2 / neu (Tanaka et al., Brit J Cancer 84: 94-9 (2001)), CEA (Nukaya et al. , Int J Cancer 80: 92-7 (1999)).

  Despite significant advances in basic and clinical research on TAA (Rosenbeg et al., Nature Med 4: 321-7 (1998); Mukherji et al., Proc Natl Acad Sci USA 92: 8078-82 (1995); Hu et al., Cancer Res 56: 2479-83 (1996)), the number of candidate TAAs for the treatment of adenocarcinoma including colorectal cancer is very limited. TAA, which is expressed in large amounts in cancer cells and at the same time is restricted to cancer cells, is considered to be a promising candidate for immunotherapy targets. In addition, the identification of new TAAs that elicit potent and specific anti-tumor immune responses would facilitate clinical use of peptide vaccination in various types of cancer (Boon and can der Bruggen, J Exp Med 183: 725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52 (1994). Shichijo et al., J Exp Med 187: 277-88 (1998); Chen et al., Proc Natl Acad Sci USA 94: 1914-8 (1997); Harris, J Natl Cancer Inst 88: 1442-5 (1996); Butterfield Cancer Res 59: 3134-42 (1999); Vissers et al., Cancer Res 59: 5554-9 (1999); van der Burg et al., J Immunol 156: 3308-14 (1996); Tanaka et al., Cancer Res 57: 4465-8 (1997); Fujie et al., Int J Cancer 80: 169-72 (1999); Kikuchi et al., Int J Cancer 81: 459-66 (1999); Oiso et al., Int J Cancer 81: 387-94 (1999). )).

Peripheral blood mononuclear cells (PBMC) from certain healthy donors, when subjected to peptide stimulation, produce significant levels of IFN-γ in response to the peptide, but HLA-A24 or HLA-A0201 in ⁵¹ Cr release assays It has been repeatedly reported that it has little cytotoxicity against tumor cells in a restrictive manner (Kawano et al., Cancer Res 60: 3550-8 (2000); Nishizaka et al., Cancer Res 60: 4830-7 (2000); Tamura et al., Jpn J Cancer Res 92: 762-7 (2001)). However, both HLA-A24 and HLA-A0201 are HLA alleles common in Japanese and are similar in the Caucasian population (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al., J Immunol 155: 4307 -12 (1995); Kubo et al., J Immunol 152: 3913-24 (1994); Imanishi et al., Proceeding of the eleventh International Histocompatibility Workshop and Conference Oxford University Press, Oxford, 1065 (1992); Williams et al., Tissue Antigens 49: 129 (1997)). Thus, the cancer antigen peptides presented by these HLAs may be particularly useful in the treatment of cancer in the Japanese and Caucasian populations. In addition, in vitro induction of low-affinity CTLs is usually a specific peptide / MHC complex that is thought to effectively activate these CTLs on the surface of antigen-presenting cells (APC) using peptides at high concentrations. It is known to occur by raising the body to a high level (Alexander-Miller et al., Proc Natl Acad Sci USA 93: 4102-7 (1996)).

SUMMARY OF THE INVENTION We have disclosed genome-wide mechanisms of prostate cancer and genome-wide in combination with laser microbeam microdissection to identify novel diagnostic markers and / or drug targets for the treatment of these tumors. A cDNA microarray was used to analyze gene expression profiles in prostate cancer. In previous studies, accurate expression profiles of prostate cancer cells (PRC) and non-invasive progenitor cells (PIN) were performed by combining genome-wide cDNA microarrays with laser microdissection. By comparing the expression profile of invasive PRC with normal prostate epithelium or non-invasive precursor PIN, we have identified a gene 88 that is commonly up-regulated in both invasive PRC and precursor PIN. Individuals and 207 genes down-regulated were identified. In the present invention, the inventors focused on one EST and identified a novel gene CCDC4 that is overexpressed in prostate cancer cells.

  As a result, CCDC4 was identified as a gene that is specifically overexpressed in prostate cancer cells. We have the effect that siRNA knockdown effect of CCDC4 attenuates proliferation of prostate cancer cells, and this molecule has the potential to be a target for drug design for new treatments of prostate cancer It shows that.

  CCDC4 encodes a 530 amino acid protein containing a coiled coil domain. Northern blot analysis showed that CCDC4 expression is restricted to the testis and prostate.

  Many anticancer drugs are toxic not only to cancer cells but also to normally proliferating cells. However, due to the fact that the normal expression of CCDC4 is restricted to the testis and prostate, drugs that suppress the expression of CCDC4 do not have a detrimental effect on other organs and are therefore not useful for the treatment or prevention of prostate cancer. Can be used conveniently for.

  Thus, the present invention provides an isolated gene CCDC4, a promising potential target useful as a candidate diagnostic marker for prostate cancer and for developing new therapeutic methods for diagnostic and effective anticancer agents. provide. Furthermore, the present invention provides polypeptides encoded by this gene and their production and use. More specifically, the present invention provides a novel human polypeptide CCDC4 or a functional equivalent thereof that is upregulated in prostate cancer cells.

  In a preferred embodiment, the CCDC4 polypeptide comprises a 530 amino acid protein encoded by the open reading frame of SEQ ID NO: 1 or a 437 amino acid protein encoded by the open reading frame of SEQ ID NO: 3. The CCDC4 polypeptide preferably comprises the amino acid sequence shown in SEQ ID NO: 2 (Gene Bank accession number: AB126828) or 4 (GeneBank accession number: AB126829). The present application provides an isolated protein encoded from at least a portion of a CCDC4 polynucleotide sequence or a polynucleotide sequence that is at least 15%, more preferably at least 25% complementary to the sequence set forth in SEQ ID NO: 1 or 3. Also provide.

  The present invention further provides a novel human gene CCDC4 whose expression is significantly elevated in the majority of prostate cancers compared to the corresponding non-cancerous prostate duct epithelium. The isolated CCDC4 gene comprises a polynucleotide sequence as set forth in SEQ ID NO: 1 or 3. In particular, CCDC4 cDNA is 8863 nucleotides (SEQ ID NO: 1) containing an open reading frame of 1593 nucleotides, or 8692 nucleotides (SEQ ID NO: 3) containing an open reading frame of 1314 nucleotides including. The present invention hybridizes to the polynucleotide sequence shown in SEQ ID NO: 1 or 3 if it encodes the CCDC4 protein or a functional equivalent thereof, and is complementary to at least 15%, more preferably at least 25%. Further included are polynucleotides that are specific. Examples of such polynucleotides are degenerate and allelic variants of CCDC4 encoded by the sequence of SEQ ID NO: 1 or 3.

  As used herein, an isolated gene is the structure of any fragment of a natural genomic polynucleotide whose structure is not identical to the structure of any natural polynucleotide and that spans more than three separate genes. Polynucleotides that are not identical to each other. Thus, the term includes, for example, (a) DNA that is naturally present in the genome of an organism and has a partial sequence of a native genomic DNA molecule, (b) which natural vector or genomic DNA the resulting molecule is Polynucleotides incorporated into vectors or prokaryotic or eukaryotic genomic DNA, (c) cDNA, genomic fragments, polymerase chain reaction (PCR) -generated fragments or restriction fragments, etc. And (d) a recombinant nucleotide sequence that is part of a hybrid gene, ie, a gene encoding a fusion polypeptide.

  Accordingly, in one aspect, the present invention provides an isolated polynucleotide that encodes a polypeptide described herein or a fragment thereof. Preferably, the isolated polynucleotide comprises a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO: 1 or 3. The isolated nucleic acid molecule has at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94 with the nucleotide sequence shown in SEQ ID NO: 1 or 3. %, 95%, 96%, 97%, 98%, 99% or more are more preferred. If the isolated polynucleotide is longer or equal in length to a reference sequence, eg, SEQ ID NO: 1 or 3, a comparison with the full length of the reference sequence is performed. If the isolated polynucleotide is shorter than the reference sequence, eg shorter than SEQ ID NO: 1 or 3, and a segment of the reference sequence of the same length (excluding the loop necessary for calculating homology) A comparison is made.

  The present invention also provides a method for producing a protein by transfecting or transforming a host cell with a polynucleotide sequence encoding a CCDC4 protein and expressing the polynucleotide sequence. In addition, the present invention also provides a vector comprising a nucleotide sequence encoding CCDC4 protein, and a host cell carrying a polynucleotide encoding CCDC4 protein. Such vectors and host cells can be used for the production of CCDC4 protein.

  A binding agent that specifically recognizes the CCDC4 protein is also provided by the present application. For example, the binding agent may be an antibody raised against CCD4 protein. Alternatively, the binding agent may be a ligand specific for the protein, or a synthetic polypeptide that specifically binds to the protein (see, for example, WO2004044011). Antisense polynucleotides (eg, antisense DNA), ribozymes, and siRNA (small interfering RNA) of the CCDC4 gene are also provided.

  The present invention is further a method for diagnosing prostate cancer, comprising determining the expression level of a gene in a biological sample from a subject, comparing the expression level of a CCDC4 gene with the expression level in a normal sample, And a method comprising defining a high expression level of the CCDC4 gene in the sample as indicating that the subject is suffering from or at risk of developing prostate cancer.

  Furthermore, a method for screening a compound for treating or preventing prostate cancer is also provided by the present invention. The method includes contacting the CCDC4 polypeptide with a test compound and selecting a test compound that binds to or alters the biological activity of the CCDC4 polypeptide.

  The present invention further relates to a method for screening a compound for treating or preventing prostate cancer, wherein a test compound is contained in a cell expressing a CCDC4 polypeptide, or a vector comprising a transcriptional regulatory region of CCDC4 upstream of a reporter gene. Also provided is a method comprising contacting the introduced cell and selecting a test compound that suppresses the expression level of the CCDC4 polypeptide.

  The application also provides pharmaceutical compositions for the treatment or prevention of prostate cancer. The pharmaceutical composition may be, for example, an anticancer agent. The pharmaceutical composition can comprise at least a portion of an antisense S-oligonucleotide, siRNA molecule or ribozyme of the CCDC4 polynucleotide sequence presented and described in SEQ ID NOs: 1 and 3, respectively. A suitable siRNA targets SEQ ID NO: 8. Accordingly, the siRNA of the present invention comprises the nucleotide sequence of SEQ ID NO: 8. This is preferably selected as a target for the treatment or prevention of prostate cancer according to the present invention. The pharmaceutical composition may comprise a compound selected by the present screening method for a compound for the treatment or prevention of cell proliferative diseases such as prostate cancer.

  The route of action of the pharmaceutical composition is desirably one that inhibits the growth of cancer cells such as prostate cancer. The pharmaceutical composition can be applied to mammals, including humans and livestock.

  The present invention further provides a method for treating or preventing prostate cancer using the pharmaceutical composition provided by the present invention.

  Furthermore, the present invention provides a method for the treatment or prevention of cancer comprising the step of administering a CCDC4 polypeptide. Anti-tumor immunity is thought to be induced by administration of CCDC4 polypeptide. Accordingly, the present invention also provides a method for inducing anti-tumor immunity comprising administering a CCDC4 polypeptide, and a pharmaceutical composition for the treatment or prevention of cancer comprising the CCDC4 polypeptide.

  It is to be understood that both the foregoing summary of the invention and the following detailed description are of preferred embodiments and are not intended to limit the invention or other alternative embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the terms “a”, “an” and “the” mean “at least one” unless otherwise specified. .

  In order to elucidate the mechanism of prostate cancer and to identify novel diagnostic markers and / or drug targets for the treatment and / or prevention of these tumors, we have identified genome-wide cDNA microarrays. In combination with laser microbeam microdissection, gene expression profiles in prostate cancer were analyzed. As a result, CCDC4 specifically overexpressed in prostate cancer cells was identified. Furthermore, suppression of CCDC4 gene expression by transfection with small interfering RNA (siRNA) caused a marked suppression of cancer cell growth. These findings suggest that CCDC4 confers oncogenic activity to cancer cells and that inhibition of the activity of these proteins can be a promising strategy for the treatment and prevention of proliferative diseases such as prostate cancer.

CCDC4
According to the present invention, two genes with similar sequences have been identified and encode CCDC4 variants. The mutant cDNA consists of 8863 nucleotides (SEQ ID NO: 1), the long form containing an open reading frame of 1593 nucleotides, and the short form consists of 8692 nucleotides (SEQ ID NO: 3), which contains an open reading frame of 1314 nucleotides. These open reading frames encode a 530 amino acid protein and a 437 amino acid protein, respectively.

  Accordingly, the present invention includes substantially pure polypeptides encoded by these genes, including polypeptides comprising the amino acid sequence of SEQ ID NO: 2 or 4, as well as functional equivalents thereof, which are also CCDC4 protein Is included in the coding range. Examples of polypeptides that are functionally equivalent to CCDC4 include, for example, homologous proteins of other organisms corresponding to human CCDC4 protein, as well as variants of human CCDC4 protein.

  In the present invention, the term “functionally equivalent” means that the subject polypeptide has the activity of promoting cell growth and imparting oncogenic activity to cancer cells, like CCDC4 protein. Whether the target polypeptide has cell growth activity is determined by introducing DNA encoding the target polypeptide into the cell, expressing each polypeptide, and detecting the promotion of cell growth or the increase in colony forming activity. Is determined by Such cells include, for example, NIH3T3, COS7 and HEK293.

  Methods for producing polypeptides that are functionally equivalent to a given protein are well known to those of skill in the art and include known methods for introducing mutations into a protein. For example, those skilled in the art can produce polypeptides functionally equivalent to human CCDC4 proteins by introducing appropriate mutations into the amino acid sequences of these proteins by site-directed mutagenesis (Hashimoto- Gotoh et al., Gene 152: 271-5 (1995); Zoller and Smith, Methods Enzymol 100: 468-500 (1983); Kramer et al., Nucleic Acids Res. 12: 9441-9456 (1984); Kramer and Fritz, Methods Enzymol 154: 350-67 (1987); Kunkel, Proc Natl Acad Sci USA 82: 488-92 (1985); Kunkel, Methods Enzymol 85: 2763-6 (1988)). Amino acid mutations can occur naturally. On the premise that the resulting mutant polypeptide is functionally equivalent to human CCDC4 protein, proteins having the amino acid sequence of human CCDC4 protein in which one or more amino acids are mutated are included in the polypeptide of the present invention. The number of amino acids to be mutated in such variants is generally 10 amino acids or less, preferably 6 amino acids or less, more preferably 3 amino acids or less.

  A modified protein that is a mutant protein or a protein having an amino acid sequence modified by substitution, deletion, insertion, and / or addition of one or more amino acid residues of a particular amino acid sequence is the original biological Known to preserve activity (Mark et al., Proc Natl Acad Sci USA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10: 6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13 (1982)).

  The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain (a method known as conservative amino acid substitution). Examples of amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G , H, K, S, T) and side chains having the following functional groups or characteristics in common: aliphatic side chains (G, A, V, L, I, P); hydroxyl group-containing side chains (S, T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); base-containing side chains (R, K, H); Group-containing side chains (H, F, Y, W). Note that letters in parentheses indicate single letter abbreviations for amino acids.

  An example of a polypeptide in which one or more amino acid residues are added to the amino acid sequence of human CCDC4 protein is a fusion protein containing human CCDC4 protein. A fusion protein is a fusion of a human CCDC4 protein with another peptide or protein and is included in the present invention. The fusion protein is obtained by ligating DNA encoding the human CCDC4 protein of the present invention in frame with DNA encoding another peptide or protein, inserting the fusion DNA into an expression vector, and expressing it in a host. Can be made by techniques well known to those skilled in the art. There is no limitation on the peptide or protein to be fused with the protein of the present invention.

  Known peptides that can be used as peptides to be fused with the protein of the present invention include, for example, FLAG (Hopp et al., Biotechnology 6: 1204-10 (1988)), 6xHis, 6xHis containing 6 His (histidine) residues, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7 tag, HSV tag, E tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B tag, protein C fragment, etc. Is included. Examples of proteins that can be fused with the protein of the present invention include GST (glutathione-S-transferase), influenza agglutinin (HA), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein) and the like.

  A fusion protein can be made by fusing a commercially available DNA encoding a fusion peptide or protein with a DNA encoding a polypeptide of the present invention and expressing the prepared fusion DNA, as discussed above.

  Alternative methods known in the art for isolating functionally equivalent polypeptides include, for example, those using hybridization methods (Sambrook et al., “Molecular Cloning” 2nd edition, 9.47. -9.58, Cold Spring Harbor Lab. Press (1989)). Those skilled in the art can readily isolate DNA having high homology to all or part of the DNA sequence encoding human CCDC4 protein (ie, SEQ ID NO: 1 or 3) and use human CCDC4 from the isolated DNA. Polypeptides functionally equivalent to proteins can be isolated. Polypeptides of the present invention include polypeptides that are encoded by DNA that hybridizes to all or part of the DNA sequence encoding human CCDC4 protein and are functionally equivalent to human CCDC4 protein. These polypeptides include mammalian homologues corresponding to human-derived proteins (eg, polypeptides encoded by monkey, rat, rabbit, and bovine genes). When isolating a cDNA highly homologous to DNA encoding human CCDC4 protein from an animal, it is particularly preferred to use tissue from the testis or prostate.

  Hybridization conditions for isolating DNA encoding a polypeptide functionally equivalent to the human CCDC4 protein can be routinely selected by those skilled in the art. For example, prehybridization at 68 ° C. for 30 minutes or longer is performed using “Rapid-hyb buffer” (Amersham LIFE SCIENCE) for 1 hour or after adding a labeled probe. Hybridization may be performed by heating at 68 ° C. for more than that. Subsequent washing steps can be performed, for example, under low stringent conditions. Low stringency conditions are, for example, 42 ° C, 2X SSC, 0.1% SDS, or preferably 50 ° C, 2X SSC, 0.1% SDS. More preferably, high stringency conditions are used. High stringency conditions include, for example, three 20-minute washes in 2X SSC and 0.01% SDS at room temperature, followed by three 20-minute washes in 1x SSC and 0.1% SDS at 37 ° C. Perform two 20 minute washes in 1x SSC, 0.1% SDS at 50 ° C. However, several factors such as temperature and salt concentration are thought to affect the stringency of hybridization and those skilled in the art can appropriately select these factors to obtain the required stringency.

  Instead of hybridization, a gene amplification method, for example, a polymerase chain reaction (PCR) method, a DNA encoding a polypeptide functionally equivalent to the human CCDC4 protein, a sequence information of the DNA encoding the protein (SEQ ID NO: 1 Alternatively, it can be used for isolation using a primer synthesized according to 3).

  Polypeptides functionally equivalent to human CCDC4 protein encoded by DNA isolated by the above hybridization or gene amplification techniques usually have a high homology with the amino acid sequence of human CCDC4 protein. . “High homology” typically means 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 85%, between a polypeptide or polynucleotide sequence and a reference sequence, 90%, 93%, 95%, 98%, 99% or more homology. The homology rate (also called the identity rate) is typically performed between two optimally aligned sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment and comparison of sequences can be performed, for example, using the algorithm of “Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30 (1983)”.

  The polypeptides of the present invention have differences in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chains, or form depending on the cell or host used for production or the purification method used. However, as long as it has a function equivalent to the polypeptide of the human CCDC4 protein of the present invention, it is within the scope of the present invention.

  The polypeptides of the present invention can be prepared as recombinant proteins or natural proteins by methods well known to those skilled in the art. The recombinant protein is obtained by inserting DNA encoding the polypeptide of the present invention (for example, DNA containing the nucleotide sequence of SEQ ID NO: 1 or 3) into an appropriate expression vector, and introducing the vector into an appropriate host cell. Obtaining the extract and subjecting the extract to chromatography, for example ion exchange chromatography, reverse phase chromatography, gel filtration, or affinity chromatography using a column immobilized with an antibody against the protein of the invention, or It can be prepared by purifying a polypeptide by combining a plurality of the above-mentioned columns.

  Similarly, when expressing the polypeptide of the present invention as a fusion protein with a glutathione-S-transferase protein in a host cell (eg, animal cell and E. coli) or as a recombinant protein supplemented with a large number of histidines. The expressed recombinant protein can be purified using a glutathione column or a nickel column. In addition, when the polypeptide of the present invention is expressed as a protein labeled with c-myc, many histidines, or FLAG, it is detected and purified using an antibody against c-myc, His, or FLAG, respectively. be able to.

  After purifying the fusion protein, it is possible to remove regions other than the target polypeptide by cleaving with thrombin or factor Xa as necessary.

  The natural protein is contacted with an extract of a tissue or cell that expresses the polypeptide of the present invention by a method known to those skilled in the art, for example, an affinity column to which an antibody that binds to the following CCDC4 protein is bound. Can be isolated by The antibody may be a polyclonal antibody or a monoclonal antibody.

  The present invention also includes partial peptides of the polypeptides of the present invention. The partial peptide has an amino acid sequence specific to the polypeptide of the present invention, and consists of at least 7 amino acids, preferably 8 amino acids or more, more preferably 9 amino acids or more. The partial peptide is used, for example, to generate an antibody against the polypeptide of the present invention, to screen for a compound that binds to the polypeptide of the present invention, and to screen for an inhibitor of the polypeptide of the present invention. be able to.

  The partial peptide of the present invention can be produced by genetic manipulation, by a known peptide synthesis method, or by digesting the polypeptide of the present invention with an appropriate peptidase. For peptide synthesis, for example, solid phase synthesis or liquid phase synthesis can be used.

  The present invention further provides a polynucleotide encoding the above-mentioned CCDC4 polypeptide. The polynucleotide of the present invention can be used for in vivo or in vitro production of the polypeptide of the present invention as described above, or a gene for a disease caused by a genetic abnormality of a gene encoding the protein of the present invention. It can also be used for treatment. Any form of the polynucleotide of the present invention can be used, including mRNA, RNA, cDNA, genomic DNA, chemically synthesized polynucleotides, as long as it encodes a polypeptide of the present invention. In addition to DNA containing a predetermined nucleotide sequence, the polynucleotide of the present invention includes its degenerate sequence as long as the resulting DNA encodes the polypeptide of the present invention.

  The polynucleotide of the present invention can be prepared by methods known to those skilled in the art. For example, the polynucleotide of the present invention is prepared by preparing a cDNA library from cells that express the polypeptide of the present invention, and using the partial sequence of the DNA of the present invention (for example, SEQ ID NO: 1 or 3) as a probe. It can produce by doing. A cDNA library can be prepared, for example, by the method described in Sambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory Press (1989); or a commercially available cDNA library may be used. A cDNA library extracts RNA from cells expressing the polypeptide of the present invention, synthesizes oligo DNA based on the sequence of the DNA of the present invention (for example, SEQ ID NO: 1 or 3), and uses the oligo DNA as a primer. Can also be used to perform PCR and amplify the cDNA encoding the protein of the invention.

  Furthermore, by sequencing the nucleotide of the obtained cDNA, the translation region encoded by the cDNA can be routinely determined, and the amino acid sequence of the polypeptide of the present invention can be easily obtained. Moreover, genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA or a portion thereof.

  More specifically, mRNA is first prepared from a cell, tissue, or organ (eg, testis or prostate) in which a subject polypeptide of the invention is expressed. Known methods can be used to isolate mRNA; for example, total RNA can be obtained from guanidine ultracentrifugation (Chirgwin et al., Biochemistry 18: 5294-9 (1979)) or AGPC (Chomczynski and Sacchi, Anal Biochem 162: 156-9 (1987)). Furthermore, mRNA may be purified from total RNA using an mRNA purification kit (Pharmacia) or the like. Alternatively, mRNA may be purified directly using a QuickPrep mRNA purification kit (Pharmacia).

  The obtained mRNA is used to synthesize cDNA using reverse transcriptase. cDNA can be purified using a commercially available kit such as an AMV reverse transcriptase first strand cDNA synthesis kit (Seikagaku Kogyo). Alternatively, 5'-RACE method (Frohman et al., Proc Natl Acad Sci USA 85: 8998-9002 using 5'-Ampli FINDER RACE kit (Clontech) and polymerase chain reaction (PCR) such as the primers described herein. (1988); Belyavsky et al., Nucleic Acids Res 17: 2919-32 (1989)) can also synthesize and amplify cDNA.

  A desired DNA fragment is prepared from the PCR product and ligated with vector DNA. This recombinant vector is used for transformation of E. coli and the like, and a desired recombinant vector is prepared from the selected colony. The nucleotide sequence of the desired DNA is verified by conventional methods such as dideoxynucleotide chain termination.

  The nucleotide sequence of the polynucleotides of the invention can also be designed to be expressed more efficiently by taking into account the codon usage in the host used for expression (Grantham et al., Nucleic Acids Res 9: 43-74 (1981)). The sequence of the polynucleotide of the present invention can also be modified by a commercially available kit or a conventional method. For example, modifying the sequence by digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate polynucleotide fragments, addition of linkers, or insertion of start codons (ATG) and / or stop codons (TAA, TGA or TAG) it can.

  Specifically, the polynucleotide of the present invention comprises DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3.

  Furthermore, the present invention hybridizes with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 or 3 under stringent conditions, and encodes a polypeptide functionally equivalent to the above-mentioned CCDC4 protein of the present invention. A polynucleotide is provided. Those skilled in the art may appropriately select stringent conditions. For example, low stringency conditions can be used. More preferably, high stringency conditions are used. These conditions are the same as described above. The DNA to be hybridized is preferably cDNA or chromosomal DNA.

  The present invention also provides a polynucleotide that is complementary to a polynucleotide (SEQ ID NO: 1 or 3) encoding human CCDC4 protein or its complementary strand and comprises at least 15 nucleotides. The polynucleotide of the present invention is preferably a polynucleotide that specifically hybridizes with DNA encoding the CCDC4 polypeptide of the present invention. The term “specifically hybridizes” as used herein means that cross-hybridization with DNA encoding other proteins is prominent under normal hybridization conditions, preferably stringent hybridization conditions. Means that it doesn't happen. Such polynucleotides include probes, primers, nucleotides and nucleotide derivatives (eg, antisense oligonucleotides and ribozymes) that specifically hybridize to DNA encoding the polypeptide of the invention or its complementary strand. . Furthermore, such a polynucleotide can be used for the production of a DNA chip.

Vectors and host cells The present invention also provides vectors and host cells into which a polynucleotide of the invention has been introduced. The vectors of the present invention are useful for storing the polynucleotides of the present invention, particularly DNA, in host cells and for expressing the polypeptides of the present invention.

  If E. coli is the host cell and the vector is amplified and produced in large quantities within E. coli (eg, JM109, DH5α, HB101, or XL1Blue), the vector is “ori” for amplification in E. coli, In addition, it is necessary to have a marker gene (for example, a drug resistance gene selected by a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol) for selecting transformed E. coli. For example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, and the like can be used. Furthermore, like the above vectors, pGEM-T, pDIRECT, and pT7 can also be used for cDNA subcloning and extraction. Expression vectors are particularly useful when using vectors for the production of the proteins of the invention. For example, an expression vector to be expressed in E. coli must have the above characteristics for amplification in E. coli. When E. coli such as JM109, DH5α, HB101, or XL1Blue is used as a host cell, the vector can be a promoter that can efficiently express a desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature 341: 544). -6 (1989); FASEB J 6: 2422-7 (1992)), araB promoter (Better et al., Science 240: 1041-3 (1988)), T7 promoter and the like. In this regard, for example, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP and pET (in this case, preferably the host is BL21 expressing T7 RNA polymerase) of the above vector. It may be used instead. In addition, the vector may include a signal sequence for polypeptide secretion. An example of a signal sequence that directs polypeptide secretion into the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169: 4379 (1987)). Means for introducing the vector into the target host cell include, for example, the calcium chloride method and the electroporation method.

  In addition to E. coli, for example, mammalian-derived expression vectors (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18 (17): 5322 (1990)), pEF, pCDM8), insect cell-derived expression vectors ( For example, “Bac-to-BAC baculovirus expression system” (Gibco BRL (GIBCO BRL)), pBacPAK8), plant-derived expression vectors (eg, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV) PAdexLcw), retrovirus-derived expression vectors (eg, pZIpneo), yeast-derived expression vectors (eg, “Pichia expression kit” (Invitrogen), pNV11, SP-Q01), and expression from Bacillus subtilis Vectors (eg, pPL608, pKTH50) can also be used for production of the polypeptides of the present invention.

  In order for a vector to be expressed in animal cells such as CHO cells, COS cells or NIH3T3 cells, the vector must be a promoter required for expression in this type of cell, eg the SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter and the like, and a marker gene for selecting a transformant (for example, It is preferable to have a drug (for example, a drug resistance gene selected by neomycin, G418). Examples of known vectors with these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Production of polypeptides The present invention further provides methods for producing the polypeptides of the present invention. The polypeptide may be prepared by culturing a host cell carrying an expression vector containing a gene encoding the polypeptide. If desired, the method can also be used to stably express the gene and at the same time to amplify the copy number of the gene in the cell. For example, a vector containing a complementary DHFR gene (eg, pCHO I) can be amplified by methotrexate (MTX) after introduction into a CHO cell lacking the nucleic acid synthesis pathway. Furthermore, in the case of transient expression of a gene, a method of transforming a vector containing an SV40 origin of replication (such as pcD) into COS cells containing a gene expressing the SV40T antigen on the chromosome can be used. .

  The polypeptide of the present invention obtained as described above can be isolated from the inside or outside of a host cell (such as a medium) and purified as a substantially pure homogeneous polypeptide. “Substantially pure” as used herein with reference to a given polypeptide means that the polypeptide is substantially free from other biopolymers. A substantially pure polypeptide is at least 75% pure (eg, at least 80, 85, 95 or 99%) by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Methods for polypeptide isolation and purification are not limited to any particular method; in practice any standard method may be used.

  For example, appropriate selection of column chromatography, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization In combination, the polypeptide may be isolated and purified.

  Examples of chromatography include, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. ("Strategies for Protein Purification and Characterization: A Laboratory Course Manual ", edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). These chromatographies may be performed by liquid chromatography such as HPLC and FPLC. Therefore, the present invention provides a high purity polypeptide prepared by the above method.

  The polypeptide of the invention can optionally be altered or partially deleted by treating it with an appropriate protein modifying enzyme before or after purification. Useful protein modifying enzymes include, but are not limited to, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like.

Antibodies The present invention provides antibodies that bind to the polypeptides of the present invention. The antibodies of the present invention can be used in any form such as monoclonal antibodies or polyclonal antibodies, including antisera obtained by immunizing animals such as rabbits with the polypeptides of the present invention, all classes of polyclonals. Antibodies and monoclonal antibodies, human antibodies, and humanized antibodies produced by genetic recombination are included.

  The polypeptide of the present invention used as an antigen for obtaining an antibody may be derived from any animal species, but is preferably derived from a mammal such as a human, mouse, or rat, more preferably from a human. Human-derived polypeptides can be obtained from the nucleotide or amino acid sequences disclosed herein.

  According to the present invention, the polypeptide used as the immunizing antigen may be a complete protein or a partial peptide of the protein. The partial peptide can include, for example, an amino (N) -terminal or carboxy (C) -terminal fragment of the polypeptide of the present invention.

  As used herein, an antibody is defined as a protein that reacts with either the full length or a fragment of a polypeptide of the invention.

  A gene encoding the polypeptide of the present invention or a fragment thereof may be inserted into a known expression vector and subsequently used for transformation of a host cell as described herein. The desired polypeptide or fragment thereof can be recovered from the inside or outside of the host cell by any standard method and later used as an antigen. Alternatively, whole cells expressing the polypeptide or a lysate thereof, or a chemically synthesized polypeptide may be used as the antigen.

  Any mammal can be immunized with the antigen, but preferably the compatibility with the parental cell used for cell fusion is taken into account. In general, rodent, Lagomorpha, or primate animals are used. Rodent animals include, for example, mice, rats, and hamsters. Rabbit eyes include, for example, rabbits. Primate animals include, for example, the Catarrhini (Old World monkey) monkey, Macaca fascicularis, rhesus monkey, sacred baboon and chimpanzee.

  Methods for immunizing animals with antigens are well known in the art. Intraperitoneal or subcutaneous injection of antigen is a standard method for immunization of mammals. More specifically, the antigen is diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline or the like. If desired, the antigen suspension may be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, to give an emulsion and administered to the mammal. Thereafter, administration of the antigen mixed with an appropriate amount of Freund's incomplete adjuvant is preferably performed several times every 4 to 21 days. A suitable carrier may be used for immunization. Following immunization as described above, sera are examined by standard methods for increasing amounts of the desired antibody.

  Polyclonal antibodies against the polypeptides of the present invention can also be prepared by collecting blood from immunized mammals examined for an increase in the desired antibody in the serum and separating the serum from the blood by any conventional method. it can. Polyclonal antibodies include serum containing polyclonal antibodies, and fractions containing polyclonal antibodies can also be isolated from the serum. Immunoglobulin G or M is obtained from a fraction that recognizes only the polypeptide of the present invention.For example, after using an affinity column to which the polypeptide of the present invention is bound, this fraction is divided into a protein A column or a protein G column. And can be further purified and prepared.

  In order to prepare a monoclonal antibody, immune cells are collected from a mammal immunized with an antigen, checked for increased levels of the desired antibody in serum as described above, and subjected to cell fusion. Immune cells used for cell fusion are preferably obtained from the spleen. Other preferred parent cells for fusing with the above immune cells include, for example, mammalian myeloma cells, more preferably myeloma cells with acquired properties for selection of fusion cells with drugs.

  The above immune cells and myeloma cells can be fused according to known methods, for example, the method of Milstein et al. (Galfre and Milstein, Methods Enzymol 73: 3-46 (1981)).

  The resulting hybridomas by cell fusion can be selected by culturing them in a standard selective medium such as HAT medium (medium containing hypoxanthine, aminopterin, and thymidine). Cell culture is usually continued in HAT medium for days to weeks, a period sufficient for all other cells (non-fused cells) except the desired hybridoma to die. This is followed by standard limiting dilution for screening and cloning of hybridoma cells producing the desired antibody.

  In addition to the above method of immunizing non-human animals with antigens for hybridoma preparation, human lymphocytes such as lymphocytes infected with EB virus are immunized in vitro with polypeptides, polypeptide-expressing cells, or lysates thereof. You can also Subsequently, lymphocytes after immunization can be fused with human-derived myeloma cells such as U266, which can divide indefinitely, to obtain hybridomas that produce the desired human antibody that can bind to the polypeptide (Japanese Patent Application Laid-Open (JP-A) No. 1993-263). Sho 63-17688).

  The obtained hybridoma is subsequently transplanted into the abdominal cavity of the mouse, and ascites is extracted. The obtained monoclonal antibody can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column to which the polypeptide of the present invention is bound. The antibody of the present invention can be used not only for purification and detection of the polypeptide of the present invention but also as a candidate for antagonist of the polypeptide of the present invention. Furthermore, this antibody can also be applied to antibody therapy for diseases associated with the polypeptide of the present invention. When the obtained antibody is administered to the human body (antibody therapy), a human antibody or a humanized antibody is preferable in order to suppress immunogenicity.

  For example, transgenic animals having a repertoire of human antibody genes can be immunized with an antigen selected from polypeptides, polypeptide-expressing cells, or lysates thereof. Subsequently, antibody-producing cells are collected from the animal, fused with myeloma cells to obtain a hybridoma, and a human antibody against the polypeptide can be prepared from the hybridoma (International Publication No. 92-03918, International Publication No. No. 93-2227, International Publication No. 94-02602, International Publication No. 94-25585, International Publication No. 96-33735, and International Publication No. 96-34096).

  Alternatively, immune cells that produce antibodies, such as immunized lymphocytes, can be immortalized by oncogenes and used to prepare monoclonal antibodies.

  Monoclonal antibodies thus obtained may be prepared recombinantly using genetic engineering techniques (eg Borrebaeck and Larrick, “Therapeutic Monoclonal Antibodies”, published by MacMillan Publishers LTD (UK) (1990)). reference). For example, a DNA encoding an antibody can be cloned from an immune cell such as an antibody-producing hybridoma or immune lymphocyte, inserted into an appropriate vector, and introduced into a host cell to prepare a recombinant antibody. . The present invention also provides a recombinant antibody prepared as described above.

Furthermore, the antibody of the present invention may be an antibody fragment or a modified antibody as long as it binds to one or more of the polypeptides of the present invention. For example, the antibody fragment may be a single chain Fv (scFv) in which Fab, F (ab ′) ₂ , Fv, or Fv fragments derived from H and L chains are linked by an appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83 (1988)). More specifically, antibody fragments can be prepared by treating an antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding an antibody fragment may be constructed and inserted into an expression vector and expressed in a suitable host cell (eg, Co et al., J Immunol 152: 2968-76 (1994); Better and Horwitz , Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); see Bird and Walker, Trends Biotechnol 9: 132-7 (1991)).

  Antibodies can also be modified by conjugation with various molecules such as polyethylene glycol (PEG). The present invention provides such modified antibodies. The modified antibody can be obtained by chemically modifying the antibody. These modification methods are routine in the art.

  Alternatively, the antibody of the present invention can be used as a chimeric antibody of a variable region derived from a non-human antibody and a constant region of a human antibody, or a complementarity determining region (CDR), framework region (FR) and human derived from a non-human antibody. It can also be obtained as a humanized antibody containing a constant region derived from a humanized antibody. Such antibodies can be prepared according to known techniques. Humanization can be performed by replacing rodent CDRs or CDR sequences with the corresponding sequences of human antibodies (see, eg, Verhoeyen et al., Science 239: 1534-1536 (1988)). Accordingly, such humanized antibodies are chimeric antibodies in which a human variable domain that is clearly shorter than its full length is replaced by the corresponding sequence from a non-human species.

  Fully human antibodies that contain human variable regions in addition to the human framework and constant regions can also be used. Such antibodies can be made using various techniques known in the art. For example, in vitro methods involve the use of recombinant libraries of human antibody fragments displayed on bacteriophages (eg, Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described, for example, in US Pat. Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016.

  The antibody obtained as described above may be purified to homogeneity. For example, antibodies can be separated and purified according to separation and purification methods used for common proteins. For example, column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing etc. are selected appropriately (but not limited to). In combination, antibodies can be separated and isolated ("A Laboratory Manual", edited by Harlow and David Lane, Cold Spring Harbor Laboratory (1988)). Protein A columns and protein G columns can be used as affinity columns. Examples of protein A columns used include, for example, Hyper D, POROS and Sepharose F.F. (Pharmacia).

  Examples of chromatography, excluding affinity chromatography, include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. ("Strategies for Protein Purification and Characterization: A Laboratory Course Manual ", edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures can also be performed by liquid phase chromatography such as HPLC and FPLC.

  To measure the antigen binding activity of the antibody of the present invention, for example, absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and / or immunofluorescence test Can be used. In the case of ELISA, the antibody of the present invention is immobilized on a plate, the polypeptide of the present invention is added to the plate, and then a sample containing a desired antibody such as a culture supernatant of antibody-producing cells or a purified antibody is added. To do. Subsequently, a secondary antibody that recognizes the primary antibody and is labeled with an enzyme such as alkaline phosphatase is added, and the plate is incubated. Next, after washing, an enzyme substrate such as p-nitrophenyl phosphate is added to the plate and the absorbance is measured to assess the antigen binding activity of the sample. A polypeptide fragment such as a C-terminal fragment or N-terminal fragment may be used as an antigen for evaluating the binding activity of an antibody. BIAcore (Pharmacia) may be used to assess the activity of the antibody according to the invention.

  In the above method, the antibody of the present invention is exposed to a sample assumed to contain the polypeptide of the present invention, and the immune complex formed by the antibody and the polypeptide is detected or measured. Allows detection or measurement of polypeptides.

  Since the method for detecting or measuring a polypeptide according to the present invention can specifically detect or measure a polypeptide, this method seems to be useful for various experiments in which the polypeptide is used.

Antisense Polynucleotides, Small Interfering RNAs, and Ribozymes The present invention includes antisense oligonucleotides that hybridize to any site within the nucleotide sequence of SEQ ID NO: 1 or 3. Preferably, the antisense oligonucleotide is for at least about 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or 3. More preferred are antisense oligonucleotides as described above comprising one start codon in the at least 15 consecutive nucleotides.

  Derivatives or modified products of antisense oligonucleotides can also be used as antisense oligonucleotides. Examples of such modified products include lower alkyl phosphonate modifications such as methyl phosphonate or ethyl phosphonate types, phosphorothioate modifications, and phosphoramidate modifications.

  As used herein, the term “antisense oligonucleotide” refers to a mismatch in one or more nucleotides as well as those in which the nucleotides corresponding to those constituting a particular region of DNA or mRNA are completely complementary. Some are included so long as the DNA or mRNA and antisense oligonucleotide can specifically hybridize to the nucleotide sequence of SEQ ID NO: 1 or 3.

  Such polynucleotides are within a “region of at least about 15 contiguous nucleotide sequences”, at least 70% or more, preferably 80% or more, more preferably about 90% or more, even more. Preferably, it is included as having about 95% or more homology. The algorithms described herein can be used to determine homology. Algorithms known in the art can be used to determine homology. Furthermore, derivatives or modified products of antisense oligonucleotides can also be used as antisense oligonucleotides in the present invention. Such modified products include lower alkyl phosphonate modifications such as methyl phosphonate type or ethyl phosphonate type, phosphorothioate modifications and phosphoramidate modifications.

  Such an antisense polynucleotide is useful as a probe for isolation or detection of DNA encoding the polypeptide of the present invention, or as a primer for amplification.

  The antisense oligonucleotide derivative of the present invention binds to DNA or mRNA encoding a polypeptide, inhibits its transcription or translation, promotes the degradation of mRNA, inhibits the expression of the polypeptide of the present invention, It thus acts on cells producing the polypeptide of the present invention by inhibiting the function of the polypeptide.

  The present invention also includes small interfering RNA (siRNA) comprising a combination of sense and antisense strand nucleic acids of the nucleotide sequence of SEQ ID NO: 1 or 3. More specifically, this type of siRNA for suppressing the expression of CCDC4 includes those targeting the nucleotide sequence of SEQ ID NO: 8.

  The term “siRNA” refers to a double-stranded RNA molecule that prevents translation of a target mRNA. Standard techniques are used to introduce siRNA into cells, including using DNA as a template to transcribe RNA. The siRNA includes a sense nucleic acid sequence and an antisense nucleic acid sequence of a polynucleotide (SEQ ID NO: 1 or 3) encoding human CCDC4 protein. siRNAs are constructed such that a single transcript (double stranded RNA) has both a sense sequence and a complementary antisense sequence from the target gene, such as a hairpin.

  Binding of siRNA to the transcript corresponding to CCDC4 in the target cell results in decreased protein production by the cell. The length of the oligonucleotide is at least 10 nucleotides and may be the same length as the naturally occurring transcript. Preferably, the oligonucleotide is about 19 to about 25 nucleotides in length. Most preferably, the oligonucleotide is less than about 75, about 50, about 25 nucleotides in length. An example of a CCDC4 siRNA oligonucleotide that inhibits the growth of cancer cells includes a target sequence containing SEQ ID NO: 8. In addition, nucleotide “u” can be added to the 3 ′ end of the antisense strand of the target sequence to enhance the inhibitory activity of the siRNA. The number of “u” added is at least 2, generally from about 2 to about 10, preferably from about 2 to about 5. The added “u” forms a single strand at the 3 ′ end of the antisense strand of the siRNA.

  CCDC4 siRNA is introduced directly into cells in a form that can bind to mRNA transcripts. In these embodiments, the siRNA molecules of the invention are typically modified as previously described for antisense molecules. Other modifications are possible, for example, siRNA conjugated to cholesterol showed improved pharmacological properties (Song et al. Nature Med. 9: 347-351 (2003)). Alternatively, DNA encoding CCDC4 siRNA is present in the vector.

  A vector can be used to clone a CCDC4 target sequence into an expression vector, eg, in a manner that allows expression of both strands (by transcription of the DNA molecule) so that the operably linked control sequences are adjacent to the CCDC4 sequence. (Lee, NS, Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A., Salvaterra, P., and Rossi, J. (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells (Nature Biotechnology 20: 500-505). An RNA molecule that is antisense to CCDC4 mRNA is transcribed by a first promoter (eg, a promoter sequence at the 3 ′ end of the cloned DNA), and an RNA molecule that is the sense strand for CCDC4 mRNA is a second promoter. Transcribed by (for example, the promoter sequence at the 5 ′ end of the cloned DNA). The sense and antisense strands hybridize in vivo to generate an siRNA construct for silencing the CCDC4 gene. Alternatively, two constructs are utilized to create the sense and antisense strands of the siRNA construct. Cloned CCDC4 can encode a construct with a secondary structure, eg, a hairpin, where a single transcript has both sense and complementary antisense sequences from the target gene.

Furthermore, in order to form a hairpin loop structure, a loop sequence consisting of an arbitrary nucleotide sequence may be located between the sense sequence and the antisense sequence. Accordingly, the present invention also provides siRNA having the general formula 5 ′-[A]-[B]-[A ′]-3 ′. In the formula, [A] is a ribonucleotide sequence corresponding to a sequence that specifically hybridizes with mRNA or cDNA from the CCDC4 gene. In a preferred embodiment, [A] is a ribonucleotide sequence corresponding to the sequence of nucleotides 1666-1684 of SEQ ID NO: 1 or 3 (SEQ ID NO: 8). [B] is a ribonucleotide sequence consisting of about 3 to about 23 nucleotides, and [A ′] is a ribonucleotide sequence consisting of the complementary sequence of [A]. The loop sequence can consist of any sequence, preferably having a length of 3-23 nucleotides. The loop sequence can be selected, for example, from the group consisting of the following sequences (http://www.ambion.com/techlib/tb/tb_506.html). In the siRNA of the present invention, nucleotide “u” can be added to the 3 ′ end of [A ′] to enhance the inhibitory activity of siRNA. The number of “u” added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5. In addition, a loop sequence of 23 nucleotides also provides active siRNA (Jacque, J.-M., Triques, K., and Stevenson, M. (2002) Modulation of HIV by RNA interference. -1 replication by RNA interference) Nature 418: 435-438).
CCC, CCACC, or CCACACC: Jacque, JM, Triques, K., and Stevenson, M. Modulation of HIV-1 replication by RNA interference Nature, Vol. 418: 435-438 (2002);
UUCG: Lee, NS, Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A., Salvaterra, P., and Rossi, J. (2002) in human cells Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells Nature Biotechnology 20: 500-505 .; Fruscoloni, P., Zamboni, M ., and Tocchini-Valentini, GP Exonucleolytic degradation of double-stranded RNA by an activity in Xenopus laevis germinal vesicles Proc. Natl. Acad. Sci. USA 100 (4): 1639-1644 (2003); and
UUCAAGAGA: Dykxhoorn, DM, Novina, CD, and Sharp, PA Messenger inactivation: Short RNAs that cause gene expression silencing (Killing the messenger: Short RNAs that silence gene expression) Nature Reviews Molecular Cell Biology 4: 457-467 (2002).

For example, preferred siRNAs having the hairpin structure of the present invention are those shown below. In the following structure, the loop sequence can be selected from the group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA. A preferred loop sequence is UUCAAGAGA ("ttcaagaga" in DNA).
gaugguucugcagcaccac- [B] -guggugcugcagaaccauc (for the target sequence of SEQ ID NO: 8)

  Expression can be regulated independently in time or space by the same or different regulatory sequences adjacent to the CCDC4 sequence. The siRNA is transcribed intracellularly, for example, by cloning the CCDC4 gene template into a vector containing the RNA polymerase III transcription unit derived from small nuclear RNA (snRNA) U6 or the human H1 RNA promoter. Transfection enhancing agents can be used to introduce the vector into the cell. FuGENE (Rochediagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as transfection enhancers.

  The siRNA nucleotide sequence can be designed using a computer program for siRNA design available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). it can. The nucleotide sequence for siRNA is selected by a computer program based on the following protocol.

siRNA target site selection:
1． Starting downstream from the AUG start codon of the transcript of interest, it is scanned downstream for the AA dinucleotide sequence. Record the occurrence of each AA and the 19 nucleotides adjacent to the 3 ′ end as potential siRNA target sites. Tuschl et al. ((Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13 (24): 3191-7 (1999)) reported that siRNA 5 'and 3' untranslated regions (UTR) and near the start codon (75 It is recommended not to design for (intrabase) regions, since these regions may contain many regulatory protein binding sites, UTR binding proteins and / or translations. The initiation complex may interfere with the binding of the siRNA endonuclease complex.
2． The potential target sites are compared to the human genome database and any target regions with apparent homology with other coding sequences are excluded from consideration. Homology searches can be performed using BLAST, which is on the NCBI server: www.ncbi.nlm.nih.gov/BLAST/.
3． Select a qualified target sequence for synthesis. Ambion can select several target sequences for evaluation along the entire length of the gene.

  Oligonucleotides of various parts of CCDC4 mRNA and oligonucleotides complementary thereto according to standard methods (for example, using PC3 or DU145 prostate cancer cell lines) with respect to the ability to reduce the production of CCDC4 in tumor cells Tested in vitro. A reduction in the CCDC4 gene product in cells contacted with the candidate siRNA composition is detected using CCDC4-specific antibodies or other detection strategies compared to cells cultured in the absence of the candidate composition. Sequences that reduce the production of CCDC4 in cell-based or cell-free assays in vitro are then tested for inhibitory effects on cell proliferation. Sequences that inhibit cell proliferation in in vitro cell-based assays are tested in vivo in rats or mice to confirm decreased CCDC4 production and decreased tumor cell proliferation in animals with malignant neoplasms.

  The present invention includes a double-stranded molecule comprising the nucleic acid sequence of the target sequence, eg, nucleotides 1666-1684 of SEQ ID NO: 1 or 3 (SEQ ID NO: 8). In the present invention, a double-stranded molecule comprising a sense strand and an antisense strand, the sense strand comprises a ribonucleotide sequence corresponding to SEQ ID NO: 8, and the antisense strand comprises a ribonucleotide sequence complementary to the sense strand. The sense strand and the antisense strand hybridize to each other to form a double-stranded molecule that, when introduced into a cell expressing the CCDC4 gene, Inhibit. In the present invention, when the isolated nucleic acid is RNA or a derivative thereof, the base “t” should be replaced with “u” in the nucleotide sequence. As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen-type base pairing between nucleotide units of a nucleic acid molecule and is referred to as “binding”. The term means a physical or chemical interaction between two nucleic acids or compounds or associated nucleic acids or compounds, or combinations thereof.

  Complementary nucleic acid sequences hybridize under appropriate conditions to form stable duplexes with little or no mismatch. Furthermore, the sense strand and the antisense strand of the isolated nucleotide of the present invention can form a double-stranded nucleotide or a hairpin loop structure by hybridization. In preferred embodiments, such duplexes contain only 1 mismatch for 10 matches. In particularly preferred embodiments where the duplex strands are completely complementary, such duplexes do not contain mismatches. The nucleic acid molecule is 8863 nucleotides long (for SEQ ID NO: 1) or less than 8692 nucleotides long (for SEQ ID NO: 3). For example, the nucleic acid molecule is less than 500, 200, or 75 nucleotides in length. The invention also includes vectors containing one or more of the nucleic acids described herein and cells containing the vectors. The isolated nucleic acids of the invention are useful for siRNA against CCDC4 or DNA encoding siRNA. When nucleic acids are used for siRNA or their coding DNA, the sense strand is preferably longer than about 19 nucleotides, more preferably longer than about 21 nucleotides.

  The antisense oligonucleotide or siRNA of the present invention inhibits the expression of the polypeptide of the present invention and is therefore useful for suppressing the biological activity of the polypeptide of the present invention. Moreover, the expression inhibitor containing the antisense oligonucleotide or siRNA of the present invention is useful in that it can inhibit the biological activity of the polypeptide of the present invention. Therefore, the composition comprising the antisense oligonucleotide or siRNA of the present invention is useful in the treatment of prostate cancer. An example of a CCDC4 siRNA oligonucleotide that inhibits expression in mammalian cells includes a target sequence containing SEQ ID NO: 8. In addition, a nucleotide “u” can be added to the 3 ′ end of the antisense strand of the target sequence to enhance the inhibitory activity of the siRNA. The number of “u” added is at least about 2, generally about 2 to about 10, preferably about 2 to about 5. The added “u” forms a single strand at the 3 ′ end of the antisense strand of the siRNA.

  Similarly, expression inhibitors comprising the antisense oligonucleotides or siRNAs of the invention are also useful in that they can inhibit the biological activity of the polypeptides of the invention. Therefore, the composition containing the antisense oligonucleotide or siRNA of the present invention is useful for the treatment of cell proliferative diseases such as prostate cancer.

  Furthermore, the present invention provides ribozymes that inhibit the expression of the CCDC4 polypeptide of the present invention.

  In general, ribozymes are classified into large ribozymes and small ribozymes. Large ribozymes are known as enzymes that cleave the phosphate ester bond of nucleic acids. After the reaction with the large ribozyme, the reacted site is composed of a 5 ′ phosphate group and a 3 ′ hydroxyl group. The large ribozyme further comprises (1) a group I intron RNA that catalyzes the transesterification reaction at the 5 ′ splice site by guanosine; (2) a group II intron RNA that catalyzes self-splicing through a two-step reaction via the lariat structure; 3) It is classified as an RNA component of ribonuclease P that cleaves the tRNA precursor at the 5 'site by hydrolysis. On the other hand, the small ribozyme is smaller in size (about 40 bp) than the large ribozyme and cleaves RNA to produce a 5 ′ hydroxyl group and a 2′-3 ′ cyclic phosphate. Hammerhead ribozymes (Koizumi et al., FEBS LErr 228: 225 (1988)) and hairpin ribozymes (Buzayan, Nature 323: 349 (1986); Kikuchi and Sasaki, Nucleic Acids Res 19: 6751 (1992)) are small ribozymes. included. Methods for ribozyme design and construction are well known in the art (Koizumi et al., FEBS Lett 228: 225 (1988); Koizumi et al., Nucleic Acids Res 17: 7059 (1989); Kikuchi and Sasaki, Nucleic Acids Res 19 : 6751 (1992)). Therefore, ribozymes that inhibit the expression of the polypeptide of the present invention can be constructed based on their sequence information (SEQ ID NO: 1 or 3) and these conventional methods.

  The ribozyme against the CCDC4 gene inhibits the expression of the overexpressed CCDC4 protein and is therefore useful for suppressing the biological activity of this protein. Accordingly, these ribozymes are useful in the treatment or prevention of prostate cancer.

Diagnosis of Prostate Cancer Further, the present invention also provides a method for diagnosing a cell proliferative disease such as prostate cancer using the expression level of the gene of the present invention as a diagnostic marker.

  The diagnostic method includes (a) detecting the expression level of the CCDC4 gene of the present invention, and (b) associating the increased expression level with prostate cancer.

  The expression level of the CCDC4 gene in the biological sample can be evaluated by quantifying mRNA corresponding to the CCDC4 gene or a protein encoded by the CCDC4 gene. Methods for quantifying mRNA are well known to those skilled in the art. For example, the level of mRNA corresponding to the CCDC4 gene can be assessed by Northern blotting or RT-PCR. Since the full length nucleotide sequence of the CCDC4 gene is shown in SEQ ID NO: 1 or 3, those skilled in the art can design the nucleotide sequence of a probe or primer for quantifying the CCDC4 gene.

  The expression level of the CCDC4 gene can also be analyzed based on the activity or amount of the protein encoded by the gene. A method for determining the amount of CCDC4 protein is shown below. For example, immunoassays are useful for the determination of proteins in biological materials. As long as the marker gene (CCDC4 gene) is expressed in the sample of prostate cancer patient, any biological material can be used as the biological sample for determining the protein or its activity. For example, prostate duct epithelium can be cited as such a biological sample. However, body fluids such as blood and urine can also be analyzed. On the other hand, in order to determine the activity of the protein encoded by the CCDC4 gene, a suitable method can be selected according to the activity of the protein to be analyzed.

  The expression level of the CCDC4 gene in the biological sample is assessed and compared to the expression level in a normal sample (eg, a sample taken from an unaffected subject). If the comparison shows that the expression level of the target gene is higher than the expression level in the normal sample, it is determined that the subject is suffering from prostate cancer. The expression level of CCDC4 gene in normal subjects and biological samples derived from subjects may be determined simultaneously. Alternatively, the normal range of expression levels may be determined by statistical methods based on the results obtained by analyzing gene expression levels in samples previously taken from the control group. The results obtained by comparing the subject samples are compared with the normal range; if the results do not fall within the normal range, the subject has or is at risk of developing prostate cancer. To be judged.

  In the present invention, a diagnostic agent for diagnosing a cell proliferative disease such as prostate cancer is also provided. The diagnostic agent of the present invention includes a compound that binds to the polynucleotide or polypeptide of the present invention. An oligonucleotide that hybridizes with the polynucleotide of the present invention or an antibody that binds to the polypeptide of the present invention is preferably used as such a compound.

  This diagnostic method for prostate cancer can also be applied to evaluate the effectiveness of prostate cancer treatment in a subject. According to this method, a biological sample, such as a test cell population, is obtained from a subject undergoing treatment for prostate cancer. The method for evaluation can be performed according to conventional diagnostic methods for prostate cancer.

  If desired, biological samples are obtained from the subject before treatment, during treatment, and at various times after treatment. Subsequently, the level of expression of the CCDC4 gene in the biological sample is determined, eg, a control level obtained from a reference cell population comprising cells with known prostate cancer status (ie, cancer cells or non-cancerous cells) and Compare. The determination of the control level is performed on an untreated biological sample.

  If the control level is derived from a biological sample that does not contain cancer cells, the similarity between the expression level in the subject-derived biological sample and the control level indicates that the treatment is effective. means. A difference between the expression level of the CCDC4 gene in the subject-derived biological sample and the control level means that the clinical outcome or prognosis is less favorable.

  The term “efficacious” means that treatment results in a decrease in the expression of a pathologically up-regulated gene (CCDC4 gene) or a decrease in prostate cancer cell size, morbidity or proliferative capacity. It means being. “Effective” when the treatment is applied prophylactically indicates that the treatment delays or prevents the development of prostate cancer. Assessment of prostate cancer can be done using standard clinical protocols. Furthermore, the effectiveness of the treatment is determined according to any known method for the diagnosis or treatment of prostate cancer.

  Furthermore, the present method for diagnosing prostate cancer is applied to the prognostic evaluation of a subject having prostate cancer by comparing the expression level of CCDC4 gene in a biological sample derived from a patient such as a test cell population with a control level. You can also. Alternatively, the level of expression of the CCDC4 gene in a patient-derived biological sample can be measured throughout the stages of the disease to assess patient prognosis.

  A higher expression level of the CCDC4 gene compared to the normal control level means that the prognosis is less favorable. A low expression level of the CCDC4 gene means that the prognosis of the patient is more favorable.

Compound screening
The CCDC4 gene, the protein encoded by this gene, or the transcriptional regulatory region of this gene can be used to screen for compounds that alter the expression of the gene or the biological activity of the polypeptide encoded by the gene. Such compounds are used as pharmaceuticals for the treatment or prevention of prostate cancer.

  Therefore, the present invention provides a method for screening a compound for treating or preventing prostate cancer using the polypeptide of the present invention. One embodiment of this screening method includes (a) contacting the test compound with the polypeptide of the present invention, (b) detecting the binding activity between the polypeptide of the present invention and the test compound, and (c) A step of selecting a compound that binds to the polypeptide of the present invention is included.

  The polypeptide of the present invention used for screening may be a recombinant polypeptide, a natural protein, or a partial peptide thereof. The polypeptide of the present invention to be contacted with a test compound can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier, or a fusion protein fused to another polypeptide.

  As a method for screening a protein, for example, a protein that binds to the polypeptide of the present invention using the polypeptide of the present invention, many methods well known to those skilled in the art can be used. Such screening can be performed by, for example, an immunoprecipitation method, specifically in the following manner. By inserting a gene into an expression vector for a foreign gene such as pSV2neo, pcDNAI, pcDNA3.1, pCAGGS, and pCD8, the gene encoding the polypeptide of the present invention is expressed in a host (eg, animal cell) or the like. . The promoter used for expression may be any commonly used promoter, such as the SV40 early promoter (Rigby, Williamson (eds.), “Genetic Engineering”, Volume 3, Academic Press, London, 83-141 (1982 )), EF-1α promoter (Kim et al., Gene 91: 217-23 (1990)), CAG promoter (Niwa et al., Gene 108: 193-200 (1991)), RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)), SRα promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), CMV early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)), SV40 Late promoters (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), adenovirus late promoters (Kaufman et al., Mol Cell Biol 9: 946 (1989)), HSV TK promoter and the like are included. Introduction of a gene into a host cell for expressing a foreign gene can be performed according to any method, such as electroporation (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), calcium phosphate (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1642-3 (1985)), Lipofectin method (Derijard, B Cell 7: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)) is there. The polypeptide of the present invention is expressed as a fusion protein containing a monoclonal antibody recognition site (epitope) by introducing an epitope of a monoclonal antibody whose specificity to the N-terminus or C-terminus of the polypeptide of the present invention is known. Can be made. A commercially available epitope-antibody system can also be used (Experimental Medicine 13: 85-90 (1995)). For example, vectors that can express a fusion protein with β-galactosidase, maltose-binding protein, glutathione S-transferase, green fluorescent protein (GFP) and the like by using a multiple cloning site are commercially available.

  A fusion protein produced by introducing only a small epitope consisting of several to a dozen amino acids so as not to change the properties of the polypeptide of the present invention by fusion has also been reported. Polyhistidine (His tag), influenza agglutinin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7 tag), human herpes simplex virus glycoprotein (HSV tag) , Epitopes such as E-tags (epitopes on monoclonal phage), and monoclonal antibodies that recognize them can be used as an epitope-antibody system for screening proteins that bind to the polypeptides of the invention (Experimental Medicine 13 : 85-90 (1995)).

  In the case of immunoprecipitation, an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant. The immune complex consists of the polypeptide of the present invention, a polypeptide capable of binding to the polypeptide, and an antibody. In addition to using antibodies against the above epitopes, immunoprecipitation can also be performed using antibodies against the polypeptides of the present invention, and these antibodies can be prepared as described above.

  If the antibody is a mouse IgG antibody, the immune complex can be precipitated by, for example, protein A sepharose or protein G sepharose. When the polypeptide of the present invention is produced as a fusion protein with an epitope such as GST, an antibody against the polypeptide of the present invention is used using a substrate that specifically binds to these epitopes, such as glutathione-sepharose 4B. Immune complexes can be formed in the same manner as time.

  Immunoprecipitation can be performed, for example, following or following 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 bound proteins can be analyzed by protein molecular weight using an appropriate concentration of gel. Since it is difficult to detect a protein bound to the polypeptide of the present invention by a general staining method such as Coomassie staining or silver staining, the detection sensitivity for the protein is the radioactive isotope ³⁵ S-methionine or ³⁵ S -It can be improved by culturing cells in a culture solution containing cysteine, labeling the protein in the cell and detecting the protein. If the molecular weight of the protein is known, the target protein can be purified directly from an SDS-polyacrylamide gel and its sequence determined.

  As a method for screening a protein that binds to the polypeptide of the present invention using the polypeptide, for example, West-Western blot analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used. Specifically, the protein that binds to the polypeptide of the present invention is a cell, tissue, organ (eg, tissue such as testis or prostate), or a cultured cell that is expected to express a protein that binds to the polypeptide of the present invention. (E.g., PC3, DU145) using a phage vector (eg, ZAP) to create a cDNA library, expressing the protein on LB-agarose, immobilizing the expressed protein on a filter, and purifying and labeling The obtained polypeptide of the present invention can be obtained by reacting with the above-mentioned filter and detecting a plaque expressing a protein bound to the polypeptide of the present invention with a label. The polypeptide of the present invention can be obtained by utilizing the binding between biotin and avidin, or an antibody that specifically binds to the polypeptide of the present invention, or a peptide or polypeptide fused with the polypeptide of the present invention (for example, GST ) Can be used for labeling. A method using a radioisotope or fluorescence may be used.

  Alternatively, in another embodiment of the screening method of the present invention, a cell-based two-hybrid system can be used (“MATCHMAKER Two-Hybrid system”) “Mammalian MATCHMAKER two-hybrid assay”. Kit (Mammalian MATCHMAKER Two-Hybrid Assay Kit), “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); reference Literature "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 expected to express a protein that binds to the polypeptide of the present invention so that when the library is expressed, it is fused with a VP16 or GAL4 transcriptional activation region. Subsequently, the cDNA library is introduced into the above yeast cell, and the cDNA derived from the library is isolated from the detected positive clone (when the protein that binds to the polypeptide of the present invention is expressed in the yeast cell, The combination of these two activates the reporter gene and allows positive clones to be detected). A protein encoded by cDNA can be prepared by introducing the cDNA isolated as described above into E. coli to express the protein.

  As the reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and the like can be used in addition to the HIS3 gene.

  Compounds that bind to the polypeptides of the invention can also be screened using affinity chromatography. For example, the polypeptide of the present invention may be immobilized on a carrier of an affinity column, and a test compound containing a protein that can bind to the polypeptide of the present invention may be applied to the column. The test compound in this specification may be, for example, a cell extract or a cell lysate. After loading the test compound, the column can be washed to prepare a compound bound to the polypeptide of the present invention.

  When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and the oligo DNA is used as a probe for obtaining a DNA encoding the protein. To screen the cDNA library.

  A biosensor utilizing the surface plasmon resonance phenomenon can also be used in the present invention as a means for detecting or quantifying the bound compound. When such a biosensor is used, the interaction between the polypeptide of the present invention and the test compound can be observed in real time as a surface plasmon resonance signal using only a very small amount of polypeptide and without labeling. (Eg BIAcore, Pharmacia). For this reason, it is possible to evaluate the binding between the polypeptide of the present invention and the test compound using a biosensor such as BIAcore.

  Methods for screening molecules that bind when the immobilized polypeptides of the invention are exposed to synthetic compounds, natural product banks, or random phage peptide display libraries, and bind to proteins of the invention Screening methods using high-throughput techniques based on combinatorial chemistry to isolate not only the proteins to be performed, but also compounds (including antagonists) (Wrighton et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11 -13 (1996); Hogan, Nature 384: 17-9 (1996)) is well known to those skilled in the art.

Alternatively, the present invention
(A) contacting the test compound with the polypeptide of the present invention;
(B) detecting the biological activity of the polypeptide of step (a); and (c) comparing the biological activity of the polypeptide to the biological activity detected in the absence of the test compound. There is also provided a method for screening a compound for treating or preventing prostate cancer using the polypeptide of the present invention, comprising the step of selecting a compound to suppress.

  Since the CCDC4 protein of the present invention has the activity of promoting cell proliferation of prostate cancer cells, compounds that inhibit this activity of the protein of the present invention can be screened using this activity as an index.

  Any polypeptide can be used for screening as long as it has the biological activity of CCDC4 protein. Such biological activity includes cell proliferating activity of human CCDC4 protein. For example, human CCDC4 protein can be used, and polypeptides functionally equivalent to these proteins can also be used. Such a polypeptide may be expressed endogenously or exogenously by the cell.

  The compound isolated by this screening is a candidate for antagonists of the polypeptide of the present invention. The term “antagonist” refers to a molecule that inhibits its function by binding to a polypeptide of the invention. In addition, compounds isolated by this screening are also candidate molecules for compounds that inhibit in vivo interactions between the polypeptides of the invention and molecules (including DNA and proteins).

  When the biological activity to be detected in the present method is cell proliferation, as described in the Examples section, for example, cells expressing the polypeptide of the present invention are prepared, and the cells are treated with the test compound. It can be detected by culturing in the presence, evaluating the rate of cell growth, measuring the cell cycle, etc., and further measuring the colony forming activity.

In yet another embodiment, the present invention provides a method for screening compounds for the treatment or prevention of prostate cancer. As discussed in detail above, prostate cancer development and progression can be controlled by controlling the expression level or activity of CCDC4. Therefore, compounds that can be used for the treatment or prevention of prostate cancer can be identified by screening using the expression level of CCDC4 as an indicator. In the context of the present invention, such screening includes, for example:
a) contacting a test compound with a cell expressing CCDC4; and
b) selecting a compound that reduces the expression level of CCDC4 relative to the expression level detected in the absence of the test compound.

  Cells expressing at least one of CCDC4 include, for example, cell lines established from prostate cancer; such cells can be used for the above screening of the present invention (eg, PC3, DU145). The expression level can be evaluated by methods well known to those skilled in the art. In the screening method, a compound that decreases the expression level of CCDC4 can be selected as a candidate substance to be used for the treatment or prevention of prostate cancer.

Alternatively, the screening method of the present invention comprises:
a) contacting a test compound with a cell into which a transcriptional regulatory region of one or more marker genes and a vector containing a reporter gene expressed under the control of the transcriptional regulatory region have been introduced (in this case, one or more The marker gene of this is CCDC4);
b) measuring the expression level or activity of said reporter gene; and
c) selecting a compound that reduces the expression level or activity of the reporter gene relative to a control.

  Suitable reporter genes and host cells are well known in the art. The reporter construct required for screening can be prepared by using the transcriptional regulatory region of the marker gene. If the transcriptional regulatory region of the marker gene is known to those skilled in the art, a reporter construct can be prepared by using prior sequence information. If the transcriptional regulatory region of the marker gene has not yet been identified, the nucleotide segment containing the transcriptional regulatory region can be isolated from the genomic 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; made from the above materials It is preferable to use commercially available beads and plates (for example, multiwell plates, biosensor chips, etc.). If beads are used, they may be packed into a column.

  The protein can be bound to the support according to conventional methods such as chemical binding and physical adsorption. Alternatively, the protein may be bound to the support via an antibody that specifically recognizes the protein. In addition, avidin and biotin can be used to bind the protein to the support.

  As long as the buffer does not inhibit the binding between proteins, for example, but not limited to, the binding between proteins is performed in a buffer solution of a phosphate buffer and a Tris buffer.

  In the present invention, a biosensor using the surface plasmon resonance phenomenon can also be used as a means for detecting or quantifying the bound protein. When such a biosensor is used, the interaction between proteins can be observed in real time as a surface plasmon resonance signal using only a very small amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). ).

  Alternatively, the CCDC4 polypeptide can be labeled and the bound protein label can be used to detect or measure the bound protein. Specifically, after labeling one of the proteins in advance, the labeled protein is brought into contact with the other protein in the presence of the test compound, and after washing, the bound protein is detected or measured with the label.

For protein labeling in this method, radioactive isotopes (eg ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S, ¹²⁵ I, ¹³¹ I), enzymes (eg alkaline phosphatase, horseradish peroxidase, Labeling substances such as β-galactosidase, β-glucosidase), fluorescent substances (eg fluorescein isothiocyanate (FITC), rhodamine) and biotin / avidin can be used. If the protein is labeled with a radioisotope, detection or measurement can be performed by liquid scintillation. Alternatively, a protein labeled with an enzyme can be detected or measured with an absorptiometer by adding an enzyme substrate in order to detect an enzymatic change of the substrate such as coloration. Furthermore, when a fluorescent substance is used as a label, the bound protein can be detected or measured using a fluorometer.

  When an antibody is used for this screening, it is preferable to label the antibody with any of the labeling substances described above and perform detection or measurement based on the labeling substance. Alternatively, an antibody against CCDC4 or actin may be used as the primary antibody as detected by a secondary antibody labeled with a labeling substance. Furthermore, the antibody bound to the protein in the screening of the present invention can also be detected or measured using a protein G or protein A column.

  Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system using cells can be used (“MATCHMAKER 2-hybrid system”, “Mammal MATCHMAKER 2-hybrid assay kit”, “MATCHMAKER 1-hybrid”). "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 CCDC4 polypeptide of the invention is fused to the SRF binding region or GAL4 binding region and expressed in yeast cells.

  As the reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and the like can be used in addition to the HIS3 gene.

  Any test compound such as cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptide compound, synthetic low molecular weight compound and natural The compound can be used in the screening method of the present invention. In addition, the test compound of the present invention can be obtained by (1) a biological library, (2) a parallel solid phase or liquid phase library that can be referred to by a spatial address, and (3) a synthetic library method that requires deconvolution. , (4) “one-bead one-compound” library methods, and (5) synthetic library methods using affinity chromatography sorting, known in the art It can also be obtained using any of a number of approaches in the rally method. Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches can be applied to peptide, non-peptide oligomer or small compound libraries (Lam ( 1997) Anticancer Drug Des. 12: 145). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad Sci. USA 91: 11422; Zuckermann et al. (1994) J. Med. Chem. 37: 2678; Cho et al. (1993) Science 261: 1303; 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). Compound libraries can be in solution (Houghten (1992) Bio / Techniques 13: 412), or beads (Lam (1991) Nature 354: 82), chips (Fodor (1993) Nature 364: 555), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698; 5,403,484 and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865) or phage (Scott and Smith (1990) Science 249: 386; Delvin (1990) Science 249: 404; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378; Felici (1991) J. Mol. Biol. 222: 301; U.S. Patent Application No. 2002103360).

  The compound isolated by the screening method of the present invention is a candidate for a drug that inhibits the activity of the polypeptide of the present invention for the purpose of treating or preventing a disease caused by a cell proliferative disease such as prostate cancer. . A compound obtained by converting a part of the structure of the compound obtained by this screening method by addition, deletion and / or substitution is included in the compound obtained by the screening method of the present invention.

Pharmaceutical Composition for the Treatment or Prevention of Prostate Cancer The present invention provides a composition for the treatment or prevention of prostate cancer comprising any of the compounds selected by the screening method of the present invention.

  Compounds isolated by the screening methods of the present invention can be used to treat cell proliferative diseases (eg, prostate cancer), humans or other mammals such as mice, rats, guinea pigs, rabbits, cats, dogs, sheep When administered as a pharmaceutical to pigs, cows, monkeys, baboons, chimpanzees, the isolated compound may be administered directly or formulated into dosage forms using known pharmaceutical preparation methods Also good. For example, if desired, the drug can be administered orally as sugar-coated tablets, capsules, elixirs, and microcapsules, or a sterile solution or suspension in water or any other pharmaceutically acceptable liquid It can also be administered parenterally as a liquid injection form. For example, the compound may be in a pharmaceutically acceptable carrier or vehicle, specifically sterile water, saline, vegetable oil, emulsifier, suspension, in the form of a unit dose necessary for the realization of a generally accepted drug. Can be mixed with agents, surfactants, stabilizers, flavoring agents, excipients, media, preservatives, binders and the like. The amount of active ingredient in these formulations will give a suitable dosage within the specified range.

  Examples of additives that can be mixed into tablets and capsules include binders such as gelatin, corn starch, gum tragacanth and gum arabic; media such as crystalline cellulose; swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate Agents; sweeteners such as sucrose, lactose or saccharin; flavoring agents such as peppermint, winter green oil and cherry. When the unit dosage form is a capsule, a liquid carrier such as oil can further be included in the above ingredients. Sterile mixtures for injection can be formulated using a medium such as distilled water for injection following the realization of normal drugs.

  Other isotonic solutions containing saline, glucose, and adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used as an aqueous solution for injection. These are used in combination with suitable solubilizers such as alcohols, specifically polyhydric alcohols such as ethanol, propylene glycol and polyethylene glycol, nonionic surfactants such as Polysorbate 80 ™ and HCO-50, etc. be able to.

  Sesame oil or soybean oil can be used as an oleaginous liquid, which can also be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer, and these can also be used in buffers such as phosphate buffer and sodium acetate buffer. Liquids; analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol and phenol; and antioxidants can also be formulated. The prepared injection can be filled into a suitable ampoule.

  To administer a pharmaceutical compound of the invention to a patient, for example, as an intraarterial injection, intravenous injection, subcutaneous injection, and also as intranasal, transbronchial, intramuscular or oral administration, to a person skilled in the art Well known methods can be used. The dosage and method of administration vary depending on the patient's weight and age and the method of administration; however, one skilled in the art can routinely select them. If the compound can be encoded by DNA, the DNA can be inserted into a gene therapy vector and the vector administered for treatment. The dose and method of administration vary depending on the patient's weight, age and symptoms, but those skilled in the art can appropriately select them.

  For example, although there are slight differences depending on the symptoms, the dose of the compound that binds to the polypeptide of the present invention and modulates its activity is about 0.1 mg when administered orally to a standard adult (body weight 60 kg). To about 100 mg / day, preferably about 1.0 mg to about 50 mg / day, more preferably about 1.0 mg to about 20 mg / day.

  When administered parenterally as a form of injection to a standard adult (body weight 60 kg), there are slight differences depending on the patient, target organ, symptoms and administration method, but about 0.01 mg to about It is convenient to inject a dose of 30 mg / day, preferably about 0.1 to about 20 mg / day, more preferably about 0.1 to about 10 mg / day. Similarly, in the case of other animals, it is possible to administer an amount converted to a body weight of 60 kg.

  Furthermore, the present invention provides a pharmaceutical composition for treating or preventing prostate cancer, comprising an active ingredient that inhibits the expression of the CCDC4 gene. Such active ingredients include antisense polynucleotides, siRNA or ribozymes against CCDC4 gene, or derivatives of antisense polynucleotides, siRNA or ribozymes (eg, expression vectors).

  These active ingredients can be in the form of external preparations such as liniments or poultices by mixing with a suitable base which is inert to the derivatives. Similarly, by adding additives, isotonic agents, solubilizers, stabilizers, preservatives, analgesics, etc., if necessary, tablets, powders, granules, capsules, liposome capsules, injections It can also be formulated as agents, solutions, nasal drops and lyophilized preparations. These can be prepared according to conventional methods.

  The active ingredient is administered to the patient either by topical application directly to the affected area or by injection into the blood vessel so that it reaches the affected area. Antisense encapsulants can also be used to increase durability and membrane permeability. Examples of encapsulating agents include liposomes, poly-L-lysine, lipids, cholesterol, lipofectin or derivatives thereof.

  The dosage of such a composition of the present invention can be adjusted appropriately according to the patient's condition and used in desired amounts. For example, a dose in the range of 0.1-100 mg / kg, preferably 0.1-50 mg / kg can be administered.

  Another embodiment of the present invention is a composition for treating or preventing prostate cancer comprising an antibody against a polypeptide encoded by the CCDC4 gene, or a fragment of an antibody that binds to the polypeptide.

  Although there are slight differences depending on the symptoms, the dose of the antibody or fragment thereof for treating or preventing prostate cancer is about 0.1 mg when administered orally to a standard adult (body weight 60 kg). To about 100 mg / day, preferably about 1.0 mg to about 50 mg / day, more preferably about 1.0 mg to about 20 mg / day.

  When administered parenterally in the form of an injection to a standard adult (body weight 60 kg), there are some differences depending on the patient, disease symptoms and administration method, but about 0.01 mg to about It is convenient to inject a dose of 30 mg / day, preferably about 0.1 to about 20 mg / day, more preferably about 0.1 to about 10 mg / day. Similarly, in the case of other animals, it is possible to administer an amount converted to a body weight of 60 kg.

Methods for the Treatment or Prevention of Prostate Cancer The present invention provides methods for the treatment or prevention of prostate cancer in a subject. The therapeutic compound is administered for therapeutic or prophylactic purposes to a subject afflicted with prostate cancer or a subject at risk of (or susceptible to) developing it. Such subjects are identified using standard clinical methods or by detecting abnormal expression levels or activity of CCDC4. Administration for prophylactic purposes occurs before the manifestation of overt clinical symptoms of the disease so that the disease or disorder is prevented or slowed down.

  The method of treatment includes reducing the expression or function of the CCDC4 gene. In these methods, an effective amount of a compound that reduces one or both of the genes overexpressed in the subject (CCDC4 gene) is administered to the subject. Administration can be systemic or local. Therapeutic compounds include compounds that reduce the expression level of genes endogenously present in prostate cancer cells (ie, compounds that down regulate the expression of overexpressed genes). It is expected that administration of such therapeutic compounds will counteract the effects of genes that are abnormally overexpressed in the subject and improve the clinical symptoms of the subject. Such a compound can be obtained by the screening method of the present invention described above.

  CCDC4 gene expression can also be inhibited by any of several methods known in the art, including administering to a subject a nucleic acid that inhibits or antagonizes gene expression. Antisense oligonucleotides, siRNA or ribozymes that interfere with gene expression can be used to inhibit gene expression.

  As indicated above, antisense oligonucleotides corresponding to the nucleotide sequence of the CCDC4 gene can be used to reduce the expression level of the CCDC4 gene. Specifically, the antisense oligonucleotide of the present invention binds to any of the polypeptides encoded by the CCDC4 gene or its corresponding mRNA, thereby inhibiting gene transcription or translation and promoting mRNA degradation. And / or appears to act by inhibiting the expression of the protein encoded by the gene and ultimately inhibiting the function of the CCDC4 protein.

  Antisense oligonucleotides and derivatives thereof are treated or prevented for prostate cancer of the present invention in the form of external preparations such as liniments or poultices by mixing with an appropriate substrate that is inert to the derivatives. Can be used for the method.

  Nucleic acids that inhibit the gene product of one or more overexpressed genes include small interfering RNA (siRNA), including combinations of sense and antisense strand nucleic acids of the nucleic acid encoding the CCDC4 gene It is. Standard techniques for introducing siRNA into cells, including template DNA from which RNA is transcribed, can be used in the treatment or prevention of the present invention. siRNAs are constructed so that a single transcript has both a sense sequence and a complementary antisense sequence from the target gene, such as a hairpin.

  This method is used to suppress gene expression in cells in which the expression of the CCDC4 gene is upregulated. Binding of the siRNA and the CCDC4 gene transcript in the target cell causes a reduction in the production of CCDC4 protein by the cell.

  Nucleic acids that inhibit the gene product of one or more overexpressed genes also include ribozymes against the overexpressed gene (CCDC4 gene).

  Furthermore, the present invention provides a method for treating or preventing a cell proliferative disease such as prostate cancer using an antibody against the polypeptide of the present invention. According to this method, a pharmaceutically effective amount of an antibody against the polypeptide of the present invention is administered. Since the expression of CCDC4 protein is up-regulated in prostate cancer cells and the suppression of the expression of these proteins leads to a decrease in cell proliferative activity, the combination of antibodies with these proteins can treat cell proliferative diseases. Or it is thought that prevention is possible. Thus, the antibody against the polypeptide of the present invention is administered at a dosage sufficient to reduce the activity of the protein of the present invention (which ranges from 0.1 to about 250 mg / kg / day). The dose range for adults is generally about 5 mg to about 17.5 g / day, preferably about 5 mg to about 10 g / day, and most preferably about 100 mg to about 3 g / day.

  Alternatively, antibodies that bind to cell surface markers specific for tumor cells can be used as a tool for drug delivery. For example, an antibody conjugated with a cytotoxic agent is administered as a dose sufficient to damage tumor cells.

  The present invention also relates to a method of inducing anti-tumor immunity comprising administering a CCDC4 protein or an immunoactive fragment thereof, or a polynucleotide encoding the protein or a fragment thereof. CCDC4 protein or immunologically active fragments thereof are useful as vaccines against cell proliferative diseases such as prostate cancer. In some cases, the protein or fragment thereof is administered in a form associated with a T cell receptor (TCR) or presented by an antigen presenting cell (APC) such as a macrophage, dendritic cell (DC) or B cell. You can also It is most preferable to use DC in APC because DC has high antigen presenting ability.

In the present invention, a vaccine against a cell proliferative disease refers to a substance having a function of inducing antitumor immunity when inoculated into an animal. In general, anti-tumor immunity includes immune responses such as:
-Induction of cytotoxic lymphocytes against the tumor-induction of antibodies recognizing the tumor, and-induction of anti-tumor cytokine production.

  For this reason, when a specific protein induces these immune responses when inoculated into an animal, it is determined that the protein has an antitumor immunity inducing action. Induction of anti-tumor immunity by a protein can be detected by observing the immune system's response to the protein in the host in vivo or in vitro.

  For example, methods for detecting the induction of cytotoxic T lymphocytes are well known. A foreign substance that enters the inside of the living body is presented to T cells and B cells by the action of antigen-presenting cells (APC). T cells that have responded in an antigen-specific manner to the antigen presented by APC proliferate after differentiation into cytotoxic T cells (or cytotoxic T lymphocytes; CTLs) for stimulation by the antigen ( This is called T cell activation). Therefore, CTL induction by a specific peptide can be evaluated by presenting the peptide to T cells by APC and detecting the induction of CTL. APC also has the effect of activating CD4 + T cells, CD8 + T cells, macrophages, eosinophils and NK cells. Since CD4 + T cells and CD8 + T cells are also important in anti-tumor immunity, the anti-tumor immunity inducing action of the peptide can be evaluated using the activation effect of these cells as an index.

A method for evaluating the inducing action of CTL using dendritic cells (DC) as APC is well known in the art. DC is a representative APC having the strongest CTL inducing action among APCs. In this method, the test polypeptide is first contacted with DC, and then this DC is contacted with T cells. If a T cell having a cytotoxic effect on a target cell is detected after contact with DC, it indicates that the test polypeptide has an activity of inducing a cytotoxic T cell. The activity of CTL against tumor can be detected using, for example, lysis of ⁵¹ Cr-labeled tumor cells as an indicator. Alternatively, a method of evaluating the degree of tumor cell damage using ³ H-thymidine uptake activity or LDH (lactose dehydrogenase) release as an index is also well known.

  In addition to DC, peripheral blood mononuclear cells (PBMC) may be used as APC. It has been reported that CTL induction can be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

  The test polypeptide confirmed to have CTL-inducing activity by these methods is a polypeptide having a DC activation action and a subsequent CTL-inducing activity. Therefore, polypeptides that induce CTL against tumor cells are useful as vaccines against tumors. Furthermore, APCs that have acquired the ability to induce CTLs against tumors by contact with polypeptides are also useful as vaccines against tumors. Furthermore, CTLs that have acquired cytotoxicity due to presentation of polypeptide antigens by APC can also be used as vaccines against tumors. Such a treatment for tumors using anti-tumor immunity resulting from APC and CTL is called cellular immunotherapy.

  In general, when using a polypeptide for cellular immunotherapy, it is known that a plurality of polypeptides having different structures are used in combination, and contacting them with DC increases the efficiency of CTL induction. For this reason, when stimulating DC with protein fragments, it is advantageous to use a mixture of many types of fragments.

  Alternatively, the induction of anti-tumor immunity by the polypeptide can be confirmed by observing the induction of antibody production against the tumor. For example, if an antibody to a polypeptide is induced in a laboratory animal immunized with the polypeptide, and if tumor cell growth is suppressed by such an antibody, the polypeptide is capable of inducing anti-tumor immunity. It can be determined that there is.

  Anti-tumor immunity is induced by administering the vaccine of the present invention, and induction of anti-tumor immunity enables treatment and prevention of cell proliferative diseases such as prostate cancer. Treatment for cancer or prevention of cancer development includes any of the following stages: inhibition of cancer cell growth, cancer regression, and suppression of cancer development. Reductions in mortality of individuals with cancer, reductions in tumor markers in the blood, alleviation of detectable symptoms associated with cancer, etc. are also included as therapeutic or prophylactic effects of cancer. Such therapeutic and prophylactic effects are preferably statistically significant. For example, at the observed value, the therapeutic effect or preventive effect of a vaccine against a cell proliferative disease is a significant level of 5% or less compared to a control without vaccine administration. For statistical analysis, for example, Student's t test, Mann-Whitney U test or ANOVA may be used.

  The above-mentioned protein having immunological activity or a vector encoding the protein may be used in combination with an adjuvant. An adjuvant refers to a compound that enhances the immune response to a protein when administered concurrently (or sequentially) with the protein having immunological activity. Examples of adjuvants include, but are not limited to, cholera toxin, salmonella toxin, alum and the like. Furthermore, the vaccine of the present invention may be appropriately blended with a pharmaceutically acceptable carrier. Examples of such carriers include sterilized water, physiological saline, phosphate buffer, culture solution, and the like. In addition, the vaccine may contain stabilizers, suspending agents, preservatives, surfactants and the like as required. The vaccine is administered systemically or locally. Vaccine administration may be performed by a single administration or boosting by multiple administrations.

  When APC or CTL is used as the vaccine of the present invention, the tumor can be treated or prevented, for example, by the ex vivo method. More specifically, PBMCs of a subject to be treated or prevented can be collected, cells are contacted with the polypeptide ex vivo, and the cells can be administered to the subject after induction of APC or CTL. APC can also be induced by ex vivo introduction of a vector encoding a polypeptide into PBMC. In vitro derived APCs or CTLs can be cloned prior to administration. Cell immunotherapy can be performed more efficiently by cloning and growing highly active cells that damage target cells. Furthermore, APCs and CTLs isolated in this way can be used not only for cellular immunotherapy in the individual from which the cells are derived, but also for similar types of tumors in other individuals.

  Further provided are pharmaceutical compositions for the treatment or prevention of cell proliferative diseases, such as prostate cancer, comprising a pharmaceutically effective amount of CCDC4 polypeptide. The pharmaceutical composition can also be used to provide anti-tumor immunity. Normal expression of CCDC4 is confined to the testis and prostate. For this reason, suppression of this gene is thought to have no detrimental effect on other organs. Accordingly, CCDC4 polypeptides are preferred for treating cell proliferative diseases, particularly prostate cancer. In addition, peptide fragments of proteins that are specifically expressed in cancer cells have been shown to induce an immune response against the cancer, so CCDC4 peptide fragments can be used to treat or prevent cell proliferative diseases such as prostate cancer. It can also be used in a pharmaceutical composition for. In the present invention, the polypeptide or fragment thereof is administered at a dosage sufficient to induce anti-tumor immunity, which ranges from 0.1 mg to 10 mg, preferably from 0.3 mg to 5 mg, more preferably from 0.8 mg to 1.5 mg. It is. Administration is repeated. In order to induce anti-tumor immunity, for example, 1 mg of a peptide or a fragment thereof may be administered 4 times every 2 weeks.

  In addition, polynucleotides encoding CCDC4, or fragments thereof, can be used to provide anti-tumor immunity. Such a polynucleotide may be incorporated into an expression vector for expressing CCDC4 or a fragment thereof within the subject to be treated. Accordingly, the present invention is a method for inducing anti-tumor immunity, wherein a polynucleotide encoding CCDC4 or a fragment thereof is afflicted with or at risk of developing a cell proliferative disease such as prostate cancer. It includes a method of administration to a subject.

  The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended to limit the scope of the invention in any way.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. Any patents, patent applications, and publications cited herein are hereby incorporated by reference.

Best Mode for Carrying Out the Invention The present invention is illustrated in detail by the following examples, but is not limited to these examples.
(1) cDNA microarray and laser microbeam microdissection
The preparation of cDNA microarray slides has already been described (Ono et al., Cancer Res 60: 5007-5011 (2000). With regard to the analysis of each expression profile, the inventors have tried to reduce experimental variation. Two sets of cDNA microarray slides each containing 23,040 cDNA spots were prepared: Briefly, prostate cancer cells, PIN cells, and normal cells that were microdissected from 20 prostate cancer tissues by laser microbeam microdissection Total RNA was purified from prostate duct epithelium, RNA amplification was performed using T7 polymerase to obtain sufficient RNA to perform microarray experiments, and an aliquot of amplified RNA from prostate cancer cells and normal prostate duct epithelium. Labeled by reverse transcription using Cy5-dCTP and Cy3-dCTP (Amersham Biosciences, Buckinghamshire, UK), respectively. Down, washing and detection were carried out as described previously (Ono et al., (2000)).

(2) Identification of a novel gene CCDC4 (coiled-coil domain-containing 4) Using a genome-wide cDNA microarray, we identified one up-regulated spot (intra-organization name B3537) representing one EST (Homo sapiens cDNA FLJ35632). Combining other EST information with sequences obtained by RACE using prostate cancer cDNA, we identified the novel gene CCDC4.

(3) Northern blot analysis The [α-32P] dCTP-labeled PCR product of B3537 was hybridized to a human multi-tissue northern blot (Clontech, Palo Alto, Calif.). A 361 bp PCR product was prepared by RT-PCR using primers: 5′-GTGACAAATCCATTGATCCTGA-3 ′ (SEQ ID NO: 5) and 5′-GAACACGTGGCATTCTAGAGGTA-3 ′ (SEQ ID NO: 6). Prehybridization, hybridization, and washing were performed according to the supplier's recommendations. Autoradiography of the blots was performed using an intensifying screen at −80 ° C. for 7 days.

  RT-PCR analysis confirmed the overexpression of CCDC4 in prostate cancer cells (FIG. 1A). By Northern blot analysis (FIG. 1B), this transcript is approximately 8.7 kb and from 6 exons encoding a 530 amino acid protein (GeneBank accession number: AB126828) (SEQ ID NO: 2) with a coiled-coil domain. Proved to be. An alternative splicing form was also identified that was expected to result in a short isoform 437 amino acid protein (GeneBank accession number: AB126829) (SEQ ID NO: 4) lacking the long form of the C-terminal region. This gene is restrictedly expressed in normal testis and prostate, as shown in Northern blot analysis (Figure 1B), indicating that targeting this molecule to normal human organs It shows almost no toxicity.

(4) siRNA expression construct and colony formation / MTT assay We used siRNA expression vector (psiU6BX) for RNAi effect on the target gene. Upstream of the gene-specific sequence (19 nt sequence from the target transcript separated from the reverse complement of the same sequence by a short spacer TTCAAGAGA (SEQ ID NO: 7)) and 5 thymidines as termination signals In addition, a neo cassette was incorporated to make it resistant to geneticin (Sigma). The target sequences for CCDC4 are 5'-GATGGTTCTGCAGCACCAC-3 '(SEQ ID NO: 8) (si # 1) and 5'-GAAGCAGCACGACTTCTTC-3' (SEQ ID NO: 9) (siEGFP) as a negative control. .

The oligonucleotides used for CCDC4 siRNA are shown below. si # 1 was prepared by cloning the following double stranded oligonucleotide into the Bbsl site of the psiU6BX vector. The corresponding nucleotide positions for the CCDC4 nucleic acid sequence of SEQ ID NO: 1 or 3 are shown below. An oligonucleotide is a combination of a sense nucleotide sequence and an antisense nucleotide sequence of the target sequence CCDC4. The nucleotide sequence of the hairpin loop structure of si # 1 is shown in SEQ ID NO: 10 (the endonuclease recognition site is excluded from each hairpin loop structure sequence).
si # 1 (nucleotide positions 1666 to 1684 of SEQ ID NO: 1 or 3 / SEQ ID NO: 8)
5'-caccgatggt tctgcagcac cacttcaaga gagtggtgct gcagaaccat c-3 '(SEQ ID NO: 11)
5'-aaaagatggt tctgcagcac cactctcttg aagtggtgct gcagaaccat c-3 '(SEQ ID NO: 12)

Prostate cancer cell lines PC3 and DU145 are seeded into 10 cm dishes (5 × 10 ⁵ cells / dish) and contain EGFP target sequence (EGFP) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions It was transfected with psiU6BX and psiU6BX containing the CCDC4 target sequence. Cells were selected for 1 week with 500 mg / ml Geneticin, and spare cells were harvested 48 hours after transfection and analyzed by RT-PCR to confirm the knockdown effect on CCDC4. RT-PCR primers were the same as above. In order to assess colony formation and cell number, respectively, these cells were stained with Giemsa solution and an MTT assay was also performed.

  RT-PCR confirmed the knockdown effect of CCDC4 mRNA by transfection of siRNA expression vector si # 1, but no effect by siEGFP. The colony formation assay showed a dramatic reduction in the number of colonies of cells after transfection with si # 1, which was confirmed by RT-PCR to knock down CCDC4 efficiently. The MTT assay also showed a dramatic reduction in proliferating cells transfected with si # 1. These findings strongly support that CCDC4 is essential for PRC cell proliferation and that molecular targeting of CCDC4 is a promising approach for developing new PRC therapies.

Construction of psiU6BX 3.0 plasmid
A DNA fragment encoding siRNA was inserted into the gap of nucleotides 485 to 490 indicated as (-) in the following plasmid sequence (SEQ ID NO: 13).

The snRNA U6 gene has been reported to be transcribed by RNA polymerase III to produce a short transcript with a uridine at the 3 ′ end. The genomic fragment of the snRNA U6 gene containing the promoter region is a set of primers,
5′-GGGGATCAGCGTTTGAGTAA-3 ′ (SEQ ID NO: 14), and
5'-TAGGCCCCACCTCCTTCTAT-3 '(SEQ ID NO: 15)
And was amplified by PCR using human placental DNA as a template. The product was purified and cloned into the pCR plasmid vector using the TA cloning kit according to the supplier protocol (Invitrogen). BamHI-XhoI containing snRNA U6 gene amplified by PCR with primer set, 5'-TGCGGATCCAGAGCAGATTGTACTGAGAGT-3 '(SEQ ID NO: 16) and 5'-CTCTATCTCGAGTGAGGCGGAAAGAACCA-3' (SEQ ID NO: 17) The fragment was purified and cloned into the nucleotide 1257-56 fragment of the pcDNA3.1 (+) plasmid. The ligated DNA is the primer
Used for PCR template with 5'-TTTAAGCTTGAAGACTATTTTTACATCAGGTTGTTTTTCT-3 '(SEQ ID NO: 18) and 5'-TTTAAGCTTGAAGACACGGTGTTTCGTCCTTTCCACA-3' (SEQ ID NO: 19).
The product was digested with HindIII and subsequently self-ligated to create a psiU6BX vector plasmid. As a control, double-stranded oligonucleotides of 5′-CACCGAAGCAGCACGACTTCTTCTTCAAGAGAGAAGAAGTCGTGCTGCTTC-3 ′ (SEQ ID NO: 20) and 5′-AAAAGAAGCAGCACGACTTCTTCTCTCTTGAAGAAGAAGTCGTGCTGCTTC-3 ′ (SEQ ID NO: 21) are cloned into the psiU6BX vector by using the BbsI psi EGFP was prepared.

Industrial Applicability The expression of the human gene CCDC4 is markedly elevated in prostate cancer compared to non-cancerous prostate duct epithelium. These genes are therefore advantageous as prostate cancer diagnostic markers and the proteins encoded by them are useful in prostate cancer diagnostic assays.

  The present inventors have also shown that cell growth is promoted by expression of the novel protein CCDC4, while cell growth is suppressed by small interfering RNA corresponding to the CCDC4 gene. These findings indicated that CCDC4 protein induces oncogenic activity. Thus, these novel oncoproteins are useful targets for the development of anticancer agents. For example, an agent that blocks the expression of CCDC4 or an agent that blocks its activity is considered to have therapeutic utility as an anticancer agent, particularly an anticancer agent for the treatment of prostate cancer. Examples of such agents include antisense oligonucleotides to CCDC4 gene, small interfering RNAs and ribozymes, and antibodies that recognize CCDC4.

  Although the invention has been described in detail with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. .

1 (A) shows a photograph showing the result of semi-quantitative PCR. CCDC4 was overexpressed in prostate cancer cells collected by microdissection from human prostate cancer tissue. 1 (B) shows a photograph of Northern blot analysis showing the expression pattern of CCDC4 in normal adult tissues. The 8.7 kb transcript CCDC4 is restrictedly expressed only in the adult testis and prostate. The effect of knocking down endogenous CCDC4 in prostate cancer cell line PRC3 by siRNA is shown. 2 (A) is a photograph showing the results of RT-PCR. This photograph confirms the knockdown effect of CCDC4 mRNA by transfection of siRNA expression vector si # 1. si # 1 was designed specifically for the CCDC4 mRNA sequence and siEGFP was designed specifically for the EGFP mRNA sequence. RNA was collected and analyzed 48 hours after transfection. ACTB was used to normalize the input cDNA. 2 (B) is a photograph showing the results of a colony formation assay. This photo shows a dramatic decrease in the number of colonies in PRC3 cells one week after transfection with si # 1, which was confirmed by RT-PCR to knock down CCDC4 efficiently. FIG. 2 (C) is a bar graph showing the results of the MTT assay. This assay also shows a dramatic reduction in the number of proliferating cells transfected with si # 1.

Claims (42)

A substantially pure polypeptide selected from the group consisting of:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;
(B) an amino acid sequence of SEQ ID NO: 2 or 4 or a polypeptide comprising a sequence having at least about 80% homology with SEQ ID NO: 2 or 4; and (c) a nucleotide of SEQ ID NO: 1 or 3 A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the sequence, and is equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide having activity.   2. An isolated polynucleotide encoding the polypeptide of claim 1.   A vector comprising the polynucleotide according to claim 2.   A host cell carrying the polynucleotide according to claim 2 or the vector according to claim 3. A method for producing a polypeptide according to claim 1 comprising the following steps:
(A) culturing the host cell of claim 4;
(B) allowing the host cell to express the polypeptide; and (c) collecting the expressed polypeptide.   An antibody that binds to the polypeptide of claim 1.   A polynucleotide that is complementary to a polynucleotide encoding the polypeptide of claim 1 or a complementary strand thereof and comprises at least 15 nucleotides.   An antisense polynucleotide or a small interfering RNA against the polynucleotide encoding the polypeptide according to claim 1.   9. The small interfering RNA according to claim 8, wherein the sense strand includes the nucleotide sequence of SEQ ID NO: 8 as a target sequence and is less than 75 nucleotides in length. A method for diagnosing prostate cancer comprising the following steps:
(A) detecting the expression level of the gene encoding the amino acid sequence of SEQ ID NO: 2 or 4 in the biological sample; and (b) associating the increased expression level with a disease. 11. The method of claim 10, wherein the expression level is detected by any of the methods selected from the group consisting of:
(A) detecting an mRNA encoding the amino acid sequence of SEQ ID NO: 2 or 4,
(B) detecting a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4, and (c) detecting a biological activity of the protein comprising the amino acid sequence of SEQ ID NO: 2 or 4. A method of screening for a compound for treating or preventing prostate cancer comprising the following steps:
(A) contacting a test compound with a polypeptide selected from the group consisting of:
(1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;
(2) an amino acid sequence of SEQ ID NO: 2 or 4 or a polypeptide comprising a sequence having at least about 80% homology with SEQ ID NO: 2 or 4; and (3) of SEQ ID NO: 1 or 3 A polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide comprising a nucleotide sequence under stringent conditions, and has a biological activity equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4 Having a polypeptide;
(B) detecting the binding activity between the polypeptide and the test compound; and (c) selecting a compound that binds to the polypeptide. A method of screening for a compound for treating or preventing prostate cancer comprising the following steps:
(A) contacting a test compound with a polypeptide selected from the group consisting of:
(1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;
(2) an amino acid sequence of SEQ ID NO: 2 or 4 or a polypeptide comprising a sequence having at least about 80% homology to SEQ ID NO: 2 or 4; and (3) a nucleotide of SEQ ID NO: 1 or 3 A polypeptide encoded by a polynucleotide that hybridizes with a polynucleotide comprising the sequence under stringent conditions, and has a biological activity equivalent to that of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4. The polypeptide in question;
(B) detecting the biological activity of the polypeptide of step (a); and (c) the biological activity of the polypeptide compared to the biological activity detected in the absence of the test compound. Selecting a compound to inhibit.   14. The method of claim 13, wherein the biological activity is cell proliferation activity. A method of screening for a compound for treating or preventing prostate cancer comprising the following steps:
(A) contacting a test compound with a cell expressing one or more polynucleotides comprising the nucleotide sequence of SEQ ID NO: 1 or 3, and (b) an expression level detected in the absence of the test compound Selecting a compound that reduces the expression level of one or more polynucleotides comprising the nucleotide sequence of SEQ ID NO: 1 or 3 as compared to.   16. The method of claim 15, wherein the cell is a prostate cancer cell. A method of screening for a compound for treating or preventing prostate cancer comprising the following steps:
(A) a vector comprising one or more marker gene transcription control regions comprising any one of the nucleotide sequences selected from the group consisting of SEQ ID NOs: 1 and 3, and a reporter gene expressed under the control of the transcription control region Contacting the test compound with a cell into which is introduced,
(B) measuring the expression level or activity of the reporter gene; and (c) selecting a compound that decreases the expression level or activity of the reporter gene compared to a control. A pharmaceutically effective amount of an antisense polynucleotide or a small interfering RNA against a polynucleotide encoding a polypeptide selected from the group consisting of, as an active ingredient, and a pharmaceutically acceptable carrier. A composition for treating or preventing prostate cancer comprising:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;
(B) Equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide having biological activity; and (c) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, SEQ ID NO: 2. Or a polypeptide having biological activity equivalent to a polypeptide consisting of the amino acid sequence of 4.   19. The composition of claim 18, wherein the small interfering RNA comprises the nucleotide sequence of SEQ ID NO: 8 as the target sequence. 20. The composition of claim 19, wherein the siRNA has the general formula 5 '-[A]-[B]-[A']-3 '
Where
[A] is a ribonucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 8;
[B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and
[A '] is a ribonucleotide sequence consisting of the complementary sequence of [A],
Composition.   19. The composition of claim 18, comprising a transfection enhancing agent. A composition for treating or preventing prostate cancer comprising a pharmaceutically effective amount of an antibody against a polypeptide selected from the group consisting of the following as a pharmaceutically effective ingredient and a pharmaceutically acceptable carrier:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;
(B) Equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide having biological activity; and (c) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, SEQ ID NO: 2. Or a polypeptide having biological activity equivalent to a polypeptide consisting of the amino acid sequence of 4.   18. Treatment or prevention of prostate cancer comprising a compound selected by the method of any one of claims 12-17 as a pharmaceutically effective amount, as an active ingredient, and a pharmaceutically acceptable carrier. Composition to do. A method for treating or preventing prostate cancer, comprising administering a pharmaceutically effective amount of an antisense polynucleotide or a small interfering RNA against a polynucleotide encoding a polypeptide selected from the group consisting of: Method:
(1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;
(2) Equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide having biological activity; and (3) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, SEQ ID NO: 2. Or a polypeptide having biological activity equivalent to a polypeptide consisting of the amino acid sequence of 4.   25. The method of claim 24, wherein the siRNA comprises the nucleotide sequence of SEQ ID NO: 8 as the target sequence. 26. The method of claim 25, wherein the siRNA has the general formula 5 ′-[A]-[B]-[A ′]-3 ′
Where
[A] is a ribonucleotide sequence corresponding to the nucleotide sequence of SEQ ID NO: 8;
[B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and
[A '] is a ribonucleotide sequence consisting of the complementary sequence of [A],
Method.   25. The method of claim 24, wherein the composition comprises a transfection enhancing agent. A method for treating or preventing prostate cancer comprising administering a pharmaceutically effective amount of an antibody against a polypeptide selected from the group consisting of:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;
(B) Equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide having biological activity; and (c) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, SEQ ID NO: 2. Or a polypeptide having biological activity equivalent to a polypeptide consisting of the amino acid sequence of 4.   A method for treating or preventing prostate cancer comprising administering a pharmaceutically effective amount of a compound selected by the method according to any one of claims 12 to 17. A method for treating or preventing prostate cancer, comprising administering a pharmaceutically effective amount of a polypeptide selected from the group consisting of (a) to (c) or a polynucleotide encoding the polypeptide. :
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4 or a fragment thereof;
(B) an organism equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide having a biological activity; and (c) encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, SEQ ID NO: 2 or A polypeptide having biological activity equivalent to a polypeptide consisting of the amino acid sequence of 4 or a fragment thereof. Contacting a polypeptide selected from the group consisting of (a) to (c) with an antigen-presenting cell, or introducing a polynucleotide encoding the polypeptide or a vector containing the polynucleotide into the antigen-presenting cell. Methods for inducing anti-tumor immunity comprising:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4 or a fragment thereof;
(B) comprising the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence of SEQ ID NO: 2 or 4 or a fragment thereof A polypeptide having biological activity equivalent to a protein;
(C) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, and comprising an amino acid sequence of SEQ ID NO: 2 or 4 or a fragment thereof A polypeptide having an equivalent biological activity.   32. The method for inducing anti-tumor immunity according to claim 31, further comprising administering antigen-presenting cells to the subject. A pharmaceutically effective amount of a polypeptide selected from the group consisting of (a) to (c) or a polynucleotide encoding the polypeptide as an active ingredient, and a pharmaceutically acceptable carrier A pharmaceutical composition for treating or preventing prostate cancer:
(A) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4, or a fragment thereof;
(B) Equivalent to a protein comprising the amino acid sequence of SEQ ID NO: 2 or 4 in which one or more amino acids are substituted, deleted, inserted and / or added, and consisting of the amino acid sequence of SEQ ID NO: 2 or 4 A polypeptide having biological activity;
(C) an organism that is encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or 3, and is equivalent to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4 Polypeptides having biological activity, or fragments thereof.   34. A pharmaceutical composition according to claim 33, wherein the polynucleotide is incorporated into an expression vector.   A diagnostic agent comprising an oligonucleotide that hybridizes to a polynucleotide encoding the polypeptide of claim 1 or an antibody that binds to the polypeptide of claim 1.   A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to SEQ ID NO: 8, the antisense strand comprises a ribonucleotide sequence complementary to the sense strand; A double-stranded molecule that inhibits the expression of a gene when the sense strand and the antisense strand hybridize with each other to form a double-stranded molecule and are introduced into a cell expressing the CCDC4 gene.   38. The double-stranded molecule of claim 36, wherein the sense strand comprises from about 19 to about 25 contiguous nucleotides from SEQ ID NO: 1.   38. The double-stranded molecule of claim 36, wherein the sense strand consists of a ribonucleotide sequence corresponding to SEQ ID NO: 8.   40. The double-stranded molecule of claim 36, further comprising a single-stranded ribonucleotide sequence linking the sense strand and the antisense strand, wherein the single ribonucleotide transcript comprises a sense strand and an antisense strand. molecule.   A vector encoding the double-stranded molecule according to claim 36.   41. The vector of claim 40, which encodes a transcript having a secondary structure, wherein the transcript comprises a sense strand and an antisense strand.   41. The vector of claim 40, wherein the transcript further comprises a single stranded ribonucleotide sequence linking the sense strand and the antisense strand.

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