JP2008178358A - Human abh8 protein, gene for encoding the same, and curative or diagnostic application of them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify a new human ABH family molecule and provide the molecule for applying it to e.g. curing, diagnosing diseases including cancers, neuropathy and developmental disorders. <P>SOLUTION: The new human ABH family molecule is provided as follows: (a) a polynucleotide comprising a specific nucleotide sequence, (b) a polynucleotide comprising a nucleotide sequence encoding a human ABH8 protein comprising a specific amino acid sequence, (c) a polynucleotide which is hybridizable with a polynucleotide comprising a polynucleotide sequence complementary to the polynucleotide in (a) or (b) under stringent hybridization conditions and comprises a nucleotide sequence encoding an ABH8 protein, or (d) a polynucleotide comprising a nucleotide sequence encoding an ABH8 protein comprising an amino acid sequence wherein at least one amino acid residue is deleted, substituted, inserted or added in the amino acid sequence (b). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to the ABH8 protein that is a member of the AlkB homolog (ABH) family, the genes that encode it, and their therapeutic or diagnostic uses. In particular, the present invention relates to human ABH8 protein, genes encoding it, and therapeutic or diagnostic uses thereof.

  Repair of DNA damage is essential to maintain genome integrity and keep cells alive [1]. In mammals, defects in the repair system not only cause changes or loss of the genetic information of the cell, but also drastically change the structure of the cell to significantly affect the reading of the genetic information encoded therein. It can lead to cancer, neurological diseases, developmental disorders, etc. [Non-Patent Document 2]. One example is DNA damage caused by alkylation.

The cell can be repaired using 3-methyladenine-DNA glycosylase for the repair of methylated bases in DNA [Non-patent document 3, Non-patent document 4], or O ⁶ -methylguanine-DNA methyltransferase. It has been known to repair the damage by transferring the alkyl group from the damaged site to the cysteine residue of the enzyme itself [Non-Patent Document 5, Non-Patent Document 6].

  In 2002, B.M. Sedgwick et al. See Seeberg et al. Reported that bacterial AlkB protein repairs DNA alkylation damage by an unprecedented mechanism [7, 8]. They show that AlkB oxidatively demethylates 1-methyladenine, 3-methylcytosine in the presence of oxygen, 2-oxoglutarate, iron and 2-oxoglutarate-Fe (II) -oxygenase Suggested to belong to the superfamily. Thereafter, E.E. Seeberg, H.C. E. It was shown by Krokan et al. That two types of AlkB human homologs ABH2 and ABH3 repair DNA and RNA alkylation damage by the same mechanism as AlkB [Non-patent Document 9]. ABH3 is the same molecule as Prostate Cancer Antigen-1 (PCA-1), which has already been identified by the present inventors as a gene highly expressed in human prostate cancer, and has high homology with AlkB at the C-terminal part. Therefore, the function of AlkB as a human homolog was estimated [Non-patent Document 10].

Also recently, J.H. M.M. Reported in a silico analysis based on the homology of the AlkB homologous region that PCA-1 together with the other six molecules constitutes the AlkB homolog (ABH) family [Non-Patent Document 11, Non-patent Document 11]. Reference 12]. In addition to the three types of human ABH reported so far, they newly revealed the existence of four molecules from ABH4 to ABH7, and these molecules were also identified as AlkB and ABH2 by homology analysis of the secondary structure of the protein. , Similar to ABH3, it was suggested to belong to the 2-oxoglutarate-Fe (II) -oxygenase superfamily. A schematic diagram of these ABH family molecules is shown in FIG. In addition, J.A. M.M. Bujnicki et al. Estimated the existence of ABH8 in Macaca fascicularis (cynomolgus monkey) and others, but in homology analysis based on the human cDNA database (NCBI library), exons 6, 9, and 10 are missing and frame shift errors are observed. It is reported that only incomplete splicing foam bodies were detected.
PC. Hanawalt, Preferred repar of damage in activated transferred DNA sequences in vivo, Genome. 1989; 31 (2): 605-11, Review. VA. Bohr, DNA repair at the level of the gene: molecular and clinical conjugations, J Cancer Res Clin Oncol. 1990; 116 (4): 384-91, Review. J. et al. Labahn, OD. Scharer, et al. Structural basis for the excision repair of alkylation-damaged DNA, Cell. 1996; 86 (2): 321-9 BP. Engelward, A.D. Dreslin, et al. Repair-defective 3-methyladenine DNA glycosylate homozygoous mutant mouse cells have been sensitized to alkylation. 1996; 15 (4): 945-52. P. Karran, T .; Lindahl, et al. Adaptive response to alkylating agents evolves in situ of O6-methylguanine residues in DNA, Nature. 1979; 280 (5717): 76-7. R. Montesano, R.A. Becker, et al. Repair of DNA alkylation add-ins in mammalian cells, Biochimie. 1985; 67 (9): 919-28, Review. S. C. Trewick, T.W. F. Hensshaw, et al. Oxidative demethylation by Escherichia coli AlkB direct reverses DNA base damage, Nature. 2002; 419 (6903): 174-8. P. O. Falnes, R.M. F. Johansenand Erring Seeberg, AlkB-mediated oxidative deversing reverses DNA damage in Escherichia coli, Nature. 2002; 419 (6903): 178-82. P. A. Aas, M.M. Otterei, et al. Human and Bacterial Oxidative Demonstrations Repair alkylation damage in both RNA and DNA, Nature. 2003; 421 (6925): 859-63. N. Konishi, M .; Nakamura, et al. High expression of a new marker PCA-1 in human prosthesis carcinoma, Clin Cancer Res. 2005; 11 (14); 5090-7 M.M. A. Kurowski, A.R. S. Bhagwat, et al. Phylogenomic identification of five new human homolog of the DNA repair enzyme AlkB, BMC Genom. 2003; 4 (1): 48. F. Drablos, E .; Feyzi, et al. Alkylation damage in DNA and RNA-repair mechanisms and medical signature, DNA Repair (Amst). 2004; 3 (11): 1389-1407. Review

  Under such circumstances, it is expected to identify a new human ABH family molecule and use it for the treatment or diagnosis of diseases such as cancer, neurological diseases and developmental disorders.

  In order to identify a new human ABH family molecule, the present inventors tried and cloned a human counterpart of ABH8, which was supposed to exist in cynomolgus monkeys. Furthermore, expression analysis of the molecule in human normal tissues and cancer cell lines, analysis of the influence of expression inhibition, etc. were performed, and it was confirmed that apoptosis of cancer cells was induced by inhibiting the expression of the human ABH gene. . In addition, ABH8 protein was confirmed to react strongly to immunohistochemical staining in several cancer tissues.

  Accordingly, the present invention provides the following polynucleotide, protein, cancer therapeutic agent, cancer diagnostic agent, screening method for human ABH8 gene expression inhibitor or human ABH8 protein activity inhibitor, and cancer therapeutic agent containing the inhibitor A method for detecting the ABH8 protein or ABH8 gene of the present invention as a cancer marker in a biological sample is provided.

(1) (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1,
(B) a polynucleotide comprising a nucleotide sequence encoding the human ABH8 protein comprising the amino acid sequence of SEQ ID NO: 2,
(C) a polynucleotide comprising a nucleotide sequence which hybridizes under stringent hybridization conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of (a) or (b) and which encodes an ABH8 protein, or d) A polynucleotide consisting of a nucleotide sequence encoding an ABH8 protein consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2.
(2) The polynucleotide of (c) or (d) described in (1) above, which has a homology of 97% or more with the nucleotide sequence of the polynucleotide of (a) or (b) A polynucleotide consisting of a nucleotide sequence.
(3) A polynucleotide containing the polynucleotide according to (1) or (2) above.
(4) A human ABH8 protein consisting of the amino acid sequence of SEQ ID NO: 2, or an ABH8 protein consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2.
(5) A polypeptide comprising the amino acid sequence of the ABH8 protein according to (4) above.
(6) A cancer therapeutic agent comprising a human ABH8 gene expression inhibitor as an active ingredient.
(7) The expression inhibitor of human ABH8 gene is
(A) a nucleic acid having an action of inhibiting the expression of the human ABH8 gene by the RNAi effect,
(B) an antisense nucleic acid for the transcription product of human ABH8 gene or a part thereof,
And (c) a nucleic acid having ribozyme activity that specifically cleaves a transcript of the human ABH8 gene,
The cancer therapeutic agent according to (6) above, comprising a substance selected from the group consisting of:
(8) The nucleic acid described in (7) above, wherein the nucleic acid comprises a polynucleotide having the base sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 Cancer treatment.
(9) A cancer therapeutic agent containing a human ABH8 protein activity inhibitor as an active ingredient.
(10) The human ABH8 protein activity inhibitor is
An antibody that binds to the human ABH8 protein;
The cancer therapeutic agent as described in said (9) containing.
(11) The cancer therapeutic agent according to any one of (6) to (10), wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colon cancer.
(12) A method for screening an expression inhibitor of human ABH8 gene,
(A) contacting a test compound with a cell expressing the human ABH8 gene;
(B) a method for measuring the expression level of the human ABH8 gene, and (c) a method for selecting a compound that reduces the expression level as compared with the case where the test compound is not contacted.
(13) A method for screening an activity inhibitor of human ABH8 protein,
(A) contacting human ABH8 protein with a test compound;
(B) a screening method comprising a step of measuring the binding activity between the human ABH8 protein and a test compound, and (c) a step of selecting a compound that binds to the human ABH8 protein.
(14) An antibody that binds to human ABH8 protein.
(15) A cancer therapeutic agent containing the antibody according to (14).
(16) The cancer therapeutic agent according to (15), further comprising a vector carrying a radioisotope, a therapeutic protein, a low molecular drug, or a therapeutic gene.
(17) The cancer therapeutic agent according to (15) or (16) above, wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colon cancer.
(18a) A cancer diagnostic agent comprising the antibody according to (14).
(18b) A cancer diagnostic agent comprising a base sequence capable of hybridizing under stringent hybridization conditions to a human ABH8 gene or a partial base sequence thereof.
(19) The cancer diagnostic agent according to (18a) or (18b), wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colon cancer.
(20a) A kit for cancer diagnosis containing the antibody according to (14).
(20b) A cancer diagnostic kit comprising a polynucleotide comprising a nucleotide sequence capable of hybridizing under stringent hybridization conditions to the human ABH8 gene or a partial nucleotide sequence thereof.
(21) The cancer diagnostic kit according to (20a) or (20b), wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colorectal cancer.
(22) A method for detecting and / or quantifying ABH8 protein in a biological sample derived from a subject as a cancer marker.
(23) (a) contacting a biological sample derived from a subject with an antibody that binds to ABH8 protein; and (b) detecting and / or quantifying the binding between the antibody and ABH8 protein in the sample. Process,
The method according to (22) above, comprising
(24) The method according to (22) or (23) above, wherein the biological sample is a cell or tissue section.
(25) The method according to (22) or (23) above, wherein the biological sample is blood (including whole blood, plasma, serum, etc.) or urine.
(26) The method according to any one of (22) to (24) above, wherein the binding between the antibody and ABH8 protein is detected and / or quantified by immunohistochemical staining.
(27) The method according to any one of (22) to (26) above, which is used for diagnosis of cancer.
(28) The method according to (27) above, wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colon cancer.
(29) A polynucleotide having the base sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21.
(30) A cancer therapeutic agent containing an ABH8 gene expression inhibitor as an active ingredient, the nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 The cancer therapeutic agent containing the polynucleotide which has this.
(31) A method for detecting and / or quantifying the ABH8 gene in a biological sample derived from a subject as a cancer marker.
(32) The method according to (31) above, wherein the biological sample is a cell or tissue section.
(33) (a) contacting a biological sample derived from a subject with a polynucleotide comprising a base sequence capable of hybridizing under stringent hybridization conditions to the base sequence of the ABH8 gene or a fragment thereof; and (b) Detecting and / or quantifying hybridization between the polynucleotide in the sample and the ABH8 gene or a fragment thereof;
The method according to (31) or (32) above, comprising:
(34) A method for detecting and / or quantifying an anti-ABH8 autoantibody in a biological sample derived from a subject as a cancer marker.
(35) The method according to (34) above, wherein the biological sample is a cell or tissue section.
(36) The method according to (34) above, wherein the biological sample is whole blood, serum, plasma, or urine.
(37) The method according to any one of (34) to (36) above, wherein the anti-ABH8 autoantibody is detected and / or quantified using an ABH8 antigen.
(38) The method according to (37), comprising the step of bringing the biological sample into contact with the ABH8 antigen, and the step of detecting and / or quantifying the binding between the anti-ABH8 autoantibody and the ABH8 antigen in the biological sample. Method.
(39) The detection and / or quantification step includes detecting and / or quantifying the binding between ABH8 and the anti-ABH8 autoantibody using a labeled antibody against the anti-ABH8 autoantibody (38) ) Method.
(40) Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, fluorescence immunoassay (FIA), time-resolved fluoroimmunoassay (TRFIA), enzyme immunoassay (EIA), luminescence immunoassay (LIA), electrochemiluminescence immunoassay (ECLIA), latex agglutination, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutinin assay The method according to any one of (34) to (39) above, according to an immunoassay selected from the group consisting of a complementation assay, a complement binding assay, an immunoradiometric assay, a fluorescence immunoassay, and a protein A immunoassay the method of.
(41) The method according to any one of (34) to (40) above, which is used for diagnosis of breast cancer, bladder cancer, prostate cancer, or colorectal cancer.
(42) A kit for detecting and / or quantifying an anti-ABH8 autoantibody in a biological sample derived from a subject containing an ABH8 antigen as a cancer marker.
(43) The method according to (42), further comprising a labeled antibody against the anti-ABH8 autoantibody, wherein the antibody is used for detecting and / or quantifying the binding between the ABH8 antigen and the anti-ABH8 autoantibody. Kit.
(44) Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, fluorescence immunoassay (FIA), time-resolved fluoroimmunoassay (TRFIA), enzyme immunoassay (EIA), luminescence immunoassay (LIA), electrochemiluminescence immunoassay (ECLIA), latex agglutination, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutinin assay (42) for performing the detection and / or quantification according to an immunoassay selected from the group consisting of a method comprising: a method of complementation, a complement binding assay, an immunoradiometric assay, a fluorescence immunoassay, and a protein A immunoassay ) Or (43).
(45) The kit according to any one of (42) to (44), which is used for diagnosis of breast cancer, bladder cancer, prostate cancer, or colorectal cancer.
(46) A cancer diagnostic agent for detecting and / or quantifying an anti-ABH8 autoantibody in a biological sample derived from a subject containing an ABH8 antigen as a cancer marker.

  According to the present invention, there are provided drugs, kits, methods and the like useful for diagnosis / treatment of diseases (eg, cancer (eg, breast cancer, prostate cancer, bladder cancer, colon cancer)) associated with high expression of ABH8 gene and ABH8 protein. Provided.

1. ABH8 protein of the present invention and polynucleotide encoding the same The present inventors succeeded in cloning a human counterpart of ABH8, a new human ABH family molecule, and determined the nucleotide sequence (SEQ ID NO: 1) of the cDNA. . The amino acid sequence deduced therefrom (SEQ ID NO: 2) was also determined.

Therefore, in one embodiment, the present invention provides the following polynucleotides (a) to (d):
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1,
(B) a polynucleotide comprising a nucleotide sequence encoding the human ABH8 protein comprising the amino acid sequence of SEQ ID NO: 2,
(C) a polynucleotide comprising a nucleotide sequence which hybridizes under stringent hybridization conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of (a) or (b) and which encodes an ABH8 protein, and ( d) A polynucleotide comprising a nucleotide sequence encoding an ABH8 protein consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2.

  In a preferred embodiment, the polynucleotide (c) or (d) consists of a nucleotide sequence having 97% or more homology to the nucleotide sequence of the polynucleotide (a) or (b).

  The present invention further provides polynucleotides containing these polynucleotides.

  The present invention also provides a human ABH8 protein consisting of the amino acid sequence of SEQ ID NO: 2 and ABH8 consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2. Provide protein.

  The invention further provides polypeptides containing the amino acid sequences of these proteins.

  In the present specification, the term “ABH8 protein of the present invention” refers to (i) not only the human ABH8 protein consisting of the amino acid sequence of SEQ ID NO: 2, but also ABH8 consisting of an amino acid sequence having high homology with the amino acid sequence of SEQ ID NO: 2. A protein, for example (ii) an ABH8 protein consisting of an amino acid sequence in which at least one amino acid residue has been deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, and (iii) of SEQ ID NO: 2 An ABH8 protein consisting of an amino acid sequence having a high identity (eg, about 80% or more) with the amino acid sequence is also included.

  Examples of the “amino acid sequence in which at least one amino acid residue is deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2” include those in the amino acid sequence of SEQ ID NO: 2, for example, 1 to 50 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1-33 1-30, 1-29, 1-28, 1-27, 1-26, 1-25, 1-24, 1-23, 1-22, 1-21 1-20, 1-19, 1-18, 1-17, 1-16, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11 1-10, 1-9, 1-8, 1-7, 1-6 (1-several), 1-5, 1-4, 1-3, Two, one amino Residues are deleted, substituted, it includes inserts and / or added in the amino acid sequence. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better.

  Examples of the “amino acid sequence having high identity with the amino acid sequence of SEQ ID NO: 2” include the amino acid sequence of SEQ ID NO: 2 of about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85 % Or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more , 99.8% or more, or 99.9% or more amino acid sequences. In general, the larger the numerical value of identity, the better. In the present specification, the terms “identity” and “homology” are used separately for amino acid sequences. For example, when referring to homology between amino acid sequences, amino acids having the same properties (such as glutamic acid and aspartic acid) are treated as one group, but are distinguished when considering identity. That is, identity refers to consistency.

  In the present specification, “hybridizes” with respect to a nucleotide sequence means that a double strand is formed when the complementarity between nucleotide sequences is high. Usually, the double strands are designed so that the nucleic acids are complementary to each other, but if necessary (for example, in the case where nucleic acids are used for detection, as long as no problem occurs for that purpose). The nucleic acid may contain one or several (for example, 2 to 3) mismatches. The number of mismatches that may be present may vary depending on, for example, the required detection accuracy and the length of the nucleotide sequence (or base sequence or nucleic acid (sequence) or polynucleotide). In addition, by appropriately changing the hybridization conditions between the nucleotide sequence and the nucleotide sequence with which it hybridizes, it is possible to eliminate the case where there is a mismatch or to adjust the number of allowable mismatches. Are well known to those skilled in the art.

  As a hybridization method, a colony hybridization method, a plaque hybridization method, a Southern hybridization method, or the like can be used (for example, Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997). reference).

  In the present specification, “stringent hybridization conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. Under these conditions, it can be expected that DNA having higher identity can be efficiently obtained as the temperature is increased. However, factors affecting the stringency of hybridization include multiple factors such as temperature, nucleic acid concentration, nucleic acid length, ionic strength, time, and salt concentration. Those skilled in the art appropriately select these factors. It is possible to achieve the same stringency.

  In addition, when using a commercially available kit for hybridization, Alkphos Direct Labeling Reagents (made by Amersham Pharmacia) can be used, for example. In this case, according to the protocol attached to the kit, after overnight incubation with the labeled probe, the membrane was washed with a primary wash buffer containing 0.1% (w / v) SDS at 55 ° C. After washing, the hybridized polynucleotide can be detected.

  Examples of “nucleotide sequence capable of hybridizing under stringent hybridization conditions” to a desired nucleotide sequence (for example, the nucleotide sequence of SEQ ID NO: 1) include homologous search software such as FASTA, BLAST, etc. About 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more with the desired nucleotide sequence 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more It may be mentioned a nucleotide sequence having 99.9% or more homology.

  The identity (or homology) of the amino acid sequence and base sequence is determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA, 87, 2264-2268, 1990; Proc. Natl. Acad. Sci by Carlin and Arthur. USA, 90, 5873, 1993). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence using BLASTX, parameters are set to score = 50 and wordlength = 3, for example. When using BLAST and Gapped BLAST programs, the default parameters of each program can be used.

2. Although the present invention contains (1) a cancer therapeutic agent containing a human ABH8 gene expression inhibitor as an active ingredient, and (2) a human ABH8 protein activity inhibitor as an active ingredient. Provide therapeutic agents.

  In this specification, the term “cancer therapeutic agent” includes anticancer agents, cancer metastasis inhibitors, cancer cell apoptosis inducers, cancer cell proliferation inhibitors, cancer cell infiltration inhibitors, cancer preventive agents, and the like. Used in meaning. In the present specification, the terms “cancer (or cancer)” and “tumor” are used as terms having the same meaning.

(Cancer therapeutic agent containing ABH8 gene expression inhibitor)
In one embodiment of the present invention, a gene encoding the ABH8 protein of the present invention (sometimes referred to simply as “ABH8 gene” in the present specification), particularly a human ABH8 gene expression inhibitor as an active ingredient. A cancer therapeutic agent is provided.

  In the present specification, the “ABH8 gene expression inhibitor” is not limited as long as it inhibits the expression of ABH8 gene. For example, (i) a substance that inhibits transcription from ABH8 gene to ABH8 mRNA, And (ii) substances that inhibit translation of ABH8 mRNA into ABH8 protein.

Examples of substances that inhibit transcription from ABH8 gene to ABH8 mRNA include:
(A) an antisense nucleic acid against the ABH8 gene or a part thereof,
(B) a decoy nucleic acid for the ABH8 gene or a part thereof,
(C) ABH8 gene mutants that act dominant negatively on the ABH8 gene or a part thereof, or (d) other transcription-inhibiting compounds.

Examples of substances that inhibit the translation of ABH8 mRNA into ABH8 protein include
(E) a polynucleotide having RNAi action on ABH8 mRNA or a part thereof (eg, siRNA),
(F) an antisense polynucleotide against ABH8 mRNA or a portion thereof;
(G) a polynucleotide having ribozyme activity against ABH8 mRNA or a part thereof, or (h) other translation inhibitory compounds.

  In the present specification, “nucleic acid” means RNA or DNA. The “nucleic acid” herein may contain not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted to.

  In the cancer therapeutic agent of the present invention, a nucleic acid having an action of inhibiting the expression of ABH8 gene by RNAi effect can be used as an active ingredient. RNAi refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, expression of the introduced foreign gene and target endogenous gene are both inhibited. Examples of RNA used herein include double-stranded RNA that causes RNA interference of 19 to 30 bases in length, such as dsRNA (double strand RNA), siRNA (small interfering RNA), or shRNA (short hairpin RNA). . Such RNA can be locally delivered to a desired site by a delivery system such as a liposome, and can be locally expressed using a vector capable of generating the double-stranded RNA. Methods for preparing and using such double-stranded RNA (dsRNA, siRNA or shRNA) are known from many documents (Japanese translations of PCT publication No. 2002-516062; US Publication No. 2002 / 086356A; Nature Genetics, 24 (2), Feb., 180-183; Genesis, 26 (4), April, 240-244; Nature, Spe. 21, 407: 6802, 319-20; Genes & Dev., Vol. ), Apr. 16, 948-958; Proc. Natl. Acad. Sci. USA., 99 (8), 16 Apr., 5515-5520 ;, Science, 296 (5567), 19 Apr., 550-553; Natl. USA, Apr. 30, 99: 9, 6047-6052; Nature Biotechnology, Vol.20 (5), May, 497-500; Nature Biotechnology, Vol.20 (5), May-50; Nucleic Acids Res., May 15, etc.).

  The length of the double-stranded RNA exhibiting the RNAi effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21 to 25 bases, and most preferably 21 to 23 bases.

  In this specification, “antisense nucleic acid” or “antisense polynucleotide” has a polynucleotide complementary to at least a part of a DNA region of interest, and the polynucleotide is at least one of the region. It means a nucleic acid capable of hybridizing with a part. The antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). The antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). They may be double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA, or even a DNA: RNA hybrid. Specific examples of modified nucleic acids include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those that are resistant to degradation of polynucleotide amides and oligonucleotide amides. .

  The antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 'side. The nucleic acid thus prepared can be transformed into a desired animal by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the animal to be transformed, but it is not completely complementary as long as the gene expression can be effectively suppressed. May be.

  For example, designing an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of the ABH8 gene is effective for inhibiting translation of the gene. A sequence complementary to the coding region or 3'-untranslated region can also be used. Antisense nucleic acid effective for inhibiting gene translation is about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more complement to the target gene transcript. Have sex.

  In order to effectively suppress the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is at least about 10 bases (for example, about 10 to 40 bases), preferably about 15 bases or more, More preferably, it is about 100 bases or more, More preferably, it is about 500 bases or more. Antisense nucleic acids can be designed with reference to known literature (for example, Hirashima and Inoue, Shinsei Kagaku Kogaku Lecture 2, Replication and Expression of Nucleic Acid IV Gene, edited by the Japanese Biochemical Society, Tokyo Chemical Dojin, 1993, p.319) -347), J. et al. Kawakami et al. Pharm Tech Japan. Vol. 8, p. 247, 1992; Vol. 8, p. 395, 1992; T.A. Crooke et al. , Ed. , Antisense Research and Applications, CRC Press, 1993, etc.).

  In the cancer therapeutic agent of the present invention, a nucleic acid having a ribozyme activity that specifically cleaves a transcription product of the ABH8 gene can be used as an active ingredient. As used herein, “ribozyme activity” refers to a nucleic acid that specifically cleaves mRNA, which is a transcription product of a target gene. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type or M1 RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Protein Nucleic Acid Enzyme, 1990, 35, p. 2191). For the hammerhead ribozyme, see, for example, FEBS Lett, 1988, 228, p. 228; FEBS Lett, 1988, 239, p. 285; protein nucleic acid enzyme, 1990, 35, p. 2191; Nucl Acids Res, 1989, 17, p. 7059 and the like can be referred to. For hairpin ribozymes, see, for example, Nature, 1986, 323, p. 349; Nucl Acids Res, 1991, 19, p. 6751; Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, p. 112 and the like can be referred to. By specifically cleaving the transcript of the ABH8 gene in the present invention using such a ribozyme, the expression of the gene can be inhibited.

  Furthermore, the present invention can use compounds other than nucleic acids that inhibit the transcriptional activity of the ABH8 gene as active ingredients. Such a compound is, for example, a compound that binds to a factor involved in the expression / transcription of the ABH8 gene. Such a compound may be a natural product or a synthetic compound. Such a compound can be obtained by a screening method described later.

(Cancer therapeutic agent containing ABH8 protein activity inhibitor)
In another embodiment, the present invention also provides a cancer therapeutic agent containing an ABH8 protein of the present invention, particularly a human ABH8 protein activity inhibitor.

In the present specification, “ABH8 protein activity inhibitor” includes, for example,
(A) an antibody that binds to the ABH8 protein,
(B) an ABH8 protein variant having a dominant negative property with respect to the ABH8 protein, or (c) a compound that binds to the ABH8 protein (excluding the above antibody and variant)
Etc. are included.

  As used herein, “antibody” means an antibody that reacts with the full length or fragment of a protein. The form of the antibody of the present invention is not particularly limited, so long as it binds to the ABH8 protein of the present invention, in addition to the above polyclonal antibody and monoclonal antibody, a human antibody, a humanized antibody by genetic recombination, an antibody fragment thereof, Antibody modifications are also included. Antibodies that bind to ABH8 protein (anti-ABH8 antibodies) can be prepared by methods known to those skilled in the art. Details of the anti-ABH8 antibody will be described later.

  The term “ABH8 protein mutant having a dominant negative property with respect to ABH8 protein” in the present specification means a function of eliminating or reducing the activity of endogenous wild-type ABH8 protein by expressing a gene encoding the same. (See, for example, Kunihiro Tsuchida, Gene Activity Inhibition Experiment, edited by Yoshikazu Tahira, Yodosha (2001) 26-32).

  Furthermore, in the present invention, as a substance capable of inhibiting the activity of ABH8 protein, a compound other than the above antibody or mutant that binds to ABH8 protein can be used as an active ingredient. Such compounds are, for example, compounds that bind to ABH8 protein and inhibit its activity. Such a compound may be a natural product or a synthetic compound. Such a compound can be obtained by a screening method described later.

  The substance capable of inhibiting the activity of the ABH8 protein of the present invention described above can be used as a cancer therapeutic agent.

3. The screening method of the substance which inhibits the activity or expression of ABH8 protein This invention also provides the screening method of the candidate compound which has a cancer suppression effect.

  One preferred embodiment is a method using the binding between the ABH8 protein and the test compound as an index. Usually, a compound that binds to ABH8 protein is expected to have an effect of inhibiting the activity of ABH8 protein. Here, the compound preferably binds to the active site of ABH8 protein. In this method, first, ABH8 protein and a test compound are contacted. The ABH8 protein can be, for example, a purified form of the ABH8 protein, a form expressed intracellularly or extracellularly, or a form bound to an affinity column, depending on the indicator for detecting binding to the test compound. . The test compound used in this method can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.

  In this method, the binding between the ABH8 protein and the test compound is then detected.

  There is no restriction | limiting in particular as a test compound used for this method. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts Products, plant extracts and the like, but are not limited thereto.

  The binding between the ABH8 protein and the test compound can be detected by, for example, a label attached to the test compound bound to the ABH8 protein. In addition, a change in the activity of the ABH8 protein caused by the binding of the test compound to the ABH8 protein expressed inside or outside the cell can also be detected as an index. The binding activity between a protein and a test compound can be measured by a known method (for example, Sullivan, F.X., et al. (1998) J. Biol. Chem. 273, 8193-8202; Ohyama, (1998) J. Biol. Chem. 273, 14582-14587; Noda, K., et al. (2003) Cancer Res. 63, 6282-6289).

  In this method, a test compound that binds to the ABH8 protein and inhibits its activity is then selected.

  The compound isolated by this method is expected to have a cancer suppressing action and is useful as a cancer therapeutic agent.

  Another embodiment of the screening method of the present invention is a method using the expression of ABH8 gene as an index. In this method, first, a test compound is brought into contact with cells expressing the ABH8 gene. Examples of the origin of the “cell” used include, but are not limited to, cells derived from humans, mice, cats, dogs, cows, sheep, birds, pets, livestock, and the like. As the “cell expressing the ABH8 gene”, a cell expressing the endogenous ABH8 gene or a cell into which the exogenous ABH8 gene has been introduced and expressing the gene can be used. A cell in which an exogenous ABH8 gene is expressed can be usually prepared by introducing an expression vector into which the ABH8 gene has been inserted into a host cell. The expression vector can be prepared by general genetic engineering techniques.

  The test compound used in this method is not particularly limited. For example, a natural compound, an organic compound, an inorganic compound, a protein, a single compound such as a peptide, a compound library, an expression product of a gene library, a cell Extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts and the like are used.

  The “contact” of the test compound to the cell expressing the ABH8 gene is usually performed by adding the test compound to the culture medium of the cell expressing the ABH8 gene, but is not limited to this method. When the test compound is a protein or the like, “contact” can be performed by introducing a DNA vector expressing the protein into the cell.

  In this method, the expression level of the ABH8 gene is then measured. Here, “gene expression” includes both transcription and translation. The gene expression level can be measured by methods known to those skilled in the art. For example, mRNA can be extracted from cells expressing the ABH8 gene according to a conventional method, and the transcription level of the gene can be measured by performing Northern hybridization or RT-PCR using this mRNA as a template. Alternatively, the promoter region of the ABH8 gene is isolated according to a conventional method, and a downstream of the labeled gene (for example, a gene that can be detected by using luminescence, fluorescence, color development, etc. of luciferase, GFP, galactosidase, etc. as an index, is limited The transcription level of the gene can also be measured by observing the activity of the marker gene. Alternatively, the translational level of the gene can be measured by collecting the protein fraction from cells expressing the ABH8 gene and detecting the expression of the ABH8 protein by electrophoresis such as SDS-PAGE. Furthermore, it is also possible to measure the translation level of a gene by detecting the expression of the protein by performing Western blotting using an antibody that binds to the ABH8 protein. The antibody used for detecting the ABH8 protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.

  In this method, a compound that decreases the expression level is then selected as compared to the case where the test compound is not contacted (control). The compound thus selected becomes a candidate compound for a cancer therapeutic agent.

4). Anti-ABH8 antibody and therapeutic agent, complex and composition containing the antibody The present invention also provides an anti-ABH8 antibody, a cancer therapeutic agent containing the antibody, and the like. In one preferred embodiment of the invention, the cancer therapeutic agent is used for cancer targeted therapy or targeted drug delivery.

(Anti-ABH8 antibody)
In the present specification, the “anti-ABH8 antibody” includes an antibody that specifically binds to the ABH8 protein of the present invention (including a fragment (partial peptide) or a salt thereof). As used herein, an antibody “specifically binds” to a protein or fragment thereof refers to the specific amino acid sequence of these proteins or fragments thereof rather than its affinity for other amino acid sequences. It means binding with substantially high affinity. Here, “substantially high affinity” means high affinity that allows the specific amino acid sequence to be detected separately from other amino acid sequences by a desired measuring device. Has _a binding constant (K _a ) of at least 10 ⁷ M ⁻¹ , preferably at least 10 ⁸ M ⁻¹ , more preferably 10 ⁹ M ⁻¹ , even more preferably 10 ¹⁰ M ⁻¹ , 10 ¹¹ It means a binding affinity such that it is M ⁻¹ , 10 ¹² M ⁻¹ or higher, for example up to 10 ¹³ M ⁻¹ or higher.

The anti-ABH8 antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody. The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification. The term “antibody” used herein is used to include any antibody fragment or derivative. For example, Fab, Fab ′ ₂ , CDR, humanized antibody, multifunctional antibody, single chain antibody (ScFv), etc. including. The antibody of the present invention can be produced by a known method. Methods for producing such antibodies are well known in the art (see, eg, Harlow E. & Lane D., Antibody, Cold Spring Harbor Laboratory Press (1988)).

(1) Preparation of antigen In the present invention, the protein used as the sensitizing antigen is usually the ABH8 protein of the present invention or a salt thereof. The ABH8 protein also includes a partial peptide thereof, which is not limited to, for example, a fragment of the amino acid sequence of SEQ ID NO: 2, for example, 20 or more, 40 or more, 60 As described above, it is a partial peptide having 80 or more and 100 or more consecutive amino acid sequence portions. As these fragments, for example, amino (N) terminal fragments and carboxy (C) terminal fragments are used. In the partial peptide used in the present invention, one or more (preferably about 1 to 10, more preferably several (1 to 6)) amino acid residues in the amino acid sequence are deleted or substituted. , Inserted and / or added. Examples of the salt of ABH8 protein or a partial peptide used herein include salts with inorganic acids (for example, hydrochloric acid and sulfuric acid) or salts with organic acids (for example, acetic acid, formic acid and propionic acid). . The ABH8 protein of the present invention used as a sensitizing antigen for antibody acquisition is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse, primate, or human, and particularly preferably a protein derived from a human. .

(2) Preparation of monoclonal antibody against ABH8 protein (i) Collection of antibody-producing cells ABH8 protein as described above, a partial peptide thereof or a salt thereof (In the present specification, these are collectively referred to as “ABH8 protein. Is administered as an antigen to mammals such as rats, mice, rabbits and the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 1 to 100 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days, preferably 1 to 14 days after the final immunization day. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells, etc., but spleen cells or local lymph node cells are preferred.

(Ii) Cell fusion Cell fusion between antibody-producing cells and myeloma cells is performed to obtain hybridomas. As myeloma cells to be fused with antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells. Those having the following are preferred. Examples of myeloma cells include X63Ag. Examples include mouse myeloma cell lines such as 8.653, NSI / 1-Ag4-1, NS0 / 1, and rat myeloma cell lines such as YB 2/0.

Next, the myeloma cell and the antibody-producing cell are fused. Cell fusion is performed by using 1 × 10 ⁶ to 1 × 10 ⁷ antibody-producing cells and 2 × 10 ⁵ to 2 × 10 ^{6 in} animal cell culture media such as serum-free DMEM and RPMI-1640 media. Cells / ml myeloma cells are mixed (cell ratio of antibody-producing cells to myeloma cells is preferably 2: 1 to 3: 1), and a fusion reaction is performed in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).

(Iii) Selection and cloning of hybridoma The target hybridoma is selected from the cells after cell fusion treatment. As the method, the cell suspension is appropriately diluted with, for example, a fetal bovine serum-containing RPMI-1640 medium, then plated on a microtiter plate at about 3 × 10 ⁵ cells / well, a selective medium is added to each well, and thereafter appropriate. The culture is carried out with the selective medium changed. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas.

  Next, it is screened whether the antibody which reacts with ABH8 protein exists in the culture supernatant of the hybridoma which has propagated. Hybridoma screening is not particularly limited, and may be performed according to ordinary methods. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay, radioimmunoassay, or the like. Cloning of fused cells is performed by the limiting dilution method or the like. Finally, a hybridoma that is a cell producing a monoclonal antibody that reacts with the ABH8 protein is established.

(Iv) Collection of monoclonal antibody As a method for collecting a monoclonal antibody from the hybridoma obtained as described above, a normal cell culture method or ascites formation method can be employed. In the cell culture method, the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium, or serum-free medium under normal culture conditions (for example, 37 ° C., 5% CO ₂ concentration). The cells are cultured for 7 to 14 days, and antibodies are obtained from the culture supernatant. In the case of ascites formation, about 1 × 10 ⁷ hybridomas are administered into the abdominal cavity of a myeloma cell-derived mammal and the same type of animal, and the hybridomas are proliferated in large quantities. And ascites is collected after 1-2 weeks. When antibody purification is required in the antibody collection method, known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography are appropriately selected, or a combination thereof is used. Can be purified.

(3) Preparation of polyclonal antibody against ABH8 protein First, the antigen described above is administered to a mammal such as a rat, mouse, rabbit or the like. The dose of the antigen per animal is 0.1 to 100 mg when no adjuvant is used, and 10 to 1000 μg when an adjuvant is used. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. The immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks. And 6 to 60 days after the last immunization, antibody titer is measured by enzyme immunoassay (ELISA (enzyme-linked immunosorbent assay) or EIA (enzyme immunoassay)), radioimmunoassay (RIA; radioimmunoassay), etc. On the day when the maximum antibody titer is shown, blood is collected to obtain antiserum.

  Next, for example, an antibody (column adsorbed fraction) that reacts with the ABH8 protein is collected by applying a polyclonal antibody in the antiserum to an affinity column fixed with the ABH8 protein. The reactivity of the polyclonal antibody in the antiserum against the ABH8 protein can be measured by an ELISA method or the like.

(4) Antibody Fragment Fab or Fab ′ ₂ fragment can be prepared by digestion with a protease (eg, pepsin or papain) by a conventional method. Humanized antibodies can be obtained by methods such as those described in Riechmann et al. (Riechmann J Mol Biol. Oct 5; 203 (3): 825-8, 1988), and Jones et al. (Jones et al. Nature 321: 522-525, 1986). Can be prepared by one of the following:

  The chimeric antibody is exemplified by “Experimental Medicine (Special Issue), Vol. 1.6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and the like, and the humanized antibody is exemplified by “Nature Genetics”. , Vol.15, p.146-156, 1997 "," Nature Genetics, Vol.7, p.13-21, 1994 ", Japanese translation of PCT publication No. 4-504365, International application publication WO94 / 25585, etc. "Nikkei Science, June issue, pages 40 to 50, 1995", "Nature, Vol. 368, p. 856-859, 1994", JP-T 6-500263, etc. be able to. The antibody that binds to the ABH8 protein of the present invention may be used for the purpose of, for example, suppressing cancer cell growth or metastasis. When the obtained antibody is used for the purpose of administering it to the human body (antibody treatment), a human antibody or a human-type antibody is preferable in order to reduce immunogenicity.

  When used as a diagnostic agent, the antibody may be labeled with a labeling substance (for example, a radioisotope, a fluorescent substance, etc.) for monitoring or the like. If necessary, it can be labeled with a radioactive substance, a fluorescent compound, or the like. Among the most common fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Similarly, the antibody ABH8 antibody can be labeled with a bioluminescent compound. The presence of the bioluminescent protein is measured by detecting the presence of fluorescence. Important bioluminescent compounds for this labeling purpose are luciferin, luciferase and aequorin.

  The antibody of the present invention can be used to specifically detect ABH8 protein or the like present in a subject such as a body fluid or tissue. It can also be used for the production of antibody columns used for purifying ABH8 protein, detection of ABH8 protein in each fraction during purification, analysis of the behavior of ABH8 protein in test cells, etc. it can.

(Complex containing anti-ABH8 antibody, etc.)
Further, the anti-ABH8 antibody used in the present invention may be an agent having neutralizing activity that attenuates the activity of the antigen in the therapeutic agent or diagnostic agent of the present invention. Accordingly, it can be used in combination with other drugs for producing a therapeutic effect. Therefore, in another aspect, the present invention provides an anti-ABH8 antibody for use in targeted therapy or targeted imaging of cancer (eg, breast cancer, bladder cancer, prostate cancer, or colon cancer). And other drugs, compositions containing such complexes, and the like are also provided. According to such an aspect, the anti-ABH8 antibody used in the present invention can be used to deliver another drug having a therapeutic effect or a diagnostic labeling agent to a target site that highly expresses the ABH8 protein. it can.

  Examples of the “other drug” used in the present invention include a viral vector or a non-viral vector for gene transfer to a target, such as a radioisotope, a therapeutic protein, or a small molecule drug.

In the present invention, examples of the “radioisotope” include radioactive halogen elements such as fluorine-18, iodine-125 ( ¹²⁵ I), and iodine-131. These radioactive halogen elements can be widely used as radiotherapeutic agents or radiodiagnostic agents by labeling antibodies and peptides in the same manner as the above-mentioned radioactive metal elements. For example, iodination with ¹²⁵ I or ¹³¹ I can be bound to an antibody or antibody fragment by a known method such as the chloramine T method. Further, as the diagnostic technetium-99m, indium-111 and gallium -67 ⁽⁶⁷ Ga), etc., also yttrium -90 ⁽⁹⁰ Y) is for the treatment, rhenium -186 ⁽¹⁸⁶ Re) or rhenium -188 ⁽¹⁸⁸ Re ) Etc. can be used. When the antibody is labeled with a radioisotope, a metal chelating agent is usually used. Known metal chelating agents include EDTA, DTPA, diaminodithio compounds, cyclam, and DOTA. These chelating agents may be preliminarily bound to the antibody and then labeled with a radioactive metal, or there may be a method in which a radiometal chelate is formed and then bound to the antibody and labeled.

  In the present invention, as examples of “therapeutic proteins”, cytokines that activate cells responsible for immunity are suitable. For example, human interleukin 2, human granulocyte-macrophage-colony stimulating factor, human macrophage colony stimulating factor, Human interleukin 12 etc. are mentioned. Moreover, in order to directly kill cancer cells (for example, breast cancer, bladder cancer, prostate cancer, or colon cancer cells), toxins such as ricin and diphtheria toxin can be used. For example, for a fusion antibody with a therapeutic protein, a cDNA encoding the fusion protein is constructed by linking the cDNA encoding the therapeutic protein to a cDNA encoding the antibody or antibody fragment, and this DNA is used for prokaryotic or eukaryotic organisms. The fusion antibody can be produced by inserting the expression vector into a prokaryotic organism or a eukaryotic organism and expressing the expression vector.

  “Small molecule drug” is used herein to mean a diagnostic or therapeutic compound other than “radioisotope”, “therapeutic protein” and the like. Examples of the “small molecule drug” include alkylating agents such as nitrogen mustard and cyclofasfamide, antimetabolites such as 5-fluorouracil and methotrexate, and antibiotics such as daunomycin, bleomycin, mitomycin C, daunorubicin and doxorubicin. Substances, plant alkaloids such as vincristine, vinblastine, vindesine, anticancer agents such as hormonal agents such as tamoxifen and dexamethasone (Clinical Oncology (1996 Clinical Oncology Society, Cancer and Chemotherapy)), or hydrocortisone, prednisone, etc. Steroids, non-steroids such as aspirin and indomethacin, immunomodulators such as gold thiomalate and penicillamine, immunosuppressants such as cyclophosphamide and azathioprine, chlor maleate And anti-inflammatory agents such as phenylamine and clemacytin (inflammation and anti-inflammatory therapy 1982, Yakuhin Shuppan Co., Ltd.). For example, as a method of binding daunomycin and antibody, a method of binding between daunomycin and the amino group of the antibody via glutaraldehyde, a method of binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carbodiimide, etc. Can be given.

  As an example of “viral vector”, a viral vector modified so that it can bind to the anti-ABH8 antibody of the present invention can be used (for example, adenoviral vector (Wang, P., et al. (1995) Somatic Cell). and Molec. Genet. 21, 429-441), retroviral vectors (Naviaux RK, et al. (1996) J. Virol 70, 5701-5705), lentiviral vectors (Naldini, L. (1998) Curr. Opin.Biotechnol. 9, 457-463)). Such viral vectors include cell growth-related genes, apoptosis-related genes, immunoregulatory genes, etc. at target sites (eg, breast cancer, bladder cancer, prostate cancer, or colon cancer), for example, cancer cell A gene (therapeutic gene) having a therapeutic effect such as inducing apoptosis is incorporated. Viral vectors that bind to anti-ABH8 antibodies can be targeted to sites where antigens recognized by anti-ABH8 antibodies (ie, ABH8) when administered to patients in need of gene therapy with anti-ABH8 antibodies. .

  The anti-ABH8 antibody and the other agent can be combined chemically or genetically. Here, “chemical bond” includes ionic bond, hydrogen bond, covalent bond, bond by intermolecular force, bond by hydrophobic interaction, etc., and “genetic bond” For example, a binding mode between an antibody and a therapeutic protein when a fusion protein composed of an antibody and a therapeutic protein is produced using a technique such as gene recombination is included.

5. Formulation and administration method of the formulation Cancer therapeutic agent containing the ABH8 gene expression inhibitor of the present invention, cancer therapeutic agent containing an ABH8 protein activity inhibitor, therapeutic agent containing the anti-ABH8 antibody of the present invention, Alternatively, the anti-ABH8 antibody used in the present invention may be chemically synthesized with any of a viral vector or a non-viral vector carrying a radioisotope, a therapeutic protein, a small molecule drug, and a therapeutic gene, or any combination thereof. Or the therapeutic agent couple | bonded by genetic engineering can be formulated based on a well-known method.

  In formulating the therapeutic agent of the present invention, a pharmaceutically acceptable carrier can be added as necessary according to a conventional method. For example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, taste masking However, the present invention is not limited thereto, and other commonly used carriers can be appropriately used. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, Examples thereof include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, and inorganic salts.

  Examples of the dosage form of the therapeutic agent of the present invention include tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film coating agents, pellets, and sublingual agents as oral preparations. And pastes such as injections, suppositories, transdermal agents, ointments, plasters, liquids for external use, etc. You can choose. ABH8 protein activity (or ABH8 gene expression) inhibitor as an active ingredient can be contained in the preparation in an amount of 0.1 to 99.9% by weight.

  The dose of the active ingredient of the drug of the present invention varies depending on the administration subject, target organ, symptom, administration method and the like, but in the case of oral administration, for example, generally for a patient (as 60 kg) per day About 0.1 mg to 1,000 mg, preferably about 1.0 to 100 mg, more preferably about 1.0 to 50 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, a patient (for 60 kg), It is convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. However, the final decision can be made as appropriate based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.

  The preparation thus obtained can be administered to, for example, humans and other mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Also in the case of animals other than humans, the amount converted per 60 kg can be administered.

  The therapeutic agent of the present invention is cancer (for example, breast cancer, bladder cancer, prostate cancer, colon cancer, stomach cancer, lung cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, Prevention / treatment of uterine cancer (eg cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc., preferably breast cancer, bladder Used for the prevention and treatment of cancer, prostate cancer, or colorectal cancer.

  Since the drug of the present invention contains an ABH8 protein activity inhibitor or ABH8 gene expression inhibitor as an active ingredient, it can be used as an anticancer agent, cancer metastasis inhibitor, cancer cell apoptosis inducer, and the like. The types of cells, tissues, organs, or cancers of interest are not limited to specific ones. The agent of the present invention may contain both an ABH8 protein activity inhibitor and an ABH8 gene expression inhibitor.

  When an antisense nucleic acid is used in the therapeutic agent of the present invention, the antisense nucleic acid is administered alone or after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., and then administered according to known means. can do. Antisense nucleic acids can be formulated alone or with a physiologically acceptable carrier and administered via a gene gun or a catheter such as a hydrogel catheter.

In the present invention, when a combination of a virus vector such as a recombinant adenovirus particle and an anti-ABH8 antibody is used for cancer treatment, these may be used alone, but are generally pharmaceutically acceptable. Used with a carrier. As such a carrier, a carrier as described above and an aqueous isotonic solution such as water, physiological saline, glucose, human albumin and the like are preferable. Furthermore, additives, preservatives, preservatives, balances and the like that are commonly used in pharmaceutics can also be added. The pharmaceutical composition thus prepared can be administered by an appropriate administration form and administration route depending on the disease to be treated. Examples of the dosage form include emulsions, syrups, capsules, tablets, granules, injections, ointments and the like. When the anti-ABH8 antibody-viral vector particle of the present invention or a pharmaceutical composition containing the same is administered for treatment, it is usually preferable to administer 10 ³ to 10 ¹⁵ viral particles once per adult. Depending on the disease state and the nature of the target cell / tissue, it may be changed. The administration frequency may be once to several times a day, the administration period may be from one day to several months or more, and one set may be one to several injections, and multiple sets may be administered intermittently over a long period of time. Good. Moreover, the viral vector particle or viral vector nucleic acid molecule used in the present invention can be used for detection of specific cells and / or tissues, or diagnosis of disease states. For example, viral vector particles obtained by incorporating a detectable marker gene into a viral vector nucleic acid molecule and transfecting it into an appropriate host cell can be used to detect and diagnose tumor cells in combination with anti-ABH8 antibodies. can do. Alternatively, a detectable label can be bound to the anti-ABH8 antibody and used to detect and diagnose tumor cells.

6). Cancer diagnostic agent, diagnostic kit and diagnostic method The present invention also provides a cancer diagnostic agent. In one preferred embodiment, the diagnostic agent for cancer of the present invention hybridizes under stringent hybridization conditions to (a) an antibody that binds to an ABH8 protein, or (b) an ABH8 gene or a partial nucleotide sequence thereof. A polynucleotide comprising a possible base sequence is contained.

(Diagnostic agent and diagnostic method using anti-ABH8 antibody)
Since the antibody that binds to the ABH8 protein can specifically recognize the ABH8 protein and the like, the ABH8 protein in the test solution can be quantified. Specifically, the diagnostic method using the anti-ABH8 antibody of the present invention includes, for example, (a) a step of bringing a subject-derived biological sample into contact with an antibody that binds to ABH8 protein, and (b) Detecting and / or quantifying the binding between the antibody and the ABH8 protein or a partial peptide thereof or a salt thereof. Preferably, in the detection and / or quantification step, the binding between the ABH8 protein or a fragment thereof and the anti-ABH8 antibody is detected and / or quantified using a labeled anti-ABH8 antibody.

  In the present specification, “subject-derived biological sample” refers to a subject-derived tissue, cell, or body fluid (for example, blood (including whole blood, plasma, serum, etc.), urine, lymph, saliva, sweat, semen, etc.) including. In addition, “subject” is a human subject who usually receives or desires to undergo cancer screening, and includes a human subject suffering from or suspected of having cancer. . Examples of such cancers include breast cancer, bladder cancer, prostate cancer, colon cancer, stomach cancer, lung cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, and uterus. (Eg, cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc., among others, breast cancer, bladder cancer, prostate Cancer or colorectal cancer is preferred.

  An immunoassay for detecting the expression of ABH8 in a biological sample derived from a subject as described above is performed by using a biological sample collected from a subject suspected of having cancer or having a risk of cancer as a specific antigen. -Contacting with an anti-ABH8 antibody under conditions that produce antibody binding and then measuring the amount of immunospecific binding by the antibody. Such antibody binding is used to detect the presence and / or increased expression of ABH8 protein. In this case, detection of increased ABH8 protein expression is an indicator of disease state. As needed, you may compare the level of ABH8 protein in a biological sample with the level of the healthy person who does not have cancer.

  In one embodiment of the immunoassay, for example, a biological sample such as a serum sample is brought into contact with a solid support or carrier such as nitrocellulose for the purpose of immobilizing all proteins present in the sample. The support is then washed with buffer and subsequently treated with detectably labeled anti-ABH8 antibody. The solid support is then washed twice with buffer to remove unbound antibody. The amount of bound antibody on the solid support is measured according to well-known methods. The detection conditions suitable for each measurement can be appropriately determined by those skilled in the art using conventional test methods.

  In one method of detectably labeling an anti-ABH8 antibody, the antibody is conjugated to an enzyme, such as that used in an enzyme immunoassay (EIA) [Voiler, A. et al. "Enzyme-Linked Immunosorbent Assay" (ELISA), 1978, Diagnostic Horizons, 2: 1-7, Microbiological Associates Quarters, Microbiological Association. Pathol., 31: 507-520, 1978: Butier, JE, Meth. Enzymol., 73: 482-523, 1981] Fluorescence measurements by visible means, for example by spectrophotometric measurement of the enzyme bound to the antibody. In a manner that produces a chemical molecule that can be detected by a suitable substrate, preferably a chromogenic substrate. Enzymes that can be used to attach a detectable label to the antibody include, but are not limited to, peroxidase and alkaline phosphatase, which is also a colorimetric method using a chromogenic substrate for the enzyme. Can be achieved.

  Other methods that can be used in the present invention include radioimmunoassay (RIA), sandwich immunoassay, immunometric method, nephrometry, fluorescence immunoassay (FIA), time-resolved fluorescence immunoassay (TRFIA), enzyme immunization. Measurement method (EIA), Luminescence immunoassay (LIA), Electrochemiluminescence immunoassay (ECLIA), Latex agglutination method, Immunoprecipitation assay, Precipitation reaction method, Gel diffusion sedimentation reaction method, Immunodiffusion assay method, Aggregation Examples include an immunoassay selected from the group consisting of an elementary assay, a complement binding assay, an immunoradiometric assay, a fluorescence immunoassay, and a protein A immunoassay (WO 00/14227, page 39). Line 25 to page 42, line 8, EP1111047A2 paragraph [0115] page 19, line 35 to page 20, line 47 Reference).

  As described above, various diseases associated with ABH8 protein dysfunction can be diagnosed by utilizing the in vivo determination method of ABH8 protein using the antibody of the present invention. For example, when an increase in the concentration of ABH8 protein is detected, for example, in a disease caused by overexpression of ABH8 protein (eg, cancer (eg, breast cancer, bladder cancer, prostate cancer, or colon cancer)) It can be diagnosed as likely or likely to be affected in the future.

  The anti-ABH8 antibody of the present invention can also be used for in vivo diagnosis. The preparation and use of antibody preparations that can be used herein are well known in the art. For example, for antibody-chelators, see Nucl. Med. Biol. 1990 17: 247-254. An antibody having a paramagnetic ion as a label used in magnetic resonance imaging is described in, for example, Magnetic Resonance in Medicine 1991 22: 339-342.

(Diagnostic agent and diagnostic method using polynucleotide (eg, DNA) probe)
In the diagnostic method of the present invention, a probe or primer designed based on the base sequence of the ABH8 gene can be used. Specifically, such a diagnostic method includes, for example, (a) a biological sample derived from a subject and a polynucleotide comprising a base sequence capable of hybridizing under stringent hybridization conditions to the base sequence of the ABH8 gene or a fragment thereof. Contacting with a nucleotide (probe), and (b) detecting and / or quantifying the hybridization between the polynucleotide in the sample and the ABH8 gene or a fragment thereof.

  In the method of the present invention, ABH8 gene DNA (or a gene fragment thereof) in a biological sample derived from a subject is detected and / or quantified using the probe. The length of the base sequence used as a probe is, for example, 12 or more bases, 15 or more bases, 18 or more bases, 21 or more bases, 24 or more bases, 27 or more bases, 30 or more bases, or a longer-length polynucleotide fragment. possible. For hybridization, the low, medium or high stringent conditions described above may be used. In the present specification, the “base sequence capable of hybridizing under stringent hybridization conditions to the base sequence of the ABH8 gene or a fragment thereof” includes a base sequence complementary to the base sequence of the ABH8 gene or a fragment thereof ( Antisense polynucleotide) is also included. Methods of probe and nucleic acid hybridization are known to those skilled in the art, for example, WO 89/06698, EP-A0200362, US Pat. No. 2,915,082, EP-A0063879, EP-A0173251, EP -A0128018.

  In the diagnostic method of the present invention, the target sequence can be detected or quantified using a known polynucleotide probe or primer for the ABH8 gene using a known technique. Examples of such known techniques include Southern hybridization, Northern hybridization, RT-PCR method, PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989)), Proceedings of the National Academy of. Sciences of the United States of America, Vol. 86, 2766-2770 (1989)), FISH method, DNA chip or array CGH (Comparative Genomic Hybridization) method, and the like can be used. Quantitative detection can be performed by quantitative RT-PCR.

  The array CGH method is a method in which a chromosomal CGH method (Kallioniemi, A. et al. (1992) Science 258, 818-821) is applied, and genomic DNA fragments (BAC, PAC, YAC, etc.) covering a chromosomal region on a slide. ) And high-density spotted DNA chip, simultaneously hybridize cancer-derived DNA labeled with different dyes and normal DNA to genomic DNA fragments on the slide to detect their binding state. This is a method for detecting an abnormal DNA copy number in cancer with high resolution (Pinkel, D. et al. (1998) Nat. Genet. 20, 207-211).

  In the present invention, in order to detect whether or not the expression of ABH8 gene is up-regulated, the mRNA level of ABH8 in the cell is determined based on the standard gene (housekeeping gene (for example, Shaper, NL et al., J. Mammary Gland Biol. Neoplasmia 3 (1998) 315-324; Wu, Y. Y. and Rees, J. L., Acta Derm. Veneel. 80 (2000) 2-3) and preferably RT- It can also be compared by PCR.

  When the target sequence (DNA, mRNA, etc.) is detected and quantified by the above-described method and overexpression of the ABH8 gene is confirmed, for example, a disease caused by overexpression of ABH8 (for example, cancer (example: Breast cancer, bladder cancer, prostate cancer, or colorectal cancer))), or is likely to be affected in the future.

(Diagnostic method using mass spectrometer)
In another embodiment of the diagnostic method of the present invention, the presence of a target protein or a fragment thereof in a test sample can be identified using a mass spectrometer (MS). That is, by using a mass spectrometer, the amino acid sequence of a target protein or a fragment thereof can be determined, and it can be determined whether ABH8 protein is present in a biological sample derived from a subject. In mass spectrometry, a sample such as a protein or peptide is ionized using MS, separated according to the obtained mass / charge (m / z), and the intensity of the sample is measured to determine the mass of the sample. It is. From the results of mass spectrometry, individual amino acids constituting the amino acid sequence of a protein or peptide can be identified.

  Various methods such as matrix assisted laser desorption ionization (MALDI), electrospray ionization (ESI), gas phase (EI, CI), and field desorption (FD) can be used for ionization. For ion separation, an ion separation method compatible with the ionization method is used. For example, in the case of MALDI, a time of flight (TOF) mass spectrometer, and in the case of ESI, a quadrupole type. Mass spectrometers such as (QMS), ion trap type, and magnetic field type are used. The mass spectrometer may be used in tandem. For example, LC-ESI MS / MS, Q-TOF MS, MALDI-TOF MS, etc. are mentioned. In addition, other amino acid sequence determination methods, for example, an amino acid sequence determination method using a sequencer (eg, a gas phase sequencer) may be used.

(Diagnostic kit)
The present invention also provides a kit for detecting and / or quantifying an ABH8 protein or a fragment thereof in a biological sample of a subject containing an anti-ABH8 antibody as a cancer marker. Furthermore, the ABH8 gene or a fragment thereof in a biological sample derived from a subject, which contains a base sequence capable of hybridizing under stringent hybridization conditions to the ABH8 gene or a part of the base sequence thereof, is detected and / or used as a cancer marker. Alternatively, a kit for quantification is also provided. These kits are used to detect cancer markers by the above-described immunological technique or hybridization method. Examples of such cancers include breast cancer, bladder cancer, prostate cancer, colon cancer, stomach cancer, lung cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, and uterus. (Eg, cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, pancreatic cancer, ovarian cancer, brain tumor, blood tumor, etc., among others, breast cancer, bladder cancer, prostate Cancer or colorectal cancer is preferred.

  In the present specification, the term “cancer marker” refers to a subject's body fluid (eg, blood, urine, lymph, saliva, sweat, semen, etc.) or cells or tissues that are not derived from normal tissues, or Refers to a molecule whose expression is significantly increased in cancer cells or tissues, and the presence of the molecule in the body fluid or cells or tissues of a subject indicates or suggests the presence of cancer.

  The kit according to the first aspect includes a component for detecting and / or quantifying ABH8 antigen (including ABH8 protein and a partial peptide thereof) in a biological sample from a subject. For example, if ABH8 protein is detected and / or quantified by ELISA, such components may be used to detect and / or quantify the level of ABH8 in, for example, tissue sections or body fluid samples such as blood and urine. Can be used. Such antibodies may be labeled with radioactivity, fluorescence, colorimetry, or enzyme label. The kit of the present invention may contain a labeled secondary antibody.

  The kit of the second aspect includes a polynucleotide having a base sequence that can hybridize under stringent hybridization conditions to the ABH8 gene or a part of the base sequence. For example, the kit of the present invention may contain the polynucleotide immobilized on a DNA chip.

  The kit of the present invention may contain a container and a label in addition to a base sequence capable of hybridizing under stringent hybridization conditions to an anti-ABH8 antibody, ABH8 gene or a partial base sequence thereof. A label on or associated with the container may indicate that the drug is used to detect a cancer marker. In addition, other items such as instructions for use may be further included.

(Method of detecting anti-ABH8 autoantibodies using ABH8 antigen)
The present invention also provides a method for detecting and / or quantifying an anti-ABH8 autoantibody in a biological sample as a cancer marker. This method can be used for cancer diagnosis. Furthermore, the progress of cancer can be predicted by monitoring the level of anti-ABH8 autoantibodies. The present invention also provides diagnostic agents and diagnostic kits containing ABH8 antigen for use in such methods.

In the present specification, “ABH8”, “ABH8 protein”, or “ABH8 antigen” used in a mode of using an anti-ABH8 autoantibody in a biological sample as a cancer marker includes not only the ABH8 protein of the present invention, Fragments that contain an epitope of ABH8 protein or that can be recognized by anti-ABH8 antibody as an epitope, and derivatives thereof are also meant to be included. Here, the length of the fragment is not limited as long as it can be recognized as an antigen specific to the anti-ABH8 antibody, but is preferably 6 amino acids or more, more preferably 8 amino acids or more, and even more preferably 10 amino acids. That's it. These degradation products or fragments may be any part of the ABH8 protein, but more preferably correspond to an epitope of the ABH8 protein or include a part corresponding to the epitope. In addition, the “derivative” includes a mutation, substitution, deletion and / or addition of one or several (for example, six) amino acid residues in the amino acid sequence of the ABH8 protein or a fragment thereof. And peptides or polypeptides having the same antigen specificity.
Detection of anti-ABH8 autoantibodies in a biological sample from a subject can be performed in any of a number of ways, but representative methods include immunoassays, such as Western blotting, radioimmunoassay, ELISA, Sandwich immunoassay, fluorescence immunoassay (FIA), time-resolved fluoroimmunoassay (TRFIA), enzyme immunoassay (EIA), luminescence immunoassay (LIA), electrochemiluminescence immunoassay (ECLIA), latex Examples include agglutination, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutinin assay, complement binding assay, immunoradiometric assay, protein A immunoassay, etc. .

  Such immunoassays can be performed in various ways. For example, one method for performing such an assay involves tethering ABH8 protein on a solid support and detecting anti-ABH8 antibodies specific thereto. The ABH8 protein used in the assay of the present invention can be prepared by recombinant DNA techniques well known in the art. For example, the ABH8 protein can be expressed on a large scale by introducing DNA encoding the ABH8 protein into a suitable expression vector by a gene recombination technique. Preferably, a fusion protein capable of facilitating labeling, immobilization or detection of ABH8 is genetically engineered (eg, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N (See the technique described in Y.). Alternatively, ABH8 protein can be purified from natural sources. For example, it is purified from cancer cells using protein separation techniques well known in the art. Such purification techniques include, but are not limited to, molecular sieve chromatography and / or ion exchange chromatography. In practice, a microtiter plate is advantageously used as a solid support for ABH8 protein.

(Diagnostic agent or diagnostic kit using ABH8 antigen)
The diagnostic agent or diagnostic kit using the ABH8 antigen of the present invention contains components necessary for detecting and / or quantifying anti-ABH8 autoantibodies in a biological sample from a subject. For example, when autoantibodies are detected and / or quantified by ELISA, such components include at least one, preferably multiple different ABH8 antigens bound to a solid support or target antigens in the form of their epitopes. And means for detecting anti-ABH8 autoantibodies that bind to the target antigen. Such detection means is, for example, an antibody directed to the constant region of an anti-ABH8 autoantibody (eg, a rabbit anti-human IgG antibody) and is labeled so as to be detectable by itself (eg, radiation) , Fluorescence, colorimetric or enzyme label) or a labeled secondary antibody (eg goat anti-rabbit antibody). These diagnostic agents or diagnostic kits are used for detecting and / or quantifying anti-ABH8 autoantibodies by the immunological technique described above. In addition, the diagnostic kit may include a container and a label. The label on or associated with the container may indicate that the agent is used for detection of anti-ABH8 autoantibodies. In addition, other items such as instructions for use may be further included.

  The above-described assay for detection of ABH8 or assay for detection of anti-ABH8 autoantibodies of the present invention can be a new method that can be used alone for cancer diagnosis and / or prognostic evaluation. It is not limited to such a form, For example, it can further improve the precision of a diagnosis by using in combination with another diagnostic method.

7). Recombinant vector of the present invention, transformant, and method for producing ABH8 protein using the same In another embodiment, the present invention also provides a recombinant vector containing a polynucleotide encoding ABH8 protein of the present invention as an insert. About. Specifically, the present invention has (i) the amino acid sequence of SEQ ID NO: 2, and (ii) at least one amino acid residue deletion, substitution, insertion, and / or addition in the amino acid sequence of SEQ ID NO: 2. There is provided a recombinant vector containing a nucleotide sequence encoding an ABH8 protein consisting of either an amino acid sequence or (iii) an amino acid sequence having 80% identity or more with the amino acid sequence of SEQ ID NO: 2.

  The invention further encodes an ABH8 protein that can hybridize under stringent hybridization conditions to (iv) the nucleotide sequence of SEQ ID NO: 1, or (v) a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising a polynucleotide consisting of any of the nucleotide sequences is provided.

  Here, the nucleotide sequence corresponding to the above (ii), (iii), and (v) preferably has about 97% or more homology with the nucleotide sequence of SEQ ID NO: 1 and encodes ABH8 protein.

  In a preferred embodiment of the invention, the recombinant vector is a recombinant expression vector capable of expressing the insert. Such a recombinant vector can be prepared by any method known in the art.

  In the present invention, the vector used for expressing the ABH8 protein of the present invention includes a cell-free expression system (in vitro transcription-translation) and protein expression using a host cell such as Escherichia coli, yeast or animal cultured cells. Vectors suitable for the system can be used, and such vectors are commercially available or can be easily prepared from known vectors. For example, vectors used for in vitro translation and expression in cultured animal cells include pTargetT vectors and SV40 enhancers that incorporate the immeadiate-early enhancer / promoter region of human cytomegalovirus and have a T7 promoter sequence / multicloning site downstream thereof. And p40 vectors having the early promoter of SV40 (Promega), pBK-CMV, CMV-Script, pCMV-Tag and pBK-RSV (Stratagene). Examples of vectors suitable for expression in microbial hosts such as E. coli and yeast include, for example, pET series vector expression systems (for example, pET3a, pET27b (+) and pET28a (+); Novagen) having an E. coli T7 promoter, and yeast. Examples of such systems include Pichia expression vector pIC series vectors (for example, pPIC9K and PIC3.5K; Invitrogen) having an alcohol oxidase promoter.

  The peptide sequence for purification can be a peptide sequence used in the technical field. For example, a histidine tag sequence having 4 or more, preferably 6 or more histidine residues, Examples include an epitope tag sequence capable of binding to a monoclonal antibody, a glutathione S-transferase binding domain to glutathione, or a sequence encoding protein A. Vectors containing DNA encoding these peptide sequences as inserts are commercially available. For example, vectors used for microbial hosts such as E. coli and yeast have an E. coli T7 promoter, 10 histidine residues fused to the amino terminus (eg, pET16b; Novagen), and 6 histidine residues. An expression vector having an amino acid at the amino terminus (eg, pHB6; Roche Diagnostics, pTrc-His series vector; Invitrogen, pHAT vector series; Clontech). As a vector used for expression in cultured animal cells, a vector (for example, pHM6 or pVM6; Roche Diagnostics) which is fused with 6 histidine residues at the amino terminus can be used. In insect cell lines, a vector that fuses and expresses six baculovirus histidine residues at the amino terminus (eg, pBacPAK-His vector; Clontech). In addition, pGEX series vectors (Pharmacia) fused with glutathione S-transferase, and pRIT2 or pEZZ18 vectors (Pharmacia) expressing protein A can be used.

  In vitro translation to produce the ABH8 protein of the present invention can be accomplished using known methods such as Spirin, AS, et.al., Science 242: 1162-1164 (1988) and Patnaik, R. & Swartz, JM Biotechniques 24 : 862-868 (1998), and a commercially available in vitro translation kit (for example, TNT-in vitro transcription-translation kit, manufactured by Promega) may be used.

  The present invention or a transformant transformed with the above-described recombinant vector (for example, host cells (eg, microorganisms (E. coli, yeast, etc.)) and animal culture cells (insect culture cells, mammalian culture cells (eg, CHO cells, COS7 cells))).

  The present invention also relates to a method for producing ABH8 protein using the recombinant vector of the present invention or the transformant of the present invention. The method for producing the ABH8 protein of the present invention comprises, for example, performing in vitro transcription-translation using the recombinant expression vector of the present invention. Alternatively, the method comprises culturing host cells transformed with the above recombinant expression vector and isolating the desired protein.

  In the method for producing the ABH8 protein of the present invention, the expressed protein can be isolated from the medium or the soluble fraction of the bacterial cells after culturing the host cells.

  In the method for producing the ABH8 protein of the present invention, the expressed protein is isolated and purified by a known method, and the purification method can be appropriately selected from any known method. For example, when the ABH8 protein of the present invention includes the peptide sequence for purification described above, it is preferable to purify using this. Specifically, nickel chelate affinity chromatography for histidine tag sequences, affinity chromatography with glutathione-binding gel for the binding domain of S-transferase to glutathione, antibody affinity for protein A or other protein sequences Chromatographic methods can be used.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely using an Example, the scope of the present invention is not limited by these Examples.

Example 1: Cloning of ABH8 << Experimental Materials >>
The following materials were used as experimental materials.

(1) Human normal pancreas cDNA:
Human Multiple Tissue cDNA (MTC) Panel 1. Human Pancreas First-strand cDNA (BD Bioscience), Lot # 1100541

(2) Primer:
hABH8-F2-TOPO 5'-CACCATGGACAGCAACCATCAAAG-3 '(SEQ ID NO: 3)
hABH8-R2 5'-ATCAGGCCTTTTGAAGAATCA-3 '(SEQ ID NO: 4)
hABH8-F3 5'-AAAAAGTGCAGTGGAAGGA-3 '(SEQ ID NO: 5)
hABH8-F4 5'-TGATGACAGGAGAATCTAGA-3 '(SEQ ID NO: 6)
hABH8-F5 5'-ATTGGATGTGGTAATGGAAAG-3 '(SEQ ID NO: 7)
M13 Forward 5'-GTAAAACGACGGCCAG-3 '(SEQ ID NO: 8)
M13 Reverse 5'-CAGGAAACAGCTATGAC-3 '(SEQ ID NO: 9)

(3) DNA polymerase:
(i) PrimeSTARHS DNA Polymerase Sample (Takara), Cat.No. R010Q
(ii) BigDye (registered trademark) Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems), Cat. No. 4337036

(4) Vector construction kit and plasmid used:
pENTR ^TM Directional TOPO (registered trademark) Clonig Kits (Invitrogen)
pENTR ^TM / D-TOPO (registered trademark) vector (Invitrogen)

(5) DNA purification kit from agarose gel:
Wizard (registered trademark) SV Gel and PCR Clean-Up System (Promega), Cat. No. A9282

"experimental method"
The experiment was performed according to the following procedure.

(1) PCR

  Samples were prepared according to the following composition using human normal pancreatic cDNA as a template, and PCR was performed using a master cycler ep gradient S Manual Lid (Eppendorf). At this time, amplification was performed for 40 cycles at 98 ° C. for 10 seconds, 52 ° C. for 5 seconds, and 72 ° C. for 2 minutes.

(Composition of PCR sample)
Human Pancreas cDNA 1.0
5x Prime STAR ^TM Buffer 5.0
dNTP Mixture (2.5 mM each) 2.0
10μM hABH -F2-TOPO 0.5
10μM hABH -R2 0.5
Prime STAR ^TM HS DNA Polymerase (2.5 U / μl) 0.25
dH ₂ O 15.75
25.0 μl

(2) Cloning
The total amount of the sample after PCR was electrophoresed on a 1% agarose gel, and a single band observed at a position of 2 kbp was excised and purified by Wizard (registered trademark) SV Gel and PCR Clean-Up System. This PCR product was ligated with pENTR ^™ / D-TOPO® using pENTR ^™ Directional TOPO® Clonig Kits, and Top10 (Invitrogen) was transformed to replicate the plasmid. The plasmid was purified by Wizard® Miniprep Kit (Promega). The plasmid thus obtained is hereinafter referred to as pENTR-ABH8.

(3) Sequence analysis
Using pENTR-ABH8 as a template, prepare 5 types of samples each containing one of 5 different primers (hABH8-F3, hABH8-F4, hABH8-F5, M13 Forward, M13 Reverse) according to the composition shown below. PCR was performed using a master cycler ep gradient S Manual Lid. In this case, pre-denaturation was performed at 96 ° C. for 1 minute, and amplification was performed for 25 cycles at 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes (the following operations were performed separately for each sample) ). The sample after PCR was transferred to a 1.5 ml tube, added with 5 μl of 125 mM EDTA and 60 μl of 100% ethanol, vortexed, and left for 15 minutes under light shielding. After centrifugation at 15000 rpm and 4 ° C. for 20 minutes and discarding the supernatant, 60 μl of 70% ethanol was added, and the mixture was centrifuged again at 15000 rpm and 4 ° C. for 15 minutes, and the supernatant was discarded and dried for 2 minutes. Thereto, 25 μl of Hi Di Formamide was added, vortexed, flushed, and incubated at 95 ° C. for 2 minutes. After incubation, the mixture was cooled on ice, flushed, transferred to a sequence tube, and sequence analysis was performed using ABI PRISM (registered trademark) 310 Genetic Analyzer (ABI). The analysis results are shown in FIG.

(Composition of PCR sample)
Ready Reaction Premix 4.0
BigDye Sequencing Buffer 2.0
10μM Primer 0.32
Template (150-300ng)
dH ₂ O to 20.0μl
20.0μl

"Experimental result"
(1) Electrophoretic photograph after PCR FIG. 2 shows an electrophoretic photograph after PCR showing the expression of ABH8 in normal human pancreas.

(2) Results of Sequence Analysis FIG. 3 shows the nucleotide sequence and amino acid sequence of cloned human ABH8 cDNA.

  The sequence of the human ABH8 cDNA after analysis was analyzed for homology with the sequence of the plasmid and the sequence of the human genome. The obtained ABH8 cDNA was 2073 bp (SEQ ID NO: 1), and the ORF was composed of 664 amino acids (SEQ ID NO: 2). ABH8 was also found on chromosome 11 and consisted of at least 12 exons (Figure 4).

(3) Structural features of ABH8 FIG. 4 is a schematic diagram of the exon structure and motif domain of ABH8. A motif-domain search of the estimated 664 amino acid sequences of ABH8 revealed an RNA-recognition motif and methyltransferase domain along with an AlkB homologous region.

  As described above, when a primer was designed from the human genome sequence and the ABH8 sequence of Macaca fascicularis and RT-PCR was performed using human pancreatic cDNA, a single band of about 2 kbp could be amplified. The amplified band of about 2 kbp was cut out and ligated to pENTR / D-TOPO, and the base sequence of human ABH8 cDNA in the obtained plasmid was determined. As a result of nucleotide sequence analysis and homology analysis, it was revealed that the obtained human ABH8 cDNA had a high homology of 96% or more with the cynomolgus ABH8 cDNA. From this result, it was determined that the obtained cDNA was human ABH8. . Furthermore, RNA recognition motif and methyltransferase domain were recognized along with AlkB homologous region from the motif / domain search of all 664 amino acid sequences, so human ABH8 can be responsible for methylation and demethylation in the same molecule It was suggested that RNA and RNA might be the target. Therefore, we next examined the expression of ABH8 mRNA in normal human tissues and various human cancer cell lines.

Example 2: Quantitative expression analysis of ABH8 by real-time PCR of human normal tissue << Experimental material >>
The following experimental materials were used.

(1) Human normal organ cDNA:
Human Multiple Tissue cDNA (MTC) Panel 1 and Panel 2 (BD Bioscience)

(2) Primer:
β-actin-F 5′-CTACGAGCTGCCTGACGGC-3 ′ (SEQ ID NO: 10)
β-actin-R 5′-GCCACAGGACTCCATGCCC-3 ′ (SEQ ID NO: 11)
HA058356-F 5'-TGCTGAGACATGAAGGCATTGAG-3 '(SEQ ID NO: 12)
HA058356-R 5'-ACGGCTTGTTAGGTGGCATTAAGA-3 '(SEQ ID NO: 13)

(3) DNA polymerase:
LightCycler FastStart DNA Master SYBR Green I (Roche), Cat. No. 2239264

"experimental method"
The experiment was performed according to the following procedure.

(1) real-time PCR
Using 16 types of human normal organ cDNAs as templates, specific primers for β-actin and ABH8 mRNA expression (β-actin: β-actin-F, β-actin-R, ABH8: HA058356-F, HA058356 -R). Samples were prepared according to the composition shown below, and real-time PCR was performed using a LightCycler (Roche). In this case, β-actin was pre-denatured at 95 ° C. for 10 minutes as a condition, and amplification was carried out for 40 cycles of amplification at 95 ° C. for 15 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 10 seconds. ABH8 was pre-denatured at 95 ° C. for 10 minutes, and subjected to 40 cycles of amplification at 95 ° C. for 10 seconds, 60 ° C. for 10 seconds, and 72 ° C. for 10 seconds.

(Composition of real-time PCR sample)
Human Tissue cDNA 1.0
25mM MgCl ₂ stock solution 0.8
10μM Primer-F 0.5
10μM Primer-R 0.5
LightCycler FastStart DNA Master SYBR Green I 1.0
dH ₂ O 6.2
10.0 μl

"Experimental result"
The expression of ABH8 mRNA was calculated as a ratio to the expression of β-actin together with ABH2 and 3 analyzed for comparison, and the expression in the small intestine was shown as 1 in FIG. Although the expression of ABH8 in human normal organs indicated by red bars is different, it was confirmed in all human normal organs examined. In addition, the expression was similar to that of ABH2 and ABH3. It was also found that the expression level was particularly high in the testis.

  In this way, the expression analysis of ABH8 mRNA in normal human organs has confirmed its expression in all tissues, so that ABH8 is constantly expressed in all normal tissues, protecting tissues and It may have an important role in homeostasis. Since the expression is particularly high in the testis, it may have some role in normal reproduction and development in humans.

Example 3: Expression analysis of ABH8 by human cancer cell line RT-PCR << Experimental material >>
The following experimental materials were used.

(1) Human cancer cell line:
DU145, PC3, LNCap, A549, AGS, HUH7, SW480, HeLa, U2OS, HL60, HO, KLM, PANAC, Paca2, PKI, PK45P (American Type Culture Collection)

(2) Primer:
hABH8-F2-TOPO 5'-CACCATGGACAGCAACCATCAAAG-3 '(SEQ ID NO: 3)
hABH8-R2 5'-ATCAGGCCTTTTGAAGAATCA-3 '(SEQ ID NO: 4)
β-actin-F 5′-CTACGAGCTGCCTGACGGC-3 ′ (SEQ ID NO: 10)
β-actin-R 5′-GCCACAGGACTCCATGCCC-3 ′ (SEQ ID NO: 11)

(3) DNA polymerase:
(i) TaKaRa LA Taq (registered trademark) (Takara), Cat. No. RR002A
(ii) LightCycler FastStart DNA Master SYBR Green I (Roche), Cat. No. 22392
64

(4) Reverse transcriptase:
M-MLV Reverse Transcriptase (Invitrogen), Cat.No. 28025-013

(5) Reagents:
Trizol (Invitrogen)
Chloroform (Wako Pure Chemicals)
pd (T) _12-18 (Amersham Bioscience), Cat.No. 27-7858-01
40 units / μl RNase inhibitor (Wako Pure Chemical Industries)
2 mM dNTP (Applied Biosystems)

"experimental method"
The experiment was performed according to the following procedure.

(1) RNA preparation of various cancer cell lines by APGC method [1] Various cancer cells were peeled off, and each was collected in an Eppendorf tube and centrifuged (all the following operations were performed for each cell line). [2] The supernatant was discarded, 1 ml of Trizol was added, and the cells were well suspended by pipetting and vortexing until no clumps were visible. [3] Leave at room temperature for 5 minutes, add 0.2 ml of chloroform and vortex, then leave at room temperature for 3 minutes. [4] After centrifugation, the supernatant was transferred to a new tube, 0.5 ml of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 minutes. [5] Centrifuge and discard the supernatant, add 1 ml of 80% ethanol, vortex, and then centrifuge again to discard the supernatant. [6] After drying, the product was dissolved in an appropriate amount of dH ₂ O and confirmed by electrophoresis to have no DNA contamination.

(2) cDNA synthesis by reverse transcription [1] 5 μg of RNA was placed in an RNse-free tube, and 1 μl of 0.5 μg / μl pd (T) _12-18 was added. [2] Cold RNase-free dH ₂ O was added and lightly vortexed, incubated at 70 ° C. for 10 minutes, and then ice-cooled. [3] The following reagents were added, vortexed briefly, and incubated at 37 ° C for 60 minutes. [4] The enzyme was inactivated by incubation at 70 ° C. for 15 minutes, and the reaction was stopped on ice.

(Reagent added in [3]: μl)
0.1 M DTT (Invitrogen) 2.5
40 units / μl Rnase inhibitor (Wako Pure Chemical Industries) 0.5
5x First-Strand Buffer (Invitrogen) 5.0
2mM dNTPs (ABI) 6.0
200 units / μl MMLV 1.0

(3) Standardization of cDNA by β-actin Using 16 synthesized cancer cell line cDNAs as templates, samples were prepared according to the following composition, and real-time PCR was performed using LightCycler (Roche). At this time, pre-denaturation was performed at 95 ° C. for 10 minutes, and amplification was carried out for 40 cycles of amplification at 95 ° C. for 15 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 10 seconds. The concentration ratio was calculated from the expression level of β-actin in each cell line analyzed, and diluted according to the concentration of cDNA derived from HL60.

(Composition of real-time PCR sample)
Human Cancer Cell Line cDNA 1.0
25 mM MgCl ₂ stock solution 0.8
β-actin-F 0.5
β-actin-R 0.5
LightCycler FastStart DNA Master SYBR Green I 1.0
dH ₂ O 6.2
10.0 μl

(4) PCR
Using 16 types of human cancer cell line cDNAs standardized with β-actin as templates, samples were prepared according to the following composition, and RT-PCR was performed. At this time, pre-denaturation was performed at 94 ° C. for 2 minutes, and amplification was carried out for 40 cycles of amplification at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 2 minutes. An electrophoretic photograph of each sample after PCR is shown in FIG. 6 of << Experimental Results >>.

(Composition of RT-PCR sample)
Human Cancer Cell Line cDNA 1.0
10x LA PCR (R) Buffer II 2.5
25 mM MgCl ₂ 2.5
dNTP Mixture (2.5 mM each) 4.0
hABH8-F2-TOPO 0.5
hABH8-R2 0.5
TaKaRa LA Taq (registered trademark) (5 units / μl) 0.25
dH ₂ O 13.75
25.0 μl

"Experimental result"
Thus, the expression of ABH8 could be confirmed in all cancer cell lines examined including prostate cancer cell lines (DU145, PC3, LNCap).

Example 4: Intracellular localization of ABH8 and ABH family molecules << Experimental Materials >>
The following experimental materials were used.

(1) Cell culture:
HeLa (ATCC), a human cervical cancer cell line, was cultured in DMEM (Sigma) supplemented with 10% non-immobilized FCS (Nichirei).

(2) Transfection reagent:
TransIT (registered trademark) -HeLa MONSTER (registered trademark) Transfection Kit (Myrus), Cat. No. MIR2904

(3) Vector construction kit and plasmid used:
Gateway (registered trademark) LR Clonase ^TM Enzyme Mix (Invitrogen), Cat. No. 11791-043
pENTR ^TM / D-TOPO (registered trademark) vector (Invitrogen) Cat. No. K2400-20
pcDNA ^TM 6.2 / N-EmGFP-DEST (Invitrogen) Cat. No. V356-20

(4) Reagents:
PBS
(Composition) NaCl 8.0 g, Na ₂ HPO ₄・ 12H ₂ O 2.9 g, KH ₂ PO ₄ 0.2 g, KCl 0.2 g dissolved in dH ₂ O to 1 L, use after autoclaving
Trypsin / EDTA solution
(Composition) Dissolve 0.04 g EDTA, 0.1 g trypsin in 200 ml PBS
Tris-EDTA Buffer (pH 8.0)
(Composition) Tris base (0.6055 g) and EDTA (0.1861 g) were dissolved in 400 ml of dH2O. The pH was adjusted to 8.0 while adding 6 N hydrochloric acid, and the mixture was made up to 500 ml and used after autoclaving.

"experimental method"
The experiment was performed according to the following procedure.

(1) Preparation of cell expression vector of ABH8 [1] Using pENTR-ABH8 as an entry clone and pcDNA ^TM 6.2 / N-EmGFP-DEST as a destination vector, a sample was placed in a 1.5 ml tube according to the composition shown below. Prepared. [2] After tapping, 4 μl of LA Clonase Enzyme Mix was added, vortexed, flushed, and incubated at 25 ° C. for 60 minutes. [3] 2 μl of Proteinase K solution was added and vortexed, flushed, and incubated at 37 ° C. for 10 minutes. [4] Nova Blue (Novagen) was transformed to replicate the plasmid, and the plasmid was purified by Wizard (registered trademark) Miniprep kit (Promega). The plasmid thus obtained is hereinafter referred to as pEGFP-ABH8.

(Sample composition)
Entry clone (100-300ng)
Destination vector (150 ng / μl) 2.0
5x LR Clonase ^TM Reaction Buffer 4.0
Tris-EDTA buffer (pH 8.0) to 16.0 μl

(2) Transfection [1] On the day before transfection, HeLa cells were seeded in a 5 × 10 ⁴ cells / ml / 12-well dish and cultured in a CO ₂ incubator until the cells reached 50-70% (about 24 hr). [2] 100 μl of Opti-MEM (Invitrogen) was added to two 1.5 ml silicon tubes, 2 μl of TransIT (registered trademark) HeLa Reagent was added, vortexed and incubated at room temperature for 5 minutes. [3] Add 1 μg of pEGFP-ABH8 and pcDNA ^™ 6.2 / N-EmGFP-DEST (hereinafter referred to as pEGFP) as a control to separate tubes, pipette, and incubate at room temperature for 5 minutes. Every time). [4] 1 μl of MONSTER Reagent was added and pipetted, and incubated at room temperature for 5 minutes. [5] After adding 1 ml of pre-warmed DMEM (Sigma) and stirring by overturning, the solution was dropped on the cells from which the culture solution had been removed, and the plate was stirred by shaking back and forth and left and right. [6] After 24 hours, the culture medium was replaced with DMEM (Sigma).

(3) Fluorescence observation with EGFP
Fluorescence of ELa of HeLa cells 24 hours after transfection with pEGFP-ABH8 and pEGFP was observed using BZ-8000 (Keyence).

"Experimental result"
In addition to the stained images of HeLa cells transfected with pEGFP-ABH8 and pEGFP, stained images of HeLa cells transfected with other ABH family molecules by the same method are also shown in FIG. 7 for comparison. As a result of observation, in HeLa transfected with pEGFP-ABH8, the fluorescence was biased to the cytoplasm compared to the control, and the nucleus was not stained. Such extreme cytoplasmic localization has not been observed in other ABH family molecules such as ABH2 and 3, suggesting that ABH8 functions as a cytoplasmic protein. Furthermore, it is speculated that there is a possibility that when a tissue suffers some kind of injury, expression is specifically induced and functional expression is accompanied by translocation into the nucleus.

Example 5: Preparation of anti-ABH8 peptide antibody << Experimental material >>
The following experimental materials were used.

(1) ELISA plate:
MULTI WELL PLATE [for ELISA] H Type Plate (Sumilon), Cat. No. MS-8596F

(2) Secondary antibody:
goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology)

(3) ELISA coloring solution
Liquid A: ABTS Peroxidase Substrate (KPL), Product Code 50-64-00
Liquid B: Peroxidase Solution B [H ₂ O ₂ ] (KPL), Product Code 50-65-00

(4) Reagents:
PBS
SKIM MILK

"experimental method"
The experiment was performed according to the following procedure.

(1) Design and synthesis of antigenic peptides
The peptide antigenicity search of the entire amino acid sequence of ABH8 was commissioned to Kitayama Labes Co., Ltd. Among the several candidate sequences obtained as a result of the analysis, the sequence of 511-524 (NKQKSKYLRGNRNS) was determined as the antigenic site, and the peptide with Cys added to the head of this sequence, and the synthesized peptide and carrier protein (IGH) was commissioned to SIGMA Aldrich Japan Co., Ltd.

(2) Production of antiserum The production of antiserum by rabbits using the synthesized antigen peptide was again commissioned to Kitayama Labes Co., Ltd. Rabbits (No.1, No.2) were immunized four times every two weeks from 2005/9/1 to 2005/10/13, starting from immunization, 0 weeks, 4 weeks, 6 weeks, Antibody titers of serum samples collected at the 7th week were measured using ELISA. As a result of the measurement, sufficient antibody titers could be confirmed in the samples of the 6th and 7th weeks, and therefore, the No.1 and No.2 rabbits were all collected at 11 weeks after the start of immunization.

(3) ELISA
[1] The antigenic peptide was adjusted to 1 mM with PBS, diluted to 10 μM, placed in an ELISA plate to 100 μl / well, and incubated overnight at 4 ° C. in a wet box. [2] After each well was washed 3 times, 200 μl / well of SKIM MILK / PBS was added and incubated in a wet chamber at room temperature for 1 hour. [3] After washing each well 3 times, a 1-fold dilution series from 16000 times each serum sample was made with 1% BSA / PBS, added at 100 μl / well, and incubated in a wet box at room temperature for 1 hour. [4] After each well was washed three times, 50 μl / well of secondary antibody diluted 1000-fold with 1% BSA / PBS was added and incubated for 1 hour at room temperature in a wet chamber. [5] After washing each well three times, ELISA coloring solution A and solution B were mixed and stirred at 1: 1, added at 100 μl / well, and incubated for 20 minutes in the dark. [6] Absorbance was measured at 415 nm.

"Experimental result"
As a result of the measurement, a sufficient antibody titer could be confirmed at the 7th week of immunization. Therefore, at the 11th week, the No. 1 rabbit and the No. 2 rabbit were all collected to obtain antiserum. In addition, as shown in the graph, a relatively high antibody titer was obtained with the antiserum obtained from No.1 compared to No.2, so the expression of ABH8 protein was then used for the expression of ABH8 protein. Analyzed by Western blot.

Example 6: Expression analysis of ABH8 protein << Experimental material >>

  The following experimental materials were used.

(1) Cell culture:
DU145 (ATCC), a human prostate cancer cell line, was cultured in RPMI 1640 (Sigma) supplemented with 10% non-immobilized FCS (Nichirei)

(2) Transfection reagent:
TransIT (R) -Prostate Transfection Kit (Miles), Cat. No. MIR2130

(3) Vector construction kit and plasmid used:
Gateway (registered trademark) LR Clonase ^TM Enzyme Mix (Invitrogen), Cat. No. 11791-043
pENTR ^TM / D-TOPO (registered trademark) vector (Invitrogen) Catalog No. K2400-20
pDEST ^TM 26 (Invitrogen) Catalog No. 11809-019

(4) Primary antibody:
Rabbit anti-ABH8 peptide antibody (antiserum) No.1

(5) Secondary antibody:
goat anti-rabbit IgG-HRP (Santa Cruz)

(6) Chemiluminescent reagent
Lumigen ^TM PS-3 detection reagent (Amersham Bioscience), Cat. No. RPN2132V

(7) Reagents:
3% BSA / TBS-T
(Composition) TBS-T: 1 M tris buffer (pH 7.5) 10 ml, 5 M NaCl 30 ml, Tween 20 1 ml was dissolved in dH ₂ O and made up to 1 L.

"experimental method"
The experiment was performed according to the following procedure.

(1) Preparation of cell expression vector for ABH8 [1] Using pENTR-ABH8 as an entry clone and pDEST ^™ 26 as a destination vector, a sample was prepared in a 1.5 ml tube according to the composition shown below. [2] After tapping, 4 μl of LA Clonase Enzyme Mix was added, vortexed, flushed, and incubated at 25 ° C. for 60 minutes. [3] 2 μl of Proteinase K solution was added and vortexed, flushed, and incubated at 37 ° C. for 10 minutes. [4] Nova Blue (Novagen) was transformed to amplify the plasmid, and the plasmid was purified by Wizard (registered trademark) Miniprep Kit (Promega). The plasmid thus obtained is hereinafter referred to as pDEST26-ABH8.

(Sample composition)
Entry clone (100-300ng)
Destination vector (150 ng / μl) 2.0
5x LR Clonase ^TM Reaction Buffer 4.0
Tris-EDTA buffer (pH 8.0) to 16.0 μl

(2) Transformation [1] On the day before transformation, DU145 cells were seeded in 2 × 10 ⁵ cells / 35 Φ dish and cultured in CO ₂ incubater until the cells reached 50-70% (about 24 hr). [2] 200 μl of Opti-MEM (Invitrogen) was placed in a 1.5 ml silicon tube, 6 μl of TransIT (registered trademark) -Prostate Transfection Reagent was added, vortexed and incubated at room temperature for 5 minutes. [3] 2 μg of pDEST26-ABH8 was added to the tube, pipetted, and incubated at room temperature for 5 minutes. [4] 2 μl of Prostate Boost Reagent was added and pipetted, and incubated at room temperature for 5 minutes. [5] After 2 ml of RPMI 1640 (Sigma) warmed in advance was added and stirred by inversion, it was dropped onto the cells except for the culture solution, and the plate was shaken by shaking back and forth and left and right. [6] After 24 hours, the cells were peeled off and the whole amount was re-wound into a 90 Φ dish and further incubated for 48 hours.

(3) Recovery of lysate [1] DU145 cells transfected with pDEST26-ABH8 and wild-type DU145 cells as a control were peeled off and suspended in 1 ml of PBS, and collected in a separate 1.5 ml tube. [2] After centrifuging at 1500 rpm for 2 minutes to remove the supernatant, 200 μl of lysis buffer and 2 μl of protein inhibitor diluted 100-fold were added and incubated on ice for 30 minutes. [3] The supernatant was collected by centrifugation at 1500 rpm for 10 minutes.

(4) Western blot [1] Each collected lysate was electrophoresed using 12% SDS-PAGE. [2] This was transferred to a nitrocellulose membrane (Scraper and Squell) and then blocked with 3% BSA / TBS-T. [3] After washing 3 times with TBS-T, add 10 ml of antiserum containing anti-ABH8 peptide antibody derived from No. 1 diluted 10,000 times as the primary antibody, shake for 1 hour, and again with TBS-T. Washed 3 times. [4] 10 ml of HRP-labeled anti-rabbit IgG antibody (Santa Cruz Biotechnology) diluted 10,000 times as a secondary antibody was added, shaken for 1 hour, and washed 3 times with TBS-T. [5] The antigen-antibody complex was detected using a chemiluminescent reagent (Amersham).

"Experimental result"
FIG. 9 shows Western results of DU145 cells transfected with pDEST26-ABH8 in the left lane, and wild-type DU145 cells in the right lane. As shown in the figure, an ABH8 high expression strain and a wild type DU145 cell were confirmed to have a 82 kDa band close to the estimated mass of ABH8 of 75 kDa.

  In Examples 1 to 6 shown above, the expression levels of ABH8 mRNA in human normal organs and various human cancer cell lines differ from each other, but their expression should be confirmed in all examined organs and cancer cell lines. I was able to. As for expression in normal human organs, since expression can be confirmed in all organs, it was assumed to play an important role in maintaining normal tissues. Moreover, since the degree of expression differs in each organ such as high expression in the testis, it may be considered that each organ plays a tissue-specific role. In addition, analysis of the localization of ABH8 in cells indicated that ABH8 is present in the cytoplasm, suggesting its function as a cytoplasmic protein.

  In Western blot analysis using DU145 cells transfected with ABH8 and wild type DU145 cells, an 82 kDa band close to the estimated mass of ABH8 of 75 kDa could be confirmed in both. This result is consistent with the result of the expression analysis of ABH8 mRNA in human cancer cell lines, and supports the presence of ABH8 functional protein in cells.

Example 7: Immunohistochemical staining of human ABH8 Immunohistochemical staining of human ABH8 was performed using a tissue array.

<Procedure>
All tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned at 4 μm for histopathological analysis. After deparaffinization, the sections were immersed in 1 mM EDTA (pH 8.0) and heated in an autoclave at 120 ° C. for 5 minutes. Sections were incubated with a 1: 3000 dilution of antiserum (concentration 1 μm / ml) at 37 ° C. for 1 hour. The reaction was subsequently visualized using a Histofine SAB-PO® kit (Nichirei) and diaminobenzidine as a dye and counterstained with hematoxylin.

<Result>
The following is represented by cancer type (positive specimen / test specimen).
Breast cancer (26/28), lung cancer (adenocarcinoma 3/13, squamous cell carcinoma 3/16; but both are extremely weak), renal cancer (9/16; normal tubules are stronger), bladder cancer (13 / 20), prostate cancer (26/50), gastric cancer (14/52); strong reaction to normal gland ducts, especially stomach specific glands), colon cancer (33/57; although normal gland ducts are weak), gallbladder Cancer (3/16; very weak), liver cancer (4/8; normal stem cells are stronger).

  From the above results, breast cancer (MMK), bladder cancer (BT), prostate cancer (PK), and colon cancer (colon) seemed to respond relatively to tumors with little background reaction. Representative stained photographs are shown in FIGS.

Example 8: RNAi analysis of ABH8 gene In this example, RNAi analysis was performed on ABH8 gene using breast cancer cell line T47D, prostate cancer cell line DU145, PC3, and colon cancer cell line HCT116, and the phenotype was determined. Analyzed.

<RNAi analysis>
Cell lines were purchased from ATCC and cultured according to the attached protocol. ABH8 siRNA selected a specific 21mer in the ABH8 gene and synthesized siRNA targeting the sequence (consignment synthesis to Japan Bioservice Co., Ltd.).

  For introduction of ABH8 siRNA into cells, Lipofectamine RNAiMAX (Invitrogen) was used, and 10 nM ABH8 siRNA was introduced into the cells according to the attached protocol. As a control, Negative Control siRNA (QIAGEN) was used.

<Quantitative RT-PCR analysis>
The effect of ABH8 siRNA was verified at the mRNA level using a quantitative RT-PCR method. Total RNA was extracted from cells 24 hours after the introduction of ABH8 siRNA using a Micro-to-Midi Total RNA Purification System (Invitrogen) according to the attached protocol. Subsequently, cDNA was synthesized using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the attached protocol.

  Quantitative RT-PCR was performed using this cDNA as a template. Quantitative PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems) according to the attached protocol. The primers used were the following sequences synthesized (commissioned to OPERON).

Primer sequence:
5′-GTTGAGCCATTTGGTCCCATAG-3 ′ (SEQ ID NO: 14)
5′-CTCACGGAACACATGGTAGTAACG-3 ′ (SEQ ID NO: 15)

  As a standard gene for calculating the relative ratio, the expression level of GAPDH was obtained using Glyceraldehyde-3 phosphate dehydrogenase (GAPDH) Control Reagents (Applied Biosystems), and the relative ratio was calculated.

<Measurement of the number of living cells>
After introduction of ABH8 siRNA, the number of viable cells on the 4th day was measured with Wallac 1420 Multilabel / Luminescence Counter ARVO (PerkinElmer) using Alamar Blue (Biosource) according to the attached protocol.

<SiRNA transfection>
First, siRNAs as shown in Table 1 specific to the sequence in the ABH8 gene were synthesized. (Hereinafter referred to as “ABH8siRNAa”).

  After this ABH8siRNAa was transfected into T47D cell which is a breast cancer cell line and DU145 cell which is a prostate cancer cell line, the number of viable cells was measured with a measuring reagent using the number of viable cells on the 4th day.

  The results are shown in FIGS. 11A and 11B. The graph showed the relative amount with respect to Negative Control (NC). As shown, the number of living cells was measured, and as a result, the number of cells was significantly reduced as compared with NC (FIGS. 11A and B). The graph shows the result of N = 2, and the reproducibility is almost ensured.

Next, as shown in Table 2, another siRNA different from ABH8 siRNAa specific to another sequence in the ABH8 gene was synthesized (hereinafter referred to as “ABH8 siRNAb”).

  After transfection of this ABH8 siRNAb into prostate cancer cell line PC3 cells and colon cancer cell line HCT116 cells, the number of viable cells on the 4th day was used to determine the number of viable cells using the measurement reagent. It was shown to. The graph showed the relative amount with respect to Negative Control (NC). As a result, the number of living cells was measured as shown in the figure, and as a result, ABH8siRNAb having a sequence different from ABH8siRNAa specific to ABH8 gene also significantly reduced the number of cells compared to NC. (FIGS. 11C and D). The graph shows the result of N = 2, and the reproducibility is almost ensured.

  At the same time, in order to verify whether ABH8 gene expression was inhibited by ABH8siRNAa and ABH8siRNAb, we measured the expression level of ABH8 gene in cells on the first day after transfection of each siRNA by quantitative RT-PCR. did. FIG. 12 shows the results after transfection of ABH8siRNAa into T47D and DU145, and transfection of ABH8siRNAb into PC3 and HCT116 cells. GAPDH was used as a standard gene. The graph showed the relative amount with respect to NC. As shown, the expression level was significantly decreased in all cell lines compared to NC (FIGS. 12A, B, C, D).

  Therefore, as a result of suppressing the expression of the ABH8 gene by the RNAi effect, it was found that the growth of cancer cells was significantly suppressed in T47D, DU145, PC3, and HCT116 cells.

  From the above, it was suggested that suppression of ABH8 gene function may be effective in treating cancer in breast cancer, prostate cancer, and colorectal cancer.

Example 8: Inhibition of proliferation and induction of apoptosis in DU145 cells by ABH8siRNA We further examined the effect of inhibition of proliferation in DU145 cells by ABH8siRNA using another ABH8siRNA sequence, and verified the induction of apoptosis as a factor of growth inhibition. .

《ABH8 siRNA sequence》
The sequence of ABH8 siRNA used is shown in Table 3 (hereinafter referred to as “ABH8 siRNAc”).

<Control siRNA>
B-Bridge negative control S5C-0600 was used as a control siRNA.

RNAi transfection protocol
The RNAi transfection protocol was as follows.
1. Inoculate 2 × 10 ⁵ cells of DU145 cells (serum (−), antibiotic (−) RPMI 1640) in 1.35 φ dish and incubate at 37 ° C., 5% CO ₂ for 24 hours.
Mix 100 μl of 2.2 μM siRNA with 100 μl of RPMI 1640 of serum (−) and antibiotic (−) ([1]).
3. 4 μl of DharmaFECT ^™ 1 (DHARAMON) and 196 μl of RPMI 1640 of serum (−) and antibiotics (−) are mixed and left at room temperature for 5 minutes ([2]).
4). [1] and [2] are mixed and left at room temperature for 20 minutes ([3]).
5. The culture medium of DU145 cells in culture is changed to RPMI 1640 of serum (+) and antibiotic (−), and [2] is added dropwise and incubated at 37 ° C. with 5% CO ₂ .

《Proliferation assay》
Proliferation assays were performed according to the following protocol.
1. 48 hours after siRNA transfection, the cells are seeded at 1 × 10 ³ cells / well in a 96-well plate (Day 0).
2. The proliferation from Day 1 to Day 6 is measured using WST-1 (DOJINDO) according to the protocol.
3. The growth rate is expressed as a ratio with an OD value of Day1 being 1.

《Apoptosis analysis》
Apoptosis analysis was performed according to the following protocol.
1. Reseeding 48 hours after siRNA transfection (Day 0).
2. The cells of Day 0 and Day 3 are collected, stained according to the protocol using Annexin V-FITC Apoptosis Detection Kit (BioVision), and analyzed with a flow cytometer.

《Caspase assay analysis》
1. The measurement was performed according to the protocol using “Caspase-Glo 3/7 Assay” commercially available from Promega.
2. 48 hours after siRNA transfection, seed 96 × well plate at 1 × 10 ⁴ cells / well.
3. After 24 hours, add 100 μl of Caspase-Glo 3/7 Assay reagent, incubate at room temperature for 1 hour, and measure luminescence with a luminometer. The graph shows the luminous intensity of cells transfected with Control siRNA as 1.

<Result>
FIG. 13 shows the results of the proliferation assay.
As shown in the figure, it was confirmed that proliferation was suppressed in DU145 cells transfected with ABH8siRNAc as compared with control siRNA.

  FIG. 14 shows the results of apoptosis analysis.

  Apoptotic cells were detected at a position negative for Annexin V positive propidium iodide. On Day 0, control siRNA was 3% and ABH8 siRNAc was 4%. In Day 3, control siRNA was 11%, while in ABH8 siRNAc, it was increased to 40%. Thus, it was confirmed that ABH8 siRNAc has an apoptosis-inducing action in DU145 cells.

  FIG. 15 shows the results of the caspase assay analysis. As shown in the figure, it was confirmed that caspase 3/7 activity was increased in DU145 cells transfected with ABH8 siRNAc as compared with the case of using control siRNA. This indicates that apoptosis is induced in DU145 cells transfected with ABH8siRNAc.

  The present invention is useful for diagnosis and treatment of diseases related to the human ABH8 gene (for example, cancer (eg, breast cancer, prostate cancer, bladder cancer, colon cancer)).

The schematic diagram of an ABH family molecule is shown. It is an electrophoresis photograph showing the expression of ABH8 in human normal pancreas. The base sequence and amino acid sequence of the cloned human ABH8 cDNA are shown. It is the schematic diagram of the exon composition of ABH8, and a motif domain. It is a graph which shows the expression of ABH2, ABH3, and ABH8 in a human normal organ. It is an electrophoresis photograph showing the expression of ABH8 in various human cancer cell lines. It is a photograph showing the intracellular localization of ABH8 and other ABH family molecules in HeLa cells. It is a graph which shows the comparison of the antibody titer of the antiserum in each week after immunization (OD in x2048000 is set to 1). It is an electrophoretogram showing Western results of DU145 cells transfected with pDEST26-ABH8 in the left lane and wild type DU145 cells in the right lane. It is a dyeing | staining photograph which shows the result of the immunohistochemical staining of human ABH8. The staining results of tissues of (A) breast cancer, (B) bladder cancer, (C) prostate cancer, and (D) colon cancer are respectively shown. It is a dyeing | staining photograph which shows the result of the immunohistochemical staining of human ABH8. The staining results of tissues of (A) breast cancer, (B) bladder cancer, (C) prostate cancer, and (D) colon cancer are respectively shown. ABH8siRNAa is a breast cancer cell line T47D cell (A), prostate cancer cell line DU145 cell (B), and ABH8siRNAb is a prostate cancer cell line PC3 cell (C), and a colon cancer cell line HCT116 cell ( D) shows the results of measuring the number of viable cells with the measurement reagent using the number of viable cells on the 4th day after transfection. ABH8siRNAa is a breast cancer cell line T47D cell (A), prostate cancer cell line DU145 cell (B), and ABH8siRNAb is a prostate cancer cell line PC3 cell (C), and a colon cancer cell line HCT116 cell ( D) shows the results of measuring the number of viable cells with the measurement reagent using the number of viable cells on the 4th day after transfection. After RTH8 siRNAa was transfected into T47D (A) and DU145 (B), and ABH8 siRNAb was transfected into PC3 (C) and HCT116 cells (D), the expression level of ABH8 gene in the cells one day after quantitative RT-PCR The result of measurement is shown. After RTH8 siRNAa was transfected into T47D (A) and DU145 (B), and ABH8 siRNAb was transfected into PC3 (C) and HCT116 cells (D), the expression level of ABH8 gene in the cells one day after quantitative RT-PCR The result of measurement is shown. It is a graph which shows the growth suppression of AHB8siRNAc introduction | transduction DU145 cell. It is a graph from the flow cytometer analysis which shows the apoptosis induction effect of DU145 cell by ABH8siRNAc. It is a graph which shows the raise of caspase 3/7 activity in ABH8siRNAc introduction | transduction DU145 cell.

Claims (43)

(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1,
(B) a polynucleotide comprising a nucleotide sequence encoding the human ABH8 protein comprising the amino acid sequence of SEQ ID NO: 2,
(C) a polynucleotide comprising a nucleotide sequence which hybridizes under stringent hybridization conditions with a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of (a) or (b) and which encodes an ABH8 protein, or d) A polynucleotide consisting of a nucleotide sequence encoding an ABH8 protein consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2.   The polynucleotide (c) or (d) according to claim 1, comprising a nucleotide sequence having a homology of 97% or more with respect to the nucleotide sequence of the polynucleotide (a) or (b). , Polynucleotides.   A polynucleotide comprising the polynucleotide according to claim 1 or 2. A human ABH8 protein consisting of the amino acid sequence of SEQ ID NO: 2, or an ABH8 protein consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2.   A polypeptide comprising the amino acid sequence of the ABH8 protein according to claim 4.   A cancer therapeutic agent comprising a human ABH8 gene expression inhibitor as an active ingredient. The expression inhibitor of human ABH8 gene is
(A) a nucleic acid having an action of inhibiting the expression of the human ABH8 gene by the RNAi effect,
(B) an antisense nucleic acid for the transcription product of human ABH8 gene or a part thereof,
And (c) a nucleic acid having ribozyme activity that specifically cleaves a transcript of the human ABH8 gene,
The cancer therapeutic agent of Claim 6 containing the substance selected from the group which consists of.   The cancer therapeutic agent of Claim 7 in which the said nucleic acid contains the polynucleotide which has the base sequence of sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, or sequence number 21.   A cancer therapeutic agent comprising an activity inhibitor of human ABH8 protein as an active ingredient. The activity inhibitor of human ABH8 protein is
An antibody that binds to the human ABH8 protein;
The cancer therapeutic agent of Claim 9 containing this.   The cancer therapeutic agent according to any one of claims 6 to 10, wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colon cancer. A method for screening a human ABH8 gene expression inhibitor,
(A) contacting a test compound with a cell expressing the human ABH8 gene;
(B) a method for measuring the expression level of the human ABH8 gene, and (c) a method for selecting a compound that reduces the expression level as compared with the case where the test compound is not contacted. A method for screening for an activity inhibitor of human ABH8 protein, comprising:
(A) contacting human ABH8 protein with a test compound;
(B) a screening method comprising a step of measuring the binding activity between the human ABH8 protein and a test compound, and (c) a step of selecting a compound that binds to the human ABH8 protein.   An antibody that binds to human ABH8 protein.   A cancer therapeutic agent comprising the antibody according to claim 14.   The cancer therapeutic agent according to claim 15, further comprising a vector carrying a radioisotope, a therapeutic protein, a small molecule drug, or a therapeutic gene.   The cancer therapeutic agent according to claim 15 or 16, wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colorectal cancer.   A cancer diagnostic agent comprising the antibody according to claim 14.   The cancer diagnostic agent according to claim 18, wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colorectal cancer.   A cancer diagnostic kit containing the antibody according to claim 14.   The cancer diagnostic kit according to claim 20, wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colorectal cancer.   A method for detecting and / or quantifying ABH8 protein in a biological sample derived from a subject as a cancer marker. (A) contacting a biological sample derived from a subject with an antibody that binds to ABH8 protein; and (b) detecting and / or quantifying the binding between the antibody in the sample and ABH8 protein;
23. The method of claim 22 comprising:   24. The method of claim 22 or 23, wherein the biological sample is a cell or tissue section.   24. The method of claim 22 or 23, wherein the biological sample is whole blood, plasma, serum, or urine. The method according to any one of claims 22 to 24, wherein the binding between the antibody and the ABH8 protein is detected and / or quantified by immunohistochemical staining.   27. A method according to any of claims 22 to 26 for use in the diagnosis of cancer.   28. The method of claim 27, wherein the cancer is breast cancer, bladder cancer, prostate cancer, or colorectal cancer.   A polynucleotide having the base sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.   A cancer therapeutic agent containing a substance that inhibits expression of the ABH8 gene as an active ingredient, comprising a nucleotide sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21 A cancer therapeutic agent containing a nucleotide.   A method for detecting and / or quantifying an anti-ABH8 autoantibody in a biological sample derived from a subject as a cancer marker.   32. The method of claim 31, wherein the biological sample is a cell or tissue section.   32. The method of claim 31, wherein the biological sample is whole blood, serum, plasma, or urine.   34. The method according to any of claims 31 to 33, wherein the anti-ABH8 autoantibody is detected and / or quantified using an ABH8 antigen. 35. The method of claim 34, comprising contacting the biological sample with ABH8 antigen, and detecting and / or quantifying the binding of anti-ABH8 autoantibodies and ABH8 antigen in the biological sample.   36. The detection and / or quantification of claim 35, comprising detecting and / or quantifying the binding of ABH8 to an anti-ABH8 autoantibody using a labeled antibody against the anti-ABH8 autoantibody. Method.   Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, fluorescence immunoassay (FIA), time-resolved fluoroimmunoassay (TRFIA), enzyme immunoassay (EIA) Luminescence immunoassay (LIA), electrochemiluminescence immunoassay (ECLIA), latex agglutination, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutinin assay, complement 37. A method according to any of claims 31 to 36, according to an immunoassay selected from the group consisting of a body binding assay, an immunoradiometric assay, a fluorescence immunoassay, and a protein A immunoassay.   The method according to any one of claims 31 to 37, which is used for diagnosis of breast cancer, bladder cancer, prostate cancer, or colorectal cancer.   A kit for detecting and / or quantifying an anti-ABH8 autoantibody in a biological sample derived from a subject containing an ABH8 antigen as a cancer marker.   40. The kit of claim 39, further comprising a labeled antibody against an anti-ABH8 autoantibody, wherein the antibody is utilized to detect and / or quantify binding between the ABH8 antigen and the anti-ABH8 autoantibody.   Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, fluorescence immunoassay (FIA), time-resolved fluoroimmunoassay (TRFIA), enzyme immunoassay (EIA) Luminescence immunoassay (LIA), electrochemiluminescence immunoassay (ECLIA), latex agglutination, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutinin assay, complement 40. The detection and / or quantification according to an immunoassay selected from the group consisting of a body binding assay, an immunoradiometric assay, a fluorescence immunoassay, and a protein A immunoassay. 40. The kit according to 40.   The kit according to any one of claims 39 to 41, which is used for diagnosis of breast cancer, bladder cancer, prostate cancer, or colorectal cancer.   A cancer diagnostic agent for detecting and / or quantifying an anti-ABH8 autoantibody in a biological sample derived from a subject containing an ABH8 antigen as a cancer marker.

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