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WO2005073374A1 - Nouvelle proteine d’antigène tumorale et son utilisation - Google Patents

Nouvelle proteine d’antigène tumorale et son utilisation Download PDF

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Publication number
WO2005073374A1
WO2005073374A1 PCT/JP2005/001453 JP2005001453W WO2005073374A1 WO 2005073374 A1 WO2005073374 A1 WO 2005073374A1 JP 2005001453 W JP2005001453 W JP 2005001453W WO 2005073374 A1 WO2005073374 A1 WO 2005073374A1
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Prior art keywords
gene
seq
melk
ttk
stk12
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PCT/JP2005/001453
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English (en)
Japanese (ja)
Inventor
Kenji Harada
Takashi Enjoji
Mikio Aoki
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Dainippon Sumitomo Pharma Co., Ltd.
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Priority to JP2005517564A priority Critical patent/JP4721903B2/ja
Publication of WO2005073374A1 publication Critical patent/WO2005073374A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the use of fl sever antigen protein and peptides derived from the protein in the field of cancer immunology. Further, the present invention provides a disease marker useful for diagnosing a cancer disease, a method for screening a substance effective as a drug for preventing, ameliorating or treating a cancer disease by using the disease marker, and a substance obtained by the method. The present invention relates to a prophylactic, ameliorating or therapeutic agent for a cancer disease, comprising as an active ingredient.
  • Topical treatments include “surgery” and “radiation therapy”, and systemic treatments mainly consist of drug treatments, such as oral or intravenous injections of drugs such as anticancer drugs and hormonal drugs.
  • drug treatments such as oral or intravenous injections of drugs such as anticancer drugs and hormonal drugs.
  • systemic treatments mainly consist of drug treatments, such as oral or intravenous injections of drugs such as anticancer drugs and hormonal drugs.
  • drug treatments such as oral or intravenous injections of drugs such as anticancer drugs and hormonal drugs.
  • Herceptin is an antibody drug against the Her2 molecule highly expressed in breast cancer
  • Iressa is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGF-R) highly expressed in lung cancer. The effect is remarkable, and a strong antitumor effect has been reported.
  • CTLs cytotoxic T cells
  • TCRs T cell receptors
  • this tumor antigen peptide or tumor antigen protein as a so-called cancer vaccine has enabled therapies to enhance tumor-specific CTL in the body of tumor patients.
  • tumor antigen protein As a tumor antigen protein, T. Boon et al. First identified a protein named MAGE from human melanoma cells in 1991 (Science, 254: 1643, 1991). Since then, several tumor antigen proteins have been identified, mainly from melanoma cells. A new tumor antigen protein that can be widely applied to adenocarcinoma (such as lung cancer), which has an overwhelming frequency compared to melano, in order to apply tumor antigen proteins and tumor antigen peptides to tumor treatment and diagnosis And the identification of tumor antigen peptides is desired.
  • adenocarcinoma such as lung cancer
  • ASK is a human homologue of Dbf4 identified in Saccharomyces cerevisiae, and was cloned as a molecule that binds to human Cdc7 by a two-hybrid method using human Cdc7. High expression of ASK has been reported in cell lines derived from leukemia, Burkitt's lymphoma, colorectal cancer, and melanoma (Kumagai, H. et al., Mol. Cell. Biol., 19, 5083-5095, (1999)).
  • GPR87 is an EST (expressed sequence tag). GPR87 is a GPCR (G-protein coupled receptor) cloned by genome database search. Its physiological function, including its ligand, is unknown. . , 'CKS1 binds to Skp2 is one component factors of the enzyme (SCF S kp 2 complex Interview bi chitin ligase (E3)) responsible for Yubikichin of p27 K ipl, the p27 K ipl and Sk P 2 It has been shown to enhance binding capacity (Ganoth, D. et al., Nature Cell Biol., 3, 321-324, (2001)). The CKS1 knockout mice (Spruck, C.
  • MELK encoding MELK (maternal embryonic leucine zipper kinase chick 1) has been identified as a gene with a different expression pattern between unfertilized mouse eggs and preimplantation embryos, and has also been identified by random sequences in myeloid leukemia cell lines. claw is-learning (Nagase, T. et al., DM Res., 3, 17-24 . (1996)).
  • MELK Yadenko the 0 ⁇ 1 J than leucine zipper waxy monounsaturated and serine / threonine kinase gloating However, nothing is known about the function.
  • the STK12 gene encoding STK12 was identified as a kinase highly expressed in colon cancer using the PCR screening method (Bischoff, JR et al., EMB0 J., 17, 3052-3065. (1998)).
  • the STK12 gene is involved in chromosome instability, and the expression of the STK12 gene in a cell, such as VW ', induces chromosome number instability and also increases tumorigenicity in vivo, resulting in cancer cells. Involvement in traits has been suggested (0, T. et al., Cancer Res., 62, 5168-5177, (2002)). However, nothing is known about the effect of STK12 on cancer cell proliferation.
  • the TTK (or ESK) gene was cloned as the human MPS1 gene of MPS1 (Monopolar Spindle 1) family. It has been clawed as a molecule involved in replication of the spindle pole body (centrosome) (Winey, M. et al., J. Cel 1 Biol., 114, 745-754. (1991)). In studies using human MPS1, nothing is known about the potential for involvement in checkpoint arrest for microtubule depolymerization, and the relevance to S, ⁇ disease.
  • the present invention relates to a disease marker useful for diagnosing and treating a cancer disease, a method for detecting a cancer disease using the disease marker (gene diagnosis method), and a method for screening a drug effective for preventing, ameliorating or treating the disease. It is intended to provide a drug effective for preventing, ameliorating or treating the disease.
  • a further object of the present invention is to provide a use of the substance (protein) relating to the disease marker and a partial peptide derived therefrom in the field of cancer immunity.
  • the present inventors have conducted intensive studies in order to solve the above problems, and found that genes whose expression levels and Z or expression frequency in cancer tissues are significantly promoted as compared to expression levels in normal tissues was identified.
  • ASKJ Activator of S-phase kinase
  • CKS1 CDC2-associated protein CKS1
  • MELK maternal emnryonic leu cine zipper kinase
  • STK12 serine / threonine protein kinase 12
  • TTK protein kinase (Hereinafter referred to as " ⁇ ” in this book)
  • GPR87 G protein-coupled receptor 87
  • the present inventors have clarified that suppressing the expression of the above gene in a cancer cell line suppresses the growth of the cancer cell line.
  • a protein-derived peptide encoded by the above gene has peptide-specific cytotoxic T cell-inducing ability in IN VIVO.
  • the present inventors have proposed a cancer disease in which ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene, or their expression products (proteins) are specifically found in cancer patients. That these genes are required for cancer cell proliferation and that their expression products (proteins) have activity as novel tumor antigen proteins, and that peptides derived from these tumor antigen proteins are I was convinced that it had activity as a tide.
  • a screening system based on the suppression of the expression of the above genes, and the suppression of the expression or function (activity) of the protein encoded by the gene (ASK, CKS1, MELK, STK12, TTK or GPR87) is a new system. Search for preventive, ameliorating or therapeutic drugs for cancer diseases based on mechanisms ,-Is valid.
  • ASK, CKS1, MELK, STK12, TTK, and GPR87, or peptides derived from these proteins can be used as a cancer vaccine, and are useful for exerting a preventive, therapeutic, or ameliorating effect on tumors such as lung cancer. Since these tumor antigens are molecules required for cancer cell proliferation, they can be used as more promising antigens, which have a low possibility of causing antigen expression loss due to immune escape of cancer cells.
  • the present invention has been completed based on the above findings.
  • the gist of the present invention is as follows.
  • TTK TTK protein kinase gene reversal Bya or GPR87 (G protein-coupled receptor 87) gene, a polynucleotide having at least 15 consecutive nucleotides and Or a disease marker for a cancer disease, which is a polynucleotide complementary to the polynucleotide,
  • a method for detecting a cancer disease comprising the following steps (a), (b) and (c):
  • step (c) Judgment of the occurrence of a cancer disease in step (c) indicates that the amount of binding of the disease marker is increased when the measurement results obtained for the subject are compared with the measurement results obtained for the normal person.
  • the disease marker according to (5) which is used as a probe in detecting a cancer disease.
  • a method for detecting a cancer disease comprising the following steps (a), (b) and (c):
  • step (c) the determination of the occurrence of a cancer disease is determined by comparing the measurement result obtained for a subject with the measurement result obtained for a normal subject with an increase in the amount of binding to a disease marker as an index.
  • a substance that suppresses the expression of any of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene including the following steps (a), (b) and (c): Screening method:
  • a method for screening for a substance that inhibits the kinase activity of TTK, STK12, MELK or Cdc7 / ASK complex including the following steps (a), (b) and (c):
  • step (b) measuring the phosphorylation amount of the substrate resulting from the step (a), and comparing the phosphorylation amount of the substrate with the phosphorylation amount of the substrate occurring in the absence of the test substance;
  • a method for screening a substance that inhibits the activity of GPR87 including the following steps (a), (b) and (c):
  • a prophylactic, ameliorating or therapeutic agent for a cancer disease comprising as an active ingredient a substance that suppresses the expression of any of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene;
  • a cancer disease wherein the substance that suppresses the expression of any of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene is obtained by the screening method according to (9).
  • CTL cytotoxic ⁇ cells
  • a peptide which is a partial peptide of any of ASK, CKS1, MELK, STK12, TTK and GPR87, and which is recognized by CTL by binding to HLA antigen;
  • amino acid at position 2 in the amino acid sequence of any of SEQ ID NO: 37 to SEQ ID NO: 225 is replaced with tyrosine, phenylalanine, methionine or tryptophan, and the Z or C-terminal amino acid is replaced.
  • the amino acid at the second position in the amino acid sequence described in any of 210 and 222 to 225 is substituted with tyrosine, phenylalanine, methionine or tryptophan, and the amino acid at the Z or C terminal is phenylalanine, leucine, isoleucine.
  • the peptide according to the above (31) which comprises an amino acid sequence substituted with tryptophan or methionine;
  • a CTL inducer comprising the peptide according to any of (27) to (32) as an active ingredient
  • an inducer of CTL comprising a nucleic acid containing a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as any of SEQ ID NOs: 13 to 18,
  • a disease marker for a cancer disease As described above, according to the present invention, a disease marker for a cancer disease, a detection system for the disease, a substance that suppresses the expression of any of the ASK gene, the GPR87 gene, the CKS1, the MELK gene, the STK12 gene, and the TTK gene Screening system, ASK, CKS1, MELK, STK12, TTK and GPR87, a screening system of substances that suppress the expression, function or activity of any of them, and prevention and improvement of cancer diseases using these substances as active ingredients And treatment An agent is provided.
  • an inducer of CTL containing ASK, GPR87, CKS1, MELK, STK12 and TTK, and peptides derived therefrom as active ingredients.
  • the ASK gene, the CKS1 gene, the MELK gene, the STK12 gene, the TTK gene, and the GPR87 gene have significantly increased expression levels and expression frequencies in tissues of cancer patients. In addition, it is based on the finding that suppressing the expression of these genes suppresses cancer cell growth.
  • these genes and their expression products [proteins, (poly) (oligo) peptides] can be effectively used for elucidation, diagnosis, prevention and treatment of cancer diseases. Useful information and means can be obtained. Furthermore, detection of the expression of the above-mentioned gene or its expression product or detection of mutation of the gene or insufficient expression thereof in an individual (living tissue) can be effectively used for elucidation and diagnosis of a cancer disease.
  • these genes suppress the expression of ASK, CKS1, MELK, STK12, TTK, or GPR87 genes.
  • Is useful for screening substances that suppress the expression level, function or activity of ASK, CKS1, MELK, STK12, TTK or GPR87, and the substances obtained by the screening are useful for preventing, ameliorating and treating cancer diseases Is effective as Further, antisense nucleic acids (antisense polynucleotides) and siRNAs of these genes are useful as preventive, ameliorating and therapeutic agents for cancer diseases.
  • ASK, CKS1, MELK, STK12, TTK, and GPR87, and peptides derived therefrom can be used as a CTL inducer, and thus are useful as preventive, ameliorating and therapeutic agents for cancer diseases.
  • FIG. 1 shows the results of examining the transfection efficiency of the siRNA used in Example 8.
  • a bright-field image (Phase-contrast) of each human lung cancer cell line (H0P62, H0P92, PC-8, 11-18) using a phase-contrast microscope and a ⁇ -field image (Fluoresce nce), and an image (Merge) obtained by superimposing the phase difference and the fluorescence are shown from left to right.
  • FIG. 2 shows the results of examining the ability of the tumor antigen peptide used in Example 14 to induce peptide-specific cytotoxic T cells (CTL).
  • the abscissa indicates the tumor antigen peptide evaluated (the katakana indicates the amino acid sequence number of the first residue of the peptide sequence), and the ordinate indicates the number of spots / 2 ⁇ 10 6 cells. .
  • FIG. 3 shows the results obtained by examining the ability of the tumor antigen peptide used in Example 14 to induce peptide-specific cytotoxic T cells (CTL), as in FIG.
  • the abscissa indicates the tumor antigen peptide evaluated (the amino acid sequence number in the first residue of the peptide sequence is shown in parentheses), and the ordinate indicates the number of spots / 2 ⁇ 10 6 cells.
  • the term “gene” or “DNA” is used to include not only double-stranded DNA but also single-stranded DNAs comprising the sense strand and antisense strand.
  • the length is not particularly limited. Therefore, in the present specification, a gene (DNA) refers to a double-stranded DNA including human genomic DNA and a single-stranded DNA including cDNA (positive strand) and a sequence complementary to the positive strand unless otherwise specified. Single-stranded DNA (complementary strand), and any of these fragments.
  • the gene or DNA does not matter which functional region is used, but may include, for example, an expression control region, a coding region, an exon, or an intron.
  • the term “gene” or “DNA” includes not only the “gene” or “DNA” having the specific nucleotide sequence shown in any of SEQ ID NOs: 1 to 12, but also the protein encoded by them. Proteins with similar functions (eg, homologs (homologs, Splice variants), mutants and derivatives).
  • the “gene” or “DNA” encoding such a homologue, mutant or derivative, specifically, under the stringent conditions described in the section (1-1) described below the aforementioned SEQ ID NO: "Gene” or “DNA” having a base sequence that hybridizes with the complementary sequence of the specific base sequence shown in any one of 1 to 12 can be mentioned.
  • a gene encoding a homolog of a human-derived protein a gene of another species such as mouse and rat corresponding to the human gene encoding the protein can be exemplified.
  • These genes can be identified by HomoloGene (http: //ww.ncbi.nlm.nih.gov/HomoloGene/).
  • HomoloGene http: //ww.ncbi.nlm.nih.gov/HomoloGene/.
  • the specific human nucleotide sequence is subjected to BLAST (Proc. Natl. Acad. Sci.
  • the human ASK gene represented by the specific nucleotide sequence (SEQ ID NO: 1) or a cognate thereof is used. It is used to include genes (DNA) that encode the body, mutants and derivatives. Specifically, the human ASK gene described in SEQ ID NO: 1 (GenBank Accession No. N_006716) and the mouse ASK gene described in SEQ ID NO: 7 which is a mouse homolog thereof (GenBank Accession No. N_013726) Etc. are included.
  • the human CKS1 gene represented by the specific nucleotide sequence (SEQ ID NO: 2) or its cognate It is used to include genes (DNA) encoding the body, mutant, derivative, and the like.
  • SEQ ID NO: 2 GenBank Accession No. NM—001826
  • mouse homolog the mouse CKS1 gene described in Tori Column No. 8 and the like.
  • MELK gene and “DNA of MELK” are also used unless specifically stated otherwise, unless otherwise specified, the human MELK gene (DM) represented by the specific nucleotide sequence (SEQ ID NO: 3) or a cognate thereof. It is used for the purpose of including genes (DNA) encoding the body, mutant, derivative and the like. Specifically, the human MELK gene of SEQ ID NO: 3
  • the human STK12 gene represented by the specific nucleotide sequence (SEQ ID NO: 4) It is used to include genes (DNA) encoding homologues, mutants, derivatives, and the like. Specifically, the human STK12 gene described in SEQ ID NO: 4 (GenBank Accession No. NM_004217) and the mouse STK12 gene described in the mouse homologous rooster sequence number: 10 (GenBank Accession No. U69107) Etc. are included.
  • TTK gene or “DNA of ⁇ ”
  • DNA represented by the specific nucleotide sequence (SEQ ID NO: 5) or a cognate thereof. It is used to include genes (DNA) that encode the body, mutants and derivatives.
  • SEQ ID NO: 5 GenBank Accession No. NM_003318
  • mouse TTK gene described in SEQ ID NO: 11 which is a mouse homologue thereof (GenBank Accession No. M86377), etc. Is included.
  • GPR87 gene or “DNA of GPR87”
  • DNA represented by the specific nucleotide sequence (SEQ ID NO: 6) It is used to include genes (DNA) encoding homologues, mutants, derivatives, and the like.
  • SEQ ID NO: 6 GenBank Accession No. NM_023915
  • mouse GPR87 gene described in SEQ ID NO: 12 which is a mouse homolog thereof (GenBank Accession No. ⁇ _032399) Etc. are included Is done.
  • polynucleotide is used to include both RA and DNA.
  • the above DNA includes any of cDNA, genomic DNA and synthetic DNA.
  • the above RA includes all of total RNA, tnRA and synthetic RNA.
  • protein or “(poly) peptide” includes not only “protein” or “(poly) peptide” having a specific amino acid sequence represented by any of SEQ ID NOs: 13 to 24, but also Homologues (homolog ⁇ splice variants), variants, derivatives and amino acid modifications are included as long as their biological functions are equivalent.
  • homologues include proteins of other species, such as mouse rat, which correspond to human proteins.These are obtained by HomoloGene (http: //www.ncbi.nlm.nih.gov/HomoloGene/). It can be determined a priori from the nucleotide sequence of the identified gene (homolog).
  • mutants include naturally occurring allelic variants, non-naturally occurring variants, and variants having an amino acid sequence modified by artificial deletion, substitution, addition and insertion. Included. Examples of such a mutant include those having at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homologous to a protein or (poly) peptide having no mutation. it can.
  • Amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and specifically include phosphorylated amino acids.
  • human ASK represented by the specific amino acid sequence (SEQ ID NO: 13) and homologues, variants, derivatives and amino acids thereof Modified form (MoL Cell. Biol., 20, 5010-5018 (2000), J. Biol. Chera., 276, 993-998 (2001)) and the like.
  • human ASK GenBank Accession No. NP_006707
  • mouse ASK GeneBank Accession No. P-038754
  • CKS1 protein or “CKS1”
  • human CK represented by the specific amino acid sequence (SEQ ID NO: 14)
  • SEQ ID NO: 14 ⁇ SI and its homologues, variants, derivatives and amino acid modifications (Mol. Cell. Biol., 20, 5010-5018 (2000), J. Biol. Chem., 276, 993-998 (2001)) It is used for the purpose including the above.
  • it includes human CKS1 (GenBank Accession No. NP_001817) described in SEQ ID NO: U and mouse CKS1 (GenBank Accession No. NP_058600) described in SEQ ID NO: 20 which is a mouse homologue thereof. You.
  • MELK protein and “MELK” are also used unless specifically stated otherwise, unless otherwise specified, human MELK represented by the specific amino acid sequence (SEQ ID NO: 15), and homologues, variants, and derivatives thereof. And modified amino acids (Mol. Cell. Biol., 20, 5010-5018 (2000), J. Biol. Chem., 276, 993-998 (2001)) and the like.
  • human MELK GenBank Accession No. NP—055 606 described in SEQ ID NO: 15
  • mouse MELK GeneBank Accession No. NP—034920
  • human STK12 represented by the specific amino acid sequence (SEQ ID NO: 16) and homologues, variants, and derivatives thereof And modified amino acids (Mol. Cell. Biol., 20, 5010-5018 (2000), J. Biol. Chem., 276, 993-998 (2001)) and the like.
  • human STK12 GenBank Accession No. NP— 004208 described in Tori Column No .: 16
  • mouse STK12 GenBank Accession No. 070126, described in SEQ ID NO: 22 which is a mouse homolog thereof
  • TTK protein and “ ⁇ ” are also used unless specifically stated otherwise, unless otherwise specified, such as human ⁇ represented by the specific amino acid sequence (SEQ ID NO: 17), and homologues, variants and derivatives thereof. And modified amino acids (Mol. Cell. Biol., 20, 5010-5018 (2000), J. Biol. Chem., 276, 993-998 (2001)) and the like.
  • human TTK GenBank Accession No. NP—003309
  • mouse TTK GeneBank Accession No. AAA37578
  • the term “antibody” includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, and Fab fragments, fragments produced by Fab expression libraries, and the like.
  • the term “disease marker” herein includes a part of the above-mentioned antibodies. The term “disease marker” is used for diagnosing the presence or absence of a cancer disease or the degree of the disease or the degree of improvement or the degree of improvement. It is used directly or indirectly to screen for candidate substances useful for prevention, amelioration or treatment.
  • cancer disease includes, for example, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, colon cancer, rectum cancer, kidney cancer, liver cancer, ovarian cancer, knee cancer, prostate cancer, and gastric cancer.
  • lung adenocarcinoma a lung squamous cell carcinoma, an ovarian cancer, a kidney cancer, more preferably a lung cancer such as a lung adenocarcinoma or a lung squamous cell carcinoma.
  • ⁇ living tissue '' to be diagnosed in the present specification refers to the expression of any one of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene associated with a cancer disease.
  • a rising tissue refers to lung, breast, colon, rectum, kidney, liver, ovary, knee, prostate and stomach.
  • the genes encoding ASK, CKS1, MELK, STK12, TTK, and GPR87 have a significantly higher expression level in cancer tissues and Z or expression frequency than in normal tissues. And further suppress the expression of these genes. Are found to inhibit the growth of cancer cell lines. Therefore, these genes and their expression products [proteins, (poly) (oligo) peptides] can be effectively used for elucidation, diagnosis, prevention and treatment of cancer diseases. Information and means can be obtained.
  • these genes, their expression products, and their derivatives eg, peptides, antibodies, etc.
  • detection of the expression of the above gene or its expression product, or detection of mutation of the gene or insufficient expression thereof can be effectively used for elucidation and diagnosis of cancer diseases.
  • the human-derived ASK gene is a known gene, and its obtaining method is described in Kumagai, H. et al., Mol. Cell. Biol., 19, 5083-5095, (1999). It is known.
  • the human CKS1 gene is also a known gene, and its obtaining method is also known as described in Richardson, HE, et al., Genes Dev., 4, 1332-1344, (1990). ⁇
  • the human MELK gene is also a known gene, and its obtaining method is also known as described in Nagase, T. et al., DNA Res., 3, 17-24. (1996).
  • the human-derived STK12 gene is also a known gene, and its obtaining method is also known as described in Bischof f, JR, et al., EMB0 J., 17, 3052-3065. (1998).
  • the human ⁇ gene is also a known gene, and its obtaining method is also described in Lindberg, RA et al., Oncogene, 8, 351-359. (1993). And Mills, GB et al., J. Biol. Chem., 267, 16000. -16006. Known as described in (1992).
  • the GPR87 gene derived from human is also a known gene, and its obtaining method is also described in Wittenberger T. et al., J. Mol. Biol., 307 (3), 799-813. (2001), and Lee DK et al., Gene , 275 (1), 83-91. (2001).
  • the present invention relates to the expression level of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, or GPR87 gene in the tissue of a patient suffering from a cancer disease as compared to a normal tissue.
  • the presence or absence of the above-mentioned cancer diseases, the degree of illness, and the degree of recovery can be determined by detecting the presence or absence and the degree of expression of these genes. It is based on the idea that it can be specifically detected and the diagnosis of the disease can be made accurately.
  • the present invention provides a useful poly (disease marker) as a tool (disease marker) capable of diagnosing the presence or absence of a cancer disease in a subject by detecting the presence or absence or the degree of the expression of the gene in the subject. It provides nucleotides.
  • polynucleotide is used for screening of ASK gene, CKS1 gene, MELK gene, STK12 in screening of candidate substances useful for prevention, amelioration or treatment of cancer diseases as described in section (3-1) below. It is also useful as a screening tool (disease marker) for detecting changes in the expression of the gene, TTK gene or GPR87 gene.
  • the disease marker of the present invention comprises a polynucleotide having at least 15 consecutive nucleotides in the nucleotide sequence of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene and / or complementary to the polynucleotide. It is characterized by consisting of a typical polynucleotide.
  • the disease marker of the present invention includes the nucleotide sequence of the ASK gene described in SEQ ID NO: 7 or SEQ ID NO: 7, the nucleotide sequence of the CKS 1 gene described in SEQ ID NO: 2 or SEQ ID NO: 8, SEQ ID NO: 3 Or the nucleotide sequence of the MELK gene described in SEQ ID NO: 9, the nucleotide sequence of the STK12 gene described in SEQ ID NO: 4 or SEQ ID NO: 10, the nucleotide sequence of the TTK gene described in SEQ ID NO: 5 or SEQ ID NO: 11, or Examples of the base sequence of the GPR87 gene shown in SEQ ID NO: 6 or SEQ ID NO: 12 include a polynucleotide consisting of a polynucleotide having at least 15 consecutive bases and a polynucleotide complementary thereto.
  • the complementary polynucleotide refers to the full-length sequence of the polynucleotide consisting of the nucleotide sequence shown in each of the above SEQ ID NOs, or at least 15 consecutive nucleotides in the nucleotide sequence.
  • the subsequence having the sequence means a polynucleotide which is in a basic complementary relationship.
  • such a complementary strand is not limited to a case where it forms a completely complementary sequence with the base sequence of the target positive strand, but is of sufficient complementarity that it can hybridize with the target positive strand under stringent conditions. They may have a relationship.
  • the stringent conditions are as follows, as described in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, there can be usually mentioned conditions of about “1 X SS (:, 0.1% SDS, 37 ° C.).
  • a more severe hybridization condition is about 0.5 X SSC, 0.1% SDS, 42 ° C, and a more severe hybridization condition is Washing conditions of about 0.1 X SSC, 0.1% SDS, and about 65 ° CJ can be mentioned.
  • a complementary strand is completely complementary to the base sequence of the target positive strand.
  • the polynucleotide on the positive strand side includes not only those having the nucleotide sequence of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene, or a partial sequence thereof, but also those of the complementary strand.
  • a strand consisting of a base sequence having a more complementary relationship to the sequence can be included.
  • Each of the above-mentioned positive-strand polynucleotide and complementary-strand (reverse-strand) polynucleotide may be used as a disease marker in a single-stranded form, or may be used as a disease marker in a double-stranded form.
  • the disease marker for a cancer disease of the present invention may specifically be a polynucleotide consisting of the nucleotide sequence (full-length sequence) of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene. Alternatively, it may be a polynucleotide comprising its complementary sequence. Further, the ASK gene or a polynucleotide derived from the gene, the CKS1 gene or a polynucleotide derived from the gene, the MELK gene or a polynucleotide derived from the gene, the STK12 gene or a polynucleotide derived from the gene.
  • the above-described full-length sequence or its complement can be used. It may be a polynucleotide consisting of a partial sequence of the sequence. In this case, examples of the partial sequence include a polynucleotide having at least 15 consecutive base lengths arbitrarily selected from the base sequence of the full-length sequence or the complementary sequence thereof.
  • “selectively (specifically) recognizes” means, for example, in the Northern plot method, the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, GPR87 gene, or a gene derived from these genes.
  • the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, GPR87 gene or a polynucleotide derived therefrom is specifically generated.
  • the present invention is not limited thereto, and any method can be used as long as a person skilled in the art can determine that the above-mentioned detected substance or product is derived from these genes.
  • the disease marker of the present invention includes, for example, the ASK gene represented by SEQ ID NO: 1, the CKS1 gene represented by SEQ ID NO: 2, the MELK gene represented by SEQ ID NO: 3, the STK12 gene represented by SEQ ID NO: 4, and the SEQ ID NO: 5 TTK gene, or a nucleotide th d ⁇ 1 J of SEQ ID NO: 6 GPR87 gene represented in the original example _ iprimer 3 (http:.. .. // www genome wi mit edu / cgi-bin / primer / pritner3. cgi) or Vector NTI (manufactured by Inforaax).
  • the primer or probe candidate sequence obtained by applying the nucleotide sequence of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene to primer 3 or vector NTI software, or at least the sequence A sequence contained in one part can be used as a primer or a probe.
  • the disease marker used in the present invention may have a length of at least 15 consecutive nucleotides as described above.Specifically, the length is appropriately selected and set according to the use of the marker. Can be.
  • the detection (diagnosis) of a cancer disease is performed by detecting ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK in a living tissue of a subject, in particular, lung, stomach, breast, prostate, liver, kidney, ovary, knee tissue and the like.
  • the presence or absence of expression of at least one gene and the GPR87 gene This is done by evaluating the current level (expression level).
  • the disease marker of the present invention is used as a primer for specifically recognizing and amplifying RA generated by expression of each of the above genes or a polynucleotide derived therefrom, or as a primer for the RNA or a polynucleotide derived therefrom. It can be used as a probe for specifically detecting nucleotides.
  • a marker having a base length of usually 15 bp to 100 bp, preferably 15 bp to 50 bp, more preferably 15 bp to 35 bp can be exemplified.
  • those having a base length of usually 15 bp-all sequences, preferably 15 bp-lkb, more preferably 100 bp-lkb can be exemplified.
  • the disease marker of the present invention can be used as a primer or a probe according to a conventional method in a known method for specifically detecting a specific gene, such as a Northern blot method, an RT-PCR method, an in situ hybridization method, and the like. .
  • This makes it possible to evaluate the presence or absence or expression level (expression level) of ASK gene, CKS1, MELK gene, STK12 gene, TTK gene or GPR87 gene related to cancer diseases.
  • the sample to be measured can be appropriately selected according to the type of detection method to be used.
  • the sample may be, for example, total RNA prepared by collecting a part of the lung tissue or the like of a subject using a biopsy and the like, and then preparing the same according to a conventional method, or various poly- mers prepared based on the RNA. It may be a nucleotide.
  • the expression level of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene in living tissue can be detected or quantified using a DNA chip.
  • the disease marker of the present invention can be used as a probe of the DNA chip (for example, in the case of Gene Chip Human Genome U95A, B, C, D, E of Affymetrix, a polynucleotide of 25 bp in length) Used as a probe).
  • the disease marker (probe) of the present invention and a labeled DNA or RNA can be obtained.
  • Is formed By detecting the complex using the label of the labeled DNA or RNA as an index, the presence or absence of the expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene can be determined. -Or expression level (expression amount) can be evaluated.
  • the DNA chip only needs to contain one or more disease markers of the present invention that can bind to the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, or GPR87 gene.
  • the use of a DNA chip containing a plurality of disease markers makes it possible to simultaneously evaluate the presence or absence or the expression level of a plurality of genes in one biological sample.
  • the disease marker of the present invention is useful for diagnosis and detection of cancer disease (diagnosis of presence / absence and degree of disease). Specifically, the diagnosis of a cancer disease using the disease marker is performed by measuring the gene expression levels of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, or GPR87 gene in the living tissue of the subject and the living tissue of a normal subject. This can be done by determining the difference.
  • the difference in the gene expression level is not only the difference between the expression and the absence of the expression, but also the difference in the expression level between the subject's living tissue and the normal person's living tissue. It includes the case of 5 times or more, preferably 2 times or more, and more preferably 3 times or more.
  • ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene have a specific increase in the expression level and / or expression frequency in cancer patients, and If the expression level is 1.5 times or more, preferably 2 times or more, and more preferably 3 times or more as compared with the expression level in living tissue of a normal subject, the subject is suspected of having a cancer disease. Be done.
  • the present invention relates to the expression product (protein) of the ASK gene as a disease marker for cancer disease (this is also referred to as “ASK” or “ASK protein” in the present specification), the expression product (protein) of the CKS1 gene (this Herein, also referred to as “CSK1” or “CKS1 protein”), the expression product (protein) of the MELK gene (also referred to as “MELK” or “MELK protein” in the present specification), STK12 Gene expression product (protein) (also referred to herein as “STK12” or “STK12 protein”), TTK gene expression product (protein) (referred to herein as "TTK” or GPR87 gene expression product (protein) (also referred to herein as “GPR87J or“ GPR87 protein ”). Is also specific)
  • the present invention provides an antibody that can be recognized.
  • the antibody include an ASK protein having the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 19, a CKS1 protein having an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 20, SEQ ID NO: 15 or SEQ ID NO: MELK protein having the amino acid sequence of SEQ ID NO: 21; STK12 protein having the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 22; TTK protein having the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 23 Or an antibody that can specifically recognize the GPR87 protein having the amino acid sequence described in SEQ ID NO: 18 or SEQ ID NO: 24.
  • ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, and GPR87 gene are more specific in cancer tissues of cancer patients than in normal cells and tissues.
  • the expression level and Z or the frequency of expression are increasing, the presence or absence of the above-mentioned cancer disease and the degree of the above-mentioned disease can be identified by detecting the presence and degree of expression of these proteins. It is based on the idea that the disease can be accurately detected and the disease can be diagnosed accurately.
  • the antibody can be used as a tool for diagnosing whether or not the subject is suffering from a cancer disease, or a degree of the disease, by detecting the presence or absence or the degree of the expression of the protein in the subject. It is useful as a disease marker.
  • the above antibody can be used for screening any candidate substance useful for the prevention, amelioration or treatment of cancer diseases as described in (3-2) below, by using any of ASK, CKS1, MELK, STK12, TTK and GPR87. It is also useful as a screening tool to detect such expression fluctuations.
  • human-derived ASK is a known protein, and its obtaining method is described in Kumagai, H. et al., Mol. Cell. Biol., 19, 5083-5095, (1999). It is well known.
  • Human-derived CKS1 is a known protein, and its obtaining method is also known as described in Richardson, HE, et al., Genes Dev., 4, 1332-1344, (1990).
  • MELK derived from human is a known protein, and its obtaining method is also known as described in Nagase, T. et al., DNA Res., 3, 17-24. (1996).
  • Human-derived STK12 is a known protein, and its obtaining method is also known as described in Bischoff, JR et al., EMBO J., 17, 3052-3065. (1998).
  • Human-derived ⁇ is a known protein, and its obtaining method is also described in Lindberg, R. A. et al., Oncogene, 8, 351-359 (1993). And Mills, GB et al., J. Biol. Chem., 267. , 16000-16006 (1992).
  • Human-derived GPR87 is a known protein, and its obtaining method is also described in Wittenberger T. et al., J. Mol. Biol., 2001, 307 (3), 799-813, and Lee DK et al., Gene, 2001. , 275 (1): 83-91.
  • the form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody using ASK, CKS1, MELK, STK12, TTK or GPR87 as an immunogen, or a monoclonal antibody thereof. Further, an antibody having an antigen-binding property to a polypeptide consisting of at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids, which is at least contiguous among the amino acid sequences constituting the protein, is also included. Are included in the antibody of the present invention.
  • the antibodies of the present invention can also be produced according to these conventional methods; and 3. (Current protocols in Molecular Biology edit. Au subel et al. (1987) Publish. John Wiley and Sons. Section 11. 12—11. 13).
  • the polyclonal antibody can be obtained using ASK, CKS1, MELK, STK12, TTK or GPR87 protein expressed and purified in E. coli or the like according to a conventional method, or using the ASK, CKS1, MELK, or the like according to a conventional method.
  • an oligopeptide having a partial amino acid sequence of STK12, TTK or GPR87 immunize a non-human animal such as a rabbit, and obtain from a serum of the immunized animal according to a conventional method.
  • a monoclonal antibody is obtained by purifying ASK, CKS1, MELK, STK12, TTK, or GPR87 expressed and purified in E. coli or the like according to a conventional method, or an oligopeptide having a partial amino acid sequence of these proteins in a non-human mouse or the like.
  • An animal is immunized, and the obtained spleen cells and myeloma cells are fused with each other to prepare a hybrid.
  • et al. (1987) Publish. John Wiley and Sons. Section 11.4-1.11.
  • the 87 protein is based on the gene sequence information of any one of SEQ ID NOs: 1 to 12, provided by the present invention, and is used for DNA cloning, construction of each plasmid, transfection into a host, culturing of a transformant and culturing of a transformant. It can be obtained by the operation of recovering proteins from the product. These operations can be performed by methods known to those skilled in the art, methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM.Glover, IRL PRESS (1985)), and the like. It can be performed according to it.
  • a recombinant DNA capable of expressing a gene encoding ASK ⁇ CKS1, MELK, STK12, TTK or GPR87 in a desired host cell is prepared, and this is introduced into a host cell for transformation. After transforming, culturing the transformant and recovering the target protein from the resulting culture, a protein as an immunizing antigen for producing the antibody of the present invention can be obtained.
  • the ASK, CKS1, MELK, STK12, TTK or GPR87 of the present invention have SEQ ID NOS: 13 to
  • the homologue includes an amino acid sequence in which one or more (usually several) amino acids have been deleted, substituted or added in the amino acid sequence of the protein encoded by the above-mentioned gene. And proteins having an immunological activity equivalent to the known function.
  • proteins having the same immunological activity include those that elicit a specific immune response in a suitable animal or its cells, and that are specific for antibodies against ASK, CKS1, MELK, STK12, TTK, or GPR87. Mention may be made of proteins having the ability to bind.
  • the number and location of amino acid mutations in the protein are not limited as long as the immunological activity is maintained. Indices for determining how many amino acid residues should be substituted, inserted or deleted without losing immunological activity can be obtained by computer programs well known to those skilled in the art, for example, DNA Star software. Can be found using For example, the number of mutations is typically within 10% of all amino acids, and Or within 5% of all amino acids, and more preferably within 1% of all amino acids.
  • the amino acid to be substituted is not particularly limited as long as the protein obtained after the substitution retains an immunological activity equivalent to that of ASK, CKS1, MELK, STK12, TTK or GPR87.
  • the amino acid to be substituted is preferably an amino acid having properties similar to those of the amino acid before substitution in terms of amino acid polarity, charge, solubility, hydrophobicity, hydrophilicity, amphiphilicity, etc., from the viewpoint of protein structure retention.
  • Ala, Val, Leu, lie, Pro, Met, Phe and Trp are amino acids classified as non-polar amino acids
  • Gly, Ser, Thr, Cys, Tyr, Asn and Gin are non-charged amino acids.
  • Asp and Glu are amino acids classified as acidic amino acids
  • Lys, Arg and His are amino acids classified as basic amino acids. Therefore, amino acids belonging to the same group can be appropriately selected using these as indices.
  • the antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of ASK, CKS1, MELK, STK12, TTK or GPR87.
  • the oligopeptide used for such antibody induction does not need to have a functional biological activity, but desirably has the same immunogenic properties as the corresponding protein.
  • Oligomers preferably having this immunogenic property and comprising at least 8 contiguous amino acids, preferably 15 amino acids, more preferably 20 amino acids in the amino acid sequence of ASK, CKS1, MELK, STK12, TTK or GPR87 Peptides can be exemplified.
  • an antibody against such an oligopeptide can also be carried out by increasing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, as well as lysolecithin, pull-mouth nickle polyols, polyanion, peptides, oil emulsions, keyhole limpet hemosyanin and dinitrophenol.
  • Surface-active substances such as BCG (Bacillus Calmette-Guerin) and human adjuvants such as Corynebataterum-Panolev.
  • Anti-MELK antibodies Goat anti-Human KIAA0175 Kinase, Serotec Ltd.
  • anti-STK12 antibodies Rabbit anti-Aurora-B, Zymed Laboratories Inc.
  • anti-TTK antibodies which are already commercially available antibodies (Mouse anti-Mpsl, Zymed Laboratories Inc.) Can be used as the antibody of the present invention.
  • the antibody of the present invention has a property of specifically binding to ASK, CKS MELK, STK12, TTK or GPR87, by using the antibody, the above protein expressed in the tissue of a subject (a homolog thereof) ) Can be specifically detected. That is, the antibody is useful as a probe for detecting the presence or absence of the expression of ASK, CKS1, MELK, STK12, TTK or GPR87 in the tissue of a subject, and the degree of the expression.
  • a part of a patient's biological tissue (lung, colon, rectum, kidney, liver, ovary, kidney, stomach, etc.) is partially collected using a biopsy, etc.
  • a detection method such as Western blotting or ELISA
  • the amount of at least one of ASK, CKS1, MELK, STK12, TTK and GPR87 present in the tissue of the subject is compared with the amount of the protein present in the tissue of a normal subject. What is necessary is just to determine the difference.
  • the difference in the amount of the protein includes the case where the difference in the amount of the protein is 2 times or more, preferably 3 times or more, with or without the protein.
  • the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, or GPR87 gene show significant expression levels and increased Z or expression frequency in cancer tissues. If the expression product is present, and it is determined that the amount of the expression product is at least twice, preferably at least three times as large as the amount of the expression product in a normal tissue, it is suspected that a cancer disease will be caused.
  • the present invention provides a method for detecting (diagnosing) a cancer disease using the disease marker of the present invention.
  • the detection method of the present invention is a method of collecting a biological sample of a subject, for example, a part of a tissue such as a lung, a colon, a rectum, a kidney, a liver, an ovary, a kidney, and a stomach by a biopsy or the like.
  • the expression level (expression amount) of at least one of ASK gene, CKS1, MELK gene, STK12 gene, TTK gene and GPR87 gene related to a cancer disease included therein, or ASK, a protein derived from the gene By detecting and measuring at least one protein among CKS1, MELK, STK12, TTK and GPR87, the presence of cancer It detects (diagnoses) nothing or its degree.
  • the detection (diagnosis) method of the present invention detects (diagnoses) the presence or absence or the degree of improvement of the disease, for example, when a therapeutic agent or the like is administered for the improvement of the disease in a cancer patient. You can also.
  • the detection method of the present invention comprises the following steps (a), (b) and (c):
  • the biological sample used here includes a sample prepared from a subject's biological tissue (lung, colon, rectum, kidney, liver, ovary, knee, stomach, etc.). Specific examples include an RA-containing sample prepared from the tissue, a sample further containing a polynucleotide prepared therefrom, and a sample containing a protein prepared from the above-mentioned tissue. Samples containing these RNAs, polynucleotides or proteins can be prepared in accordance with a conventional method, for example, after a part of a living tissue of a subject is collected by biopsy or the like.
  • the diagnostic method of the present invention is implemented as follows, depending on the type of biological sample used as a measurement target.
  • RNA When RNA is used as the measurement target, the detection of a cancer disease can be specifically carried out by a method comprising the following steps (a), (b) and (c):
  • RNA prepared from a biological sample of a subject or a complementary polynucleotide transcribed therefrom, and the disease marker of the present invention ASK gene, CKS1 gene, MELK gene
  • the detection method (diagnosis method) of the present invention detects and measures the expression level of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene in the RNA. It is implemented by.
  • the disease marker of the present invention comprising the aforementioned polynucleotide (ASK gene, CKS1 gene, MELK gene, STK12 gene, polynucleotide having at least 15 bases consecutive in the base sequence of the TTK gene or GPR87 gene and / or Or its complementary polynucleotide) as a primer or probe, and can be carried out by a known method such as Northern blotting, RT-PCR, DNA chip analysis, or in situ hybridization analysis. .
  • the presence or absence of the expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene in RNA can be determined by using the disease marker of the present invention as a probe. Can be detected and measured.
  • the disease marker (complementary strand) of the present invention is first labeled with a radioisotope (32P, 33P, etc .: RI), a fluorescent substance, or the like.
  • the obtained labeled disease marker is hybridized with RNA derived from a living tissue of a subject, which is transferred to a nylon membrane or the like according to a conventional method.
  • the formed duplex of the labeled disease marker (DNA) and RA is converted to a signal derived from the label (RI, fluorescent substance, etc.) of the labeled disease marker using a radiation detector (BAS-1800II, Fujifilm Corporation). ), A method of detecting and measuring with a fluorescence detector and the like. Further, using an AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharamcia Biotech), a disease marker (probe DNA) was labeled according to the protocol and hybridized with RNA derived from a living tissue of the subject. A method of detecting and measuring a signal derived from a marker of a disease marker using a multi-bioimager ST0RM860 (manufactured by Amersham Pharmacia Biotech) can also be employed.
  • a multi-bioimager ST0RM860 manufactured by Amersham Pharmacia Biotech
  • cDNA is prepared from RNA derived from a living body tissue of a subject according to a conventional method, and is used as a type I gene to target ASK, CKS1, MELK, STK12, TTK, or GPR87.
  • a pair of primers prepared from the disease marker of the present invention (a positive strand binding to the above cDNA (-strand) and a reverse strand binding to the + strand) are hybridized with the gene so that the gene region can be amplified.
  • PCR is performed according to a conventional method, and the obtained amplified double-stranded DNA is detected.
  • a method for detecting labeled double-stranded DNA produced by performing the above PCR using primers that have been labeled with RI, fluorescent substance, etc.
  • the double-stranded DNA thus obtained can be transferred to a nylon membrane or the like according to a conventional method, and a method can be used in which a labeled disease marker is used as a probe, hybridized with the probe, and detected.
  • the generated labeled double-stranded DNA product can be measured using an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems).
  • RT-PCR reaction solution was prepared using SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems) according to the protocol, and the reaction mixture was centrifuged with an ABI PRIME 7700 Sequence Detection System (manufactured by Applied Biosystems). Can also be detected.
  • a DNA chip on which the disease marker of the present invention is attached as a DNA probe (single-stranded or double-stranded).
  • the prepared cRNA may be hybridized, and a double strand of the formed DNA and cRNA may be bound to a labeled probe prepared from the disease marker of the present invention for detection.
  • a DNA chip capable of detecting and measuring the gene expression level of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene can also be used as the above DNA chip.
  • Examples of the DNA chip capable of detecting and measuring the expression level of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene include, for example, Gene Chip Human Genome U95 A, B, C, D of Affymetrix. , E. The detection and measurement of the gene expression level of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene in subject RNA using such a DNA chip will be described in detail in Examples.
  • the detection (diagnosis) method of the present invention detects ASK, CKS1, MELK, STK12, TTK or GPR87 in a biological sample. It is implemented by measuring the amount (level). Specifically, the method including the following steps (a), (b) and (c) Therefore, it can be implemented.
  • Western blotting after using the above antibody is an invention disease marker as a primary antibody, that binds to the 1 2 5 radioisotope such as 1, labeled antibody (primary antibody labeled with a fluorescent substance as a secondary antibody Using a labeled antibody), the signal derived from the radioisotope, fluorescent substance, etc. of the obtained labeled conjugate is detected and measured with a radiometer (BAS-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, etc. It can be implemented by doing.
  • a radiometer BAS-1800II: manufactured by Fuji Film Co., Ltd.
  • detection was performed using the ECL Plus Western Blotting Dection System (manufactured by Amersham Pharmacia Biotech) to detect the protocol, and the Manoreti Bio-Imager ST0RM860 was used. (Amersham Pharmacia Biotech).
  • the function or activity of ASK, CKS1, MELK, STK12, TTK or GPR87 to be measured in the above is already known, and the amount of the protein and the functional activity have a certain correlation. Therefore, the cancer disease of the present invention can be detected (diagnosed) also by measuring the function or activity of the protein instead of measuring the amount of the protein. That is, the present invention uses the function or activity of ASK, CKS1, MELK, STK12, TTK or GPR87 as an index, and measures it according to a known method (specifically, see (3-3) below), The present invention also includes a method for detecting (diagnosing) a cancer disease, which comprises evaluating.
  • Diagnosis of cancer disease is based on the biological tissue of the subject (lung, colon, rectum, kidney, liver, ovary, kidney
  • Gene expression level of ASK, CKS1, MELK, STK12, TTK or GPR87 gene, or the amount, function or activity of ASK, CKS1, MELK, STK12, TTK or GPR87 protein in These may be referred to as “protein level” together with the gene expression level or the protein level in the corresponding normal tissue to determine the difference between the two.
  • RNA or protein RNA or protein
  • normal tissues such as cancerous parts can be collected by biopsy, etc., or obtained from tissues collected after death with the consent of the individual.
  • a person who does not suffer from a cancer disease means that the subject has at least no subjective symptoms of the cancer disease, and is preferably diagnosed as a non-cancer disease as a result of another test method, for example, an X-ray test.
  • the “person not suffering from a cancer disease” may be simply referred to as a “normal person” in the present specification.
  • the level of gene expression or protein level between the subject's living tissue and normal living tissue (the living tissue of a person without a cancer disease or the normal non-cancerous tissue of a person with a cancer disease)
  • the comparison can be performed by performing measurements on the subject's biological sample and the normal subject's biological sample in parallel.
  • ASK gene and CKS1 gene obtained by measuring a plurality of (at least 2, preferably 3 or more, more preferably 5 or more) normal living tissues under uniform measurement conditions without performing the measurement in parallel.
  • Gene expression level of MELK, STK12, TTK or GPR87 gene, or average or statistical mean of ASK, CKS1, MELK, STK12, TTK or GPR87 protein level Can be used for comparison.
  • the determination of whether the subject has a cancer disease is based on the expression level of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, or GPR87 gene in the tissue of the subject, or ASK, CKS1 which is an expression product thereof. It can be performed using as an index that the protein level of MELK, STK12, TTK or GPR87 is at least two times, preferably at least three times as high as that of normal subjects. Subject's gene expression level or protein If the quality level is higher than those of normal subjects, the subject can be determined to have a cancer disease or be suspected of having the disease.
  • the expression level or protein level of the MELK gene among the above genes is higher in the lung, kidney, liver, ovary or stomach tissue of the subject than in the corresponding tissue, cancer disease Is suspected. If the gene expression level or protein level of the TTK gene is higher in the subject's lung, colon, rectum, kidney, liver, ovary, knee or stomach tissue than in the corresponding normal tissue, a cancer disease is detected. Suspected. If the gene expression level or protein level of the STK12 gene is high in the subject's lung, liver, ovary or stomach tissue compared to the normal corresponding tissue, a cancer disease is suspected.
  • the gene expression level or protein level of the ASK gene is higher in the lung and rectum tissues of the subject than in the normal corresponding tissues, a cancer disease is suspected. If the gene expression level or the amount of protein of the CKS 1 gene is higher in the lung and ovarian tissues of the subject than in the corresponding normal tissues, a cancer disease is suspected. If the gene expression level of the GPR87 gene or the amount of the protein is higher in the lung, ovary, and knee tissues of the subject than in the normal corresponding tissues, a cancer disease is suspected.
  • the present invention provides a method for screening a substance that suppresses the expression of any of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene, and GPR87 gene.
  • the screening method of the present invention comprises the following steps (a), (b) and (c):
  • Cells used for such screening include cells capable of expressing any of endogenous and exogenous ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene. Gene expression of any of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene can be easily performed by detecting the expression of these genes by the known Northern plot method ⁇ RT-PCR method. Can be confirmed.
  • Such cells include, for example, lung, colon, rectum, kidney, liver, ovary, spleen or stomach in which any of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene can be expressed. Examples include cells obtained from tissues or cells derived from these tissues. Specifically, human lung cancer cell lines, H0P62 cells (Natinal Cancer Institute), H0P92 cells (Natinal Cancer Institute), PC-8 cells and 11-18 cells (Tohoku University Institute of Aging Medicine Cell line: Cat. No. TKG0177).
  • the cells used in the screening method of the present invention also include tissues that are aggregates of cells.
  • test substance candidate substance
  • the test substance (candidate substance) screened by the screening method of the present invention is not limited, and nucleic acids (including antisense polynucleotides of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene), Peptides, proteins, organic compounds, inorganic compounds, and the like.
  • the screening of the present invention involves contacting these test substances or a sample containing them (test sample) with the cells, Z or tissue. It is performed by.
  • the test sample includes, but is not limited to, a cell extract, a gene library expression product, a synthetic low-molecular compound, a synthetic peptide, a natural compound, and a mixture thereof containing the test substance.
  • the conditions under which the test substance is brought into contact with the cells are not particularly limited, but the cells are not killed and any one of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene is used. It is preferable to select culture conditions (temperature, PH, medium composition, etc.) that can express E. coli.
  • the cancer tissues of patients suffering from cancer diseases have ASK, CKS1, MELK, STK12, TTK and GTK genes compared to the corresponding normal tissues.
  • Increase in expression level and Z or expression frequency of PR87 gene is observed.
  • suppressing the expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene suppresses the growth of cancer cell lines.
  • the screening method of the present invention uses a gene expression level of any one of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene as an index, (Returning material).
  • this screening method it is possible to provide a candidate substance that can be used as an active ingredient of a drug for preventing, ameliorating, or treating a cancer disease.
  • the screening method of the present invention provides a drug for preventing or improving cancer diseases by searching for a substance that suppresses the expression of any of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene. It provides a candidate substance to be an active ingredient of a drug or therapeutic agent.
  • test cells when using cells expressing any of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene
  • the expression level of each of the above-mentioned genes in the cells to which the test substance (candidate substance) is not added is lower than that in cells to which the test substance (candidate substance) is not added.
  • a human lung cancer cell line H0P62 cell, H0P92 cell, PC-8 cell or 11-18 cell as a candidate to be an active ingredient of a prophylactic, ameliorating or therapeutic agent for a cancer disease
  • To screen for a substance add a test substance and select candidate substances based on a decrease in the expression level of any of the ASK, CKS1, MELK, STK12, TTK, and GPR87 genes. Can be.
  • the detection and quantification of the expression level of the gene in the screening method of the present invention is carried out by using the RNA prepared from the above-described cells or a complementary polynucleotide transcribed from the RNA, and the disease marker of the present invention.
  • the method can be carried out according to a known method such as a Northern plot method, an RT-PCR method, a method using a DNA chip, or the like.
  • the reduction (suppression / reduction) of 10%, preferably 30%, particularly preferably 50% or more of the expression level in cells can be exemplified.
  • the detection and quantification of the gene expression level of any of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene is performed in the gene region (expression control region) that controls the expression of the gene. It can also be carried out by measuring the activity of a marker gene-derived protein using a cell line into which a fusion gene linked to a marker gene such as a luciferase gene has been introduced.
  • the method for screening a gene expression-suppressing substance of any one of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene of the present invention comprises the steps of: Searching methods are also included. In this sense, the claims
  • ASK gene includes a fusion gene of a gene expression control region and a marker gene.
  • the marker gene is preferably a structural gene of an enzyme that catalyzes a luminescence reaction or a color reaction.
  • Specific examples include the above-mentioned luciferase gene, secretory alkaline phosphatase gene, chloramphenicol 'acetyltransferase gene, e-glucuronidase gene, 0 galactosidase gene, and etaolin gene.
  • the expression control region of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene can be obtained, for example, by the (i) 5'-RACE method (for example, 5, full Race Core Kit (Takara Shuzo) Step of determining the 5 'end by a conventional method such as oligocapping method, S1 primer mapping, etc .; (ii) using Genome Walker Kit (Clontech) And obtaining the 5, -upstream region, and measuring the promoter activity with respect to the obtained upstream region.
  • 5'-RACE method for example, 5, full Race Core Kit (Takara Shuzo) Step of determining the 5 'end by a conventional method such as oligocapping method, S1 primer mapping, etc .
  • Genome Walker Kit Genome Walker Kit
  • Preparation of the fusion gene and measurement of the activity derived from the marker gene can be performed by known methods.
  • the substance selected by the screening method of the present invention can be positioned as a gene expression inhibitor for any of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene. These substances suppress or reduce the expression of any of the ASK, CKS1, MELK, STK12, TTK, and GPR87 genes in vivo, thereby alleviating, suppressing (improving, and treating) cancer diseases. ) Is a potential candidate for a drug.
  • the present invention provides a method for screening a substance that suppresses the expression level of any of ASK, CKS1, MELK, STK12, TTK, and GPR87.
  • the screening method of the present invention comprises the following steps (a), (b) and (c):
  • the cells used in the screening of the present invention express any of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene and GPR87 gene regardless of endogenous or exogenous, and ASK as an expression product
  • Cells having any of CKS1, MELK, STK12, TTK and GPR87 can be mentioned.
  • the expression of any of ASK, CKS MELK, STK12, TTK and GPR87 can be easily confirmed by detecting the gene product protein by a known Western method.
  • Specific examples of the cells include H0P62 cells, H0P92 cells, PC-8 cells, and 11-18 cells, which are the lung cancer cell lines described in the section (3-1).
  • the cells refer to various fractions derived from cells, and include, for example, a cell membrane fraction, a cytoplasmic fraction, a cell nucleus fraction, and the like.
  • the expression and / or expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene in cancer tissues of patients suffering from cancer diseases is higher than in normal tissues. An increase in frequency is observed.
  • ASK Suppression of the expression of the gene, CKS1, MELK, STK12, TTK, or GPR87 gene suppresses cancer cell line growth. From these findings, it is considered that the expression level of ASK, CKS1, MELK, STK12, TTK or GPR87 has a causal relationship with cancer diseases.
  • the screening method of the present invention includes a substance that decreases the expression level of the protein using the protein expression level of any one of ASK, CKS1, MELK, STK12, TTK, and GPR87 as an index (a substance that returns the expression level to normal).
  • a method of searching for is included.
  • the screening method of the present invention searches for a substance that reduces the protein expression level of any of ASK, CKS1, MELK, STK12, TTK and GPR87, and thereby provides an effective drug for preventing, ameliorating, or treating cancer diseases. It provides a candidate substance as a component.
  • candidate substances specifically, when cells that express or produce any of ASK, CKS1, MELK, STK12, TTK and GPR87 are used, cells to which a test substance (candidate substance) has been added ASK, CKS1, MELK, STK12, TTK, or GPR87 protein level (level) in the cell The index is lower than the level (cell level) of cells to which the test substance (candidate substance) is not added. Can be done as
  • a test substance is added, and ASK, ASK according to the screening method of the present invention, which compares the protein production levels (levels) of CKS1, MELK, STK12, TTK, and GPR87, and can select candidate substances using the decrease in the production level (level) as an index.
  • the production amount of any of CKS1, MELK, STK12, TTK and GPR87 can be determined, for example, by using the antibody (eg, human ASK protein or homologue thereof, human CKS1 protein or homologous protein) as the disease marker of the present invention.
  • Human MELK protein or its homologue Human STK12 protein or its homologue, human TTK protein or its homologue or human GPR87 protein or its homologue
  • the antibody can be quantified according to a known method such as a Western plot method using an antibody recognizing the antigen.
  • Western blotting after using the present invention the disease marker one as a primary antibody, a radioactive isotope, such as 1 2 5 1 as a secondary antibody, primary antibody labeled with a fluorescent substance Labeling is carried out using an antibody that binds to the antibody, and signals derived from these labeling substances are measured with a radiometer (BAS-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, or the like.
  • a radiometer BAS-1800II: manufactured by Fuji Film Co., Ltd.
  • test substance candidate substance screened by the screening method of the present invention is not limited, but nucleic acids (including ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene antisense polynucleotide) , Peptides, proteins, organic compounds, inorganic compounds, and the like.
  • the screening of the present invention involves contacting these test substances or a sample containing them (test sample) with the cells or cell fractions described above. Done.
  • the test sample includes, but is not limited to, a cell extract, a gene library expression product, a synthetic low-molecular compound, a synthetic peptide, a natural compound, etc., containing the test substance.
  • the present invention provides a method for screening a substance that suppresses the activity (function) of ASK, CKS1, MELK, STK12, TTK or GPR87.
  • the screening method of the present invention comprises the following steps (a), (b) and (c):
  • (c) a step of selecting a test substance that reduces the activity (function) of ASK, CKS1, MELK, STK12, TTK or GPR87 based on the comparison result of (b) above.
  • any method for measuring the function and activity based on the known function and activity of ASK, CKS1, MELK, STK12, TTK or GPR87 can be used. That is, the known function of ASK, CKS1, MELK, STK12, TTK or GPR87 is added to a test substance in an activity measurement system to suppress or inhibit the known function or activity of ASK, CKS MELK, STK12, TTK or GPR87. Prevent and improve test substances against cancer (malignant tumor) Any screening method selected as a candidate substance having a therapeutic effect is included in the category of the screening method of the present invention.
  • the screening of the present invention can be performed by contacting an aqueous solution containing ASK, CKS1, MELK, STK12, TTK or GPR87, cells or a cell fraction prepared from the cells with a test substance.
  • the aqueous solution containing ASK, CKS1, MELK, STK12, TTK or GPR87 includes, for example, a normal aqueous solution containing ASK, CKS1, MELK, STK12, TTK or GPR87, a cell lysate containing these proteins, and a cell. Examples include a crushed liquid, a nuclear extract, and a cell culture supernatant.
  • examples of the cells used in the screening method of the present invention include cells capable of expressing ASK, CKS1, MELK, STK12, TTK, or GPR87 regardless of endogenous or exogenous.
  • the cells specifically, human cultured primary cells or cell lines derived from human and other animals expressing the gene of the present invention, as described in the above (3-1), may be used. Can be.
  • a transformed cell transformed with an expression vector containing the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene can be used.
  • Examples of host cells used for the transformation include well-known cells such as C0S, CH0, and Sf9.
  • the cell fraction refers to various fractions derived from the above cells, and includes, for example, a cell membrane fraction, a cytoplasmic fraction, a cell nucleus fraction, and the like.
  • ASK, CKS1, MELK, STK12, TTK or GPR87 used in the screening are all known proteins.
  • sequence information of the gene provided by the present invention SEQ ID NOS: 1 to 12
  • the DNA can be obtained by the procedures of DNA cloning, construction of each plasmid, transfection to a host, culture of transformed cells, and, if necessary, recovery of protein from the culture. These procedures can be performed by methods known to those skilled in the art, or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRES S (1985)) and the like. It can be performed according to it.
  • the cancer tissues of patients suffering from cancer are specifically different from the normal corresponding tissues in the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene. Increased gene expression and increased Z or expression frequency . In addition, suppressing the expression of ASK gene, CKS1, MELK gene, STK12 gene, TTK gene or GPR87 gene suppresses cancer cell line growth. From these findings, it is considered that the enhancement of the function (activity) of the expression products (proteins) of these genes is related to cancer (malignant tumor) cell proliferation.
  • the screening method of the present invention includes a method of searching for a substance that suppresses the function (activity) of the protein using the function (activity) of ASK, CKS1, MELK, STK12, TTK or GPR87 as an index.
  • ADVANTAGE OF THE INVENTION According to the screening method of this invention, the substance which suppresses the function or activity of ASK, CKS1, MELK, STK12, TTK or GPR87 can be searched for.
  • Candidate substances providing improved therapeutic effects against cancer are provided. That is, the screening method of the present invention comprises ASK, CKS1, MELK, STK12, TTK or G
  • test substances candidate substances
  • the test substances (candidate substances) screened by the screening method of the present invention are not limited, but include nucleic acids, peptides, proteins (including antibodies against ASK, CKS1, MELK, STK12, TTK or GPR87), organic compounds, inorganic compounds and the like.
  • the screening of the present invention is carried out by bringing these test substances or a sample containing them (test sample) into contact with the above aqueous solution, cell or cell fraction.
  • the test sample includes, but is not limited to, a cell extract, a gene library expression product, a synthetic low-molecular compound, a synthetic peptide, a natural compound, etc., containing the test substance.
  • the screening method based on the function or activity of each protein of the present invention will be specifically exemplified.
  • TTK, STK12 and ⁇ MELK are all known to belong to the kinase family (Winey, M. et al., Oncogene, 21, 6161-6169. (1991), Bischoff, J. et al., Trends Cell Biol. , 9, 454-459. (1999), Heyer, BS et al., Mol. Reprod. Dev., 47, 148-156. (1997).). Therefore, screening for test substances (candidate substances) that decrease the known function or activity of TTK, STK12 or MELK should be performed using the phosphorylation amount of the substrate caused by the kinase activity of TTK, STK12 or MELK as an index. Can be.
  • the Cdc7 / ASK complex exhibits kinase activity. Screening can be performed using the ase activity as an index.
  • Candidate substances include, for example, the amount of phosphoric acid in a substrate produced by reacting a substrate with ATP in the presence of a test substance (candidate substance) to TTK, STK12, MELK, or Cdc7 / ASK complex. It can be selected when it decreases (decreases) compared to the phosphorylation amount of the substrate generated by the reaction in the absence of (candidate substance).
  • Examples of the screening method of the present invention based on the kinase activity of TTK, STK12, MELK or Cdc7 / ASK complex include those comprising the following steps (a), (b) and (c):
  • step (b) measuring the phosphorylation amount of the substrate resulting from the step (a), and comparing the phosphorylation amount of the substrate with the phosphorylation amount of the substrate occurring in the absence of the test substance;
  • the TTK, STK12, MELK or Cdc7 / ASK complex used herein includes (i) a purified product of TTK, STK12, MELK or Cdc7 / ASK complex (isolate), and (ii) TTK, STK12, MELK.
  • a cell containing the Cdc7 / ASK complex or a cell fraction thereof can be exemplified.
  • the cells containing the TTK, STK12, MELK or Cdc7 / ASK complex of (ii) include, for example, cells that naturally express TTK, STK12, MELK or Cdc7 / ASK complex, or TTK gene or STK12 gene.
  • Transformed cells prepared by introducing the MELK gene or the Cdc7 / ASK complex gene into cells can be mentioned, but TTK, STK12, MELK or the Cdc7 / ASK complex have a distinct substrate-specific kinase activity. If not, it is desirable to use a purified product (isolate).
  • the transformed cells can be prepared by a method known to those skilled in the art according to a basic book such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).
  • TTK gene, STK12 gene, MELK or Cdc7 / ASK complex gene can be converted to known TTK, ST such as pcDNA3.1 derivative (Invitrogen), FLAG expression vector (Sigmanored Rich), pGEX vector (Amersham Bioscience), etc.
  • transformed cells expressing a protein corresponding to the DNA of the introduced TTK, STK12, MELK or Cdc7 / ASK complex can be produced.
  • the host include, but are not limited to, CHO cells, C127 cells, BHK21 cells, and COS cells, which are generally and widely used, but are not limited to yeast, bacteria, and insects. Cells or the like may be used.
  • the tag include conventionally known tags such as a Myc tag, a His tag, a FLAG tag, and a GST tag.
  • any method can be used as long as it is a method for introducing a known expression vector into host cells.
  • a calcium phosphate method J. Virol., 52, 456-467 (1973)
  • a method using LT-1 manufactured by Panvera
  • a method using a lipid for gene transfer Lipofectamine, Lipofectin; manufactured by Invitrogen
  • TTK, STK12, MELK, or Cdc7 / ASK complex Cells that naturally express the TTK, STK12, MELK, or Cdc7 / ASK complex, or transformed cells that express the TTK, STK12, MELK, or Cdc7 / ASK complex, can be directly used for screening.
  • a purified product (isolate) of TTK, STK12, MELK or Cdc7 / ASK complex isolated and purified from these cells can also be used for screening.
  • the TTK, STK12, MELK or Cdc7 / ASK complex must be isolated and purified by a known general method.
  • a lysate is added to the cells to solubilize the cells, homogenized, and centrifuged to obtain a solubilized kinase-containing fraction (supernatant).
  • the obtained solubilized fraction can be isolated and purified by a conventional method using a column or beads to which the kinase-specific antibody or the antibody specifically recognizing the tag portion of the fusion protein is bound.
  • Substrates used in the screening include known enzyme-specific substrates, MBP (myelin basic protein), and peptides.
  • the recombinant protein may be recovered from the transformed cells prepared in the same manner as the TTK, STK12, MELK or Cdc7 / ASK complex by a conventional method. ,-You can get more.
  • These substrates may be used without labeling, or may be labeled with any labeling substance.
  • the substrate When using the substrate with unlabeled, it can also be used y 3 2 P-ATP or ⁇ 3 3 ⁇ - ATP as a tracer.
  • a labeling substance a radioactive isotope (32 ⁇ , 3 3 ⁇ etc.), fluorescent • light material (Chromagen3 ⁇ 4hNorthernLight (registered trademark), Starbright (registered trademark), Gree n Fluorophore Protein Labeling Kit, etc.) and Biochin (Pierce Inc. EZ- Link Biot inylation Kits).
  • TTK, STK12 measurement of kinase activity of MELK or Cdc7 / ASK complex
  • TTK, STK12 an aqueous solution containing the ME LK or Cdc7 / ASK complex.
  • test compound solution prepared at an appropriate concentration of 1 QM, water or buffer is usually used as the solvent, ethanol-DMS0 can be added depending on the solubility).
  • specific substrate protein or peptide 0. lm y 3 2 P -ATP, 0. ImM ATP, 10mM Mg (C 2 H 3 0 2) 2, 8mM Mops, pH7. 0, 0. 2m EDTA was added, the reaction ( Usually 30. 10 minutes to 1 hour at C). Then, the supernatant is spotted on phosphocellulose paper, washed several times with 50 mM phosphoric acid, dried, and the radioactivity is measured, whereby the phosphorylated substrate mass can be measured.
  • test compound inhibited the kinase kinase activity of the kinase By comparing the above values with the values obtained when a solvent was used as a blank in place of the test compound (control phosphorylation amount), it was determined whether or not the test compound inhibited the kinase kinase activity of the kinase. Can be evaluated. In other words, screening of candidate substances can be performed by using as an index whether or not the mass of phosphorylated groups in the presence of the test substance is reduced compared to the mass of phosphorylated groups in the absence of the test substance. .
  • the screening of the present invention based on the kinase activity of TTK includes the following steps (a) ⁇ Examples including (b) and (c) include:
  • step (b) measuring the phosphorylation amount of the substrate resulting from the step (a), and comparing the phosphorylation amount of the substrate with the phosphorylation amount of the substrate occurring in the absence of the test substance;
  • examples of the TTK substrate include myeline basic protein and poly (tyr-Glu) peptide.
  • Examples of the screening of the present invention based on the kinase activity of STK12 include those comprising the following steps (a), (b) and (c):
  • step (b) measuring the phosphorylation amount of the substrate resulting from the step (a), and comparing the phosphorylation amount of the substrate with the phosphorylation amount of the substrate occurring in the absence of the test substance;
  • the STK12 substrate includes histone H3 or a peptide derived therefrom.
  • the screening of the present invention based on the kinase activity of MELK includes the following steps (a
  • step (b) measuring the phosphorylation amount of the substrate resulting from the step (a), and comparing the phosphorylation amount of the substrate with the phosphorylation amount of the substrate occurring in the absence of the test substance;
  • examples of the MELK substrate include myeline basic protein.
  • the screening of the present invention based on the kinase activity of ASK includes the following steps (a) , (B) and (c) are included:
  • examples of the substrate of the Cdc7 / ASK complex include MCM.
  • E3 / CKS1 complex is know a property of Yubikichin the p27 K ipl. Therefore, screening of the test substance (candidate substance) to result in a decrease in the known function of CKS1 (activity) can be carried out by take advantage of the property of E3 / CKS1 complex is Yubikichin the substrate p27 K ipl.
  • the screening of the present invention can be carried out by measuring whether or not the test substance (candidate substance) inhibits (suppresses) the ubiquitination activity of the E3 / CKS1 complex on the substrate p27Kipl. Examples include the following steps (a), (b) and (c):
  • Each molecule of the E3 / CKS1 complex used in the screening of the present invention is a purified product (isolate). Is desirable.
  • the cells can be purified by a known method as described above from cells that naturally express the E3 / CKS1 complex or from transformed cells prepared by introducing the gene encoding the E3 / CKS1 complex into cells. .
  • the transformed cells can be prepared by a method known to those skilled in the art according to a basic book such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).
  • the cDNA of each factor of the E3 / CKS1 complex is converted to a known cDNA such as pcDNA3.1 derivative (Invitrogen), FLAG expression vector (Sigma Aldrich), pGEX vector (Amersham Bioscience), etc. Insert into a vector capable of expressing only each factor protein of the E3 / CKS1 complex, or an expression vector capable of expressing a fusion protein with a purified product (isolate) tag for purification.
  • the cells are introduced into an appropriate host and cultured, whereby a transformed cell expressing a protein corresponding to the DNA of the introduced receptor can be produced.
  • CHO cells, C127 cells, BHK21 cells, COS cells, and the like which are generally widely used, can be used, but are not limited to yeast, bacteria, and insects. Cells and the like can also be used.
  • the tag include conventionally known tags such as Myc tag, His tag, FLAG tag, and GST tag.
  • any method may be used as long as it is a method for introducing a known expression vector into a host cell.
  • Method J. Virol., 52, 456-467 (1973)
  • LT-1 manufactured by Panvera
  • lipid for gene transfer Lipofectamine, Lipofectin; manufactured by Invitrogen
  • the substrate used in the screening of the present invention can also be obtained by recovering a recombinant protein from a transformed cell prepared by the same method as that for the E3 / CKS1 complex by a conventional method.
  • those labeled with any labeling substance can be used.
  • the labeling substance a radioactive isotope (3 5 S, etc.), fluorescent substances (ChromagenftNort hernLight (registered trademark), Starbright (registered trademark), Green fluorophore Protein L abeling Kit).
  • GPR87 is known to be a GPCR (G-protein coupled receptor) (Wittenberger T. et al., J. Mol. Biol., 2001, 307 ( 3): 799-813, Lee DK et al., Gene, 2001, 275 (1): 83-91), but the ligand is currently unknown.
  • GPCR G-protein coupled receptor
  • a screening method for a test substance (candidate substance) that causes a decrease in the function or activity of the protein GPR87 of the present invention includes, for example, screening based on the binding activity between GPR87 and GTP, or GPCR of GPR87. Screening based on the physiological activity of the above.
  • Screening based on the binding activity between GPR87 and GTP can be performed using the property of GPR87 binding to GTP yS, a GTP analog, in the presence of GDP. That is, the screening of the present invention determines whether the test substance inhibits (suppresses) the binding between GPR87 and GTPyS (more specifically, the binding between GPR87 and G protein and the complex with GTPyS).
  • the following steps (a), (b) and (c) are exemplified:
  • step (b) measuring the amount of GTP yS binding generated in the above step (a), and measuring the amount of GTP yS binding obtained by contacting GPR87 and PGTP yS in the absence of a test substance. Comparing with the quantity, and
  • the ligand of the protein GPR87 of the present invention is unknown, a transformed cell expressing a fusion protein of GPR87 and G protein, a transformed cell containing mutaion in GPCR, or a transformed cell overexpressing GPR87 is used.
  • screening can be performed in the absence of a ligand. Specifically, it can be carried out according to the method described in Methods in Enzymology. 343: 260-73, 2002, J Biol Chem, 276: 35883-90, 2001.
  • the G protein used here is usually the ⁇ subunit of the G protein, and is classified into four classes (ctS, ai / 0, aq, ⁇ 12). ,.
  • Specific G proteins include Ge 16 of the Gq family, Gci2 of the Gi family, and GaS2 of the GS family.
  • GPCR activation such as the activity to increase intracellular cAMP
  • an expression vector for the GPCR is prepared, and a transformed cell overexpressed in CH0-K1 cells, HEK293 cells, and the like is prepared.
  • a G protein expression vector can be co-expressed.
  • the cell membrane fraction prepared by centrifugation or the like from the cells is mixed with a test substance in a solvent such as an appropriate buffer, and incubated for a certain period of time (for example, 30 minutes to 2 hours).
  • the G protein used here is usually the ⁇ subunit of the G protein, and includes four classes (a S, Q! I / 0, and aq ⁇ 12).
  • Specific G proteins include Get 16 of the Gq family, Go: i2 of the Gi family, and Ga S2 of the GS family. That is, the fusion protein of each G protein and GPR87 is expressed, and the physiological activity associated with the activation of GPCR such as the activity to increase intracellular cAMP is measured to select the most suitable G protein for screening. be able to.
  • a screening based on the biological activity of GPR87 as a GPCR is exemplified by one that includes the following steps (a), (b) and (c): -(a) contacting GPR87 with the test substance,
  • step (b) measuring the physiological activity via the GPCR caused by the step (a) above, and comparing the physiological activity with the physiological activity via the GPCR in the absence of the test substance;
  • GPR87 is a GPCR, it has physiological activities via GPCR, such as arachidonic acid release, acetylcholine release, intracellular calcium ion concentration, intracellular cAMP concentration, intracellular cGMP concentration, inositol "phosphate production, cell membrane Screening can be performed using potential fluctuation, phosphorylation of intracellular protein or c-fos activation as an index.
  • the physiological activity can be measured using a known method or a commercially available measurement kit. That is, cells containing GPR87 are cultured in a multiwell plate or the like. Replace with fresh medium or an appropriate buffer that does not show cytotoxicity, add the test compound, incubate for a certain period of time, extract the cells, or collect the supernatant to collect the product according to the appropriate method. Quantify. When the production of a substance (for example, arachidonic acid) as an indicator of a physiological activity cannot be measured by a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme can be used in combination. In addition, activities such as suppression of cAMP production can be measured with high sensitivity by increasing the amount of cells produced with forskolin or the like.
  • a substance for example, arachidonic acid
  • the screening method comprises the following steps 1) to 3).
  • Gq protein means Gq protein in which 5 C-terminal amino acid residues of Gs protein are substituted at the C-terminal.
  • Gqi 5 protein Gqi5
  • An expression vector is prepared.
  • cell lines CH0-K1 cells, etc.
  • the above expression vector can be co-transfected using a gene transfection reagent Lipofectoamine (Invitrogen) or the like.
  • the cells are then fluorescently activated by calcium ions such as robenecid (used as an inhibitor of multiple drug-resistance pumps to retain the dye inside the cells) and Fluo-3 AM (Molecular Probe). Incubate in a C02 incubator for a certain period of time (for example, 30 minutes to 2 hours) in a F12 culture medium containing a dye that shows
  • Selection of a candidate substance can be performed by using as an index whether or not the amount of calcium ions in the presence of the test substance decreases as compared to the amount of calcium ions in the absence of the test substance.
  • cAMP which is activated by signal transduction generated by the binding of ligand to GPR87, is located downstream of cAMP-responsive element (CRE), ⁇ / Cyferase, chloramphenicone acetyltransferase, alkaline phosphatase, growth hormone, GFP, etc.
  • CRE cAMP-responsive element
  • the reporter genes can be ligated and screened using Reporter Gene Atssay.
  • the screening method comprises the following steps 1) to 3).
  • GPR87 gene GPR87 and G protein fusion gene, gene with mutaion in GPCR, etc.
  • G protein Gs, Gi
  • CRE cAMP responsive element
  • a reporter gene such as luciferase, chloramphenicol acetyltransferase, alkaline phosphatase, growth hormone, or GFP.
  • the cell line CH0-K1 cells or the like can be used, and the above expression vector can be co-transfected using a gene transfer reagent, Lipofectoamine (Invitrogen) or the like. When cells expressing the G protein are used, only the GPCR gene expression vector and reporter vector are introduced.
  • the cells are placed in an appropriate medium in a C02 incubator for a certain period of time (for example, Culture for ⁇ 8 hours.
  • luciferase After replacing the medium, add a solution in which the test substance is dissolved in an appropriate culture medium, and after a certain period of time (4 to 24 hours), measure the reporter activity by a well-known conventional method.
  • luciferase cells are lysed with a cell lysate, and a portion of the lysate is reacted with a luciferase substrate solution (eg, Promega Luciferase assay system) to measure luminescence with a luminometer.
  • a luciferase substrate solution eg, Promega Luciferase assay system
  • Selection of a candidate substance can be performed based on whether or not the reporter activity in the presence of the test substance decreases as compared to the reporter activity in the absence of the test substance.
  • the candidate substances selected by the screening method of the present invention described in the above (3, 1) to (3-3-3) are further subjected to a drug efficacy test, a safety test, and a clinical test for a cancer disease patient using a cancer disease model animal. They may be subjected to tests, and by performing these tests, more practical cancer disease ameliorating or therapeutic agents can be obtained.
  • the substances thus selected can be industrially produced by chemical synthesis, biological synthesis (fermentation) or genetic manipulation based on the results of the structural analysis.
  • the screening methods of the present invention described in the above (3-1) to (3-3-3) not only select candidate substances for improving or treating a cancer disease, but also improve or treat a therapeutic agent for a cancer disease ( Candidate drug) 1 Whether to suppress the expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene, or expression or function or activity of ASK, CKS1, MELK, STK12, TTK or GPR87 It can also be used to evaluate and confirm whether or not to suppress. That is, the category of the screening method of the present invention includes not only the search for candidate substances but also those for the purpose of such evaluation or confirmation.
  • the antisense polynucleotide of the present invention comprises a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene, or a part thereof. Any antisense polynucleotide as long as it has an action capable of suppressing the expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene. , Antisense RNA, antisense DNA and the like.
  • the substantially complementary nucleotide sequence is, for example, about 70% or more of the nucleotide sequence complementary to the nucleotide sequence of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene.
  • Base sequences having homology of preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
  • it is complementary to the complementary sequence of the nucleotide sequence encoding the N-terminal part of the nucleotide sequence of the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene (for example, the base sequence near the start codon).
  • Antisense polynucleotides having a homology of 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more are suitable.
  • antisense polynucleotide having a sequence complementary to or substantially complementary to the base sequence described in any of SEQ ID NOS: 1 to 12, or an antisense polynucleotide having a part thereof.
  • the antisense polynucleotide is usually composed of about 10 to 1000 bases, preferably about 15 to 500 bases, and more preferably about 16 to 30 bases.
  • the phosphate residues (phosphates) of each of the nucleotides constituting antisense DNA are chemically modified with, for example, phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted with a phosphate residue.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • Such an antisense polynucleotide can hybridize to RNA of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene, and has the ability to inhibit the synthesis or function of the RNA or Through the interaction with RNA, the expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene can be regulated and controlled.
  • the antisense polynucleotide of the present invention is useful for regulating and controlling the expression of the protein gene of the present invention in vivo and in vitro, and is also useful for treating or diagnosing diseases and the like.
  • a terminal hairpin loop or the like can be selected as a preferred region of interest, but any region within a protein gene can be selected as a target.
  • the target nucleic acid when the target nucleic acid can hybridize with the target region, the target nucleic acid is used for the polynucleotide in the target region. It can be said to be "antisense”.
  • Antisense polynucleotides include polynucleotides containing 2-dexoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-daricosides of purine or pyrimidine bases, and non-polynucleotides.
  • Other polymers having a nucleotide skeleton eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers having a special bond where the polymer is a base such as that found in DNA or RNA
  • Including nucleotides having a configuration that allows base attachment and base attachment include, but not limited to base attachment and base attachment.
  • RNA DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known Modified, e.g., labeled in the art, capped, methylated, substituted for one or more natural nucleotides by analogs, intramolecular nucleotides Modified, eg, having uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), charged or sulfur-containing bonds (eg, phosphorothioate, phosphorodi (Eg, nucleases, nucleases' inhibitors, toxins, antibodies, proteins, etc.) Compounds having side-chain groups such as signal peptides, poly-L-lysine, etc.) or sugars (eg, monosaccharides), compounds having an interactive compound (eg, at
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications include methylated purines and pyrimidines, acylated It may contain a purine and a pyrimidine or another nitrogen ring. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with a halogen and a force, an aliphatic group, or a functional group such as an ether or amine. It may be converted to a group.
  • the antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include a nucleic acid sulfur derivative, a thiophosphate derivative, and a polynucleoside amide that is resistant to polynucleoside amide.
  • the antisense polynucleotide of the present invention can be designed, for example, as follows. In other words, it makes the antisense polynucleotide more stable in the cell, enhances the cell permeability of the antisense polynucleotide, increases the affinity for the target sense strand, and In some cases, the toxicity of the antisense polynucleotide is reduced. Many such modifications have been reported, for example, in Pharm Tech Japan, vol. 8, p. 247 or p. 395, 1992, Antisense Research and Applications, CRC Press, 1993.
  • the antisense polynucleotide of the present invention may be provided in a special form such as ribosome or microsphere, or may be provided in an added form more suitable for gene therapy.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase nucleic acid uptake.
  • polycations such as polylysine, which act to neutralize the charge on the phosphate backbone
  • lipids which enhance interaction with cell membranes or increase nucleic acid uptake.
  • phospholipids, cholesterol, etc. phospholipids, cholesterol, etc.
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside linkage.
  • capping groups specifically located at the 3 'or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as dalicol such as polyethylene dalicol and tetraethylene dalicol.
  • Antise The inhibitory activity of a polynucleotide can be determined using the screening method of the present invention.
  • the antisense polynucleotide of the present invention may be double-stranded as described above, and binds to RNA encoding ASK, CKS1, MELK, STK12, TTK or GPR87 to destroy the RNA or suppress its function. Anything should do. That is, the antisense polynucleotide of the present invention also includes a double-stranded RNA having a part of the RNA encoding ASK, CKS1, MELK, STK12, TTK or GPR87 and a complementary RNA, and the like.
  • double-stranded RNA examples include SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, or SEQ ID NO: 33 and SEQ ID NO: 34
  • the present invention provides an agent for improving and treating a cancer disease.
  • the present invention relates to ASK genes, CKS1 genes, MELK genes, STK12 genes, TTK genes, GPR87 genes, and new findings that proteins encoded by these genes are associated with cancer diseases.
  • Substances that suppress the expression of CKS1, MELK, STK12, TTK, or GPR87 gene, or substances that suppress the expression or function (activity) of ASK, CKS1, MELK, STK12, TTK, or GPR87 It is based on the belief that it is effective for improvement or treatment.
  • the therapeutic agent for improving cancer disease of the present invention is a substance that suppresses the expression of ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene, or ASK, CKS1, MELK, STK12, TTK.
  • a substance that suppresses GPR87 protein expression or function (activity) is used as an active ingredient.
  • ASK, CKS1, MELK, STK12, TTK or GPR87 expression or activity (activity) of the active ingredient ASK, CKS1, MELK, STK12, TTK or GPR87 gene Inhibitors are not only selected using the above-described screening method of the present invention, but also in accordance with ordinary methods based on information on the selected substances, by chemical and biochemical methods, or industrially. It may be manufactured in
  • the active ingredient may be used as it is or as a pharmaceutically acceptable carrier (excipient) known per se. ,., Extenders, binders, lubricants, etc.), conventional additives and the like to prepare a pharmaceutical composition.
  • the pharmaceutical composition is prepared in the form to be prepared (oral administration such as tablet, pill, capsule, powder, granule, syrup, etc .; parenteral administration such as injection, infusion, external preparation, suppository) Oral administration or parenteral administration can be performed depending on the conditions.
  • the dose varies depending on the kind of the active ingredient, the route of administration, the subject of administration or the age, weight, symptoms, etc. of the patient, and cannot be unconditionally specified. Usually, about several mg to 2 g, preferably about several tens mg, can be administered once a day or several times a day as a daily dose.
  • the DNA is incorporated into a vector for gene therapy and gene therapy is performed.
  • gene therapy can be carried out as it is or by incorporating it into a vector for gene therapy.
  • the dose and administration method of the gene therapy composition vary depending on the patient's body weight, age, symptoms and the like, and can be appropriately selected by those skilled in the art.
  • an antisense oligonucleotide or a chemically modified product thereof can be directly transferred into a patient.
  • a method of controlling the expression of a target gene by introducing antisense RNA into a target cell of a patient by introducing the antisense RNA into a target cell of a patient.
  • the chemically modified form can bind to the sense strand mRNA in the cell and regulate the expression of the target gene, that is, the expression of ASK, CKS1, MELK, STK12, TTK or GPR87.
  • the function (activity) of CKS 1, MELK, STK12, TTK or GPR87 can be controlled.
  • the antisense polynucleotide or the chemically modified product used is preferably 10 to 1000 bases, more preferably 15 to 500 bases, and most preferably. Should have a length of 16 to 30 bases.
  • antisense oligo Nucleotides or chemically modified nucleotides can be formulated using commonly used stabilizers, buffers, solvents and the like.
  • the antisense RNA used preferably has a length of at least 10 bases, more preferably at least 15 bases, and even more preferably at least 16 bases.
  • This method also includes an in vivo method in which an antisense gene is introduced into cells in a living body and an ex vivo method in which an antisense gene is introduced into cells once taken out of the body, and the cells are returned to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Special Edition, 12 (15), all pages (1994)).
  • the in vivo method is preferable, and includes a viral transfer method (a method using a recombinant virus) and a non-viral transfer method (see the above-mentioned references).
  • a vinoresogenome such as a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpes virus, a Sendai virus, a vaccinia virus, a polio virus, and a simbis virus
  • a method of incorporating an antisense polynucleotide into a living body and introducing it into a living body may be used.
  • a method using a retrovirus, an adenovirus, an adeno-associated virus or the like is particularly preferable.
  • non-viral transfer method examples include a ribosome method and a lipofectin method, and the ribosome method is particularly preferable.
  • Other non-viral introduction methods include, for example, a microinjection method, a canopy phosphate method, and an electroporation method.
  • the pharmaceutical composition for gene therapy contains, as an active ingredient, the above-mentioned antisense polynucleotide or a chemically modified product thereof, a recombinant virus containing the same, and an infected cell into which the virus has been introduced.
  • the administration form, administration route and the like of the composition to a patient can be appropriately determined depending on the disease, symptom and the like to be treated. For example, it can be administered intravenously, intraarterially, subcutaneously, intramuscularly, or the like in a suitable administration form such as an injection, or can be directly administered or introduced into a patient at a target site of disease.
  • the composition for gene therapy may be prepared, for example, by administering a viral vector containing the antisense polynucleotide of the present invention to a ribosome in addition to a dosage form such as an injection containing the antisense polynucleotide of the present invention.
  • a dosage form such as an injection containing the antisense polynucleotide of the present invention.
  • the form embedded in membrane fusion ribosome Sendai ,-Virus (HVJ)-ribosome, etc.
  • These ribosome formulations include suspensions, cryogens, and centrifuged concentrated cryogens.
  • the composition for gene therapy may be in the form of a cell culture solution infected with a virus into which the vector containing the antisense polynucleotide of the present invention has been introduced.
  • the dosage of the active ingredient in these various forms of preparations can be appropriately adjusted depending on the degree of the disease to be treated, the age and weight of the patient, and the like.
  • an antisense polynucleotide against the ASK, CKS1, MELK, STK12, TTK or GPR87 gene about 0.0001 to 100 mg, preferably about 0.001 to 100 mg per adult patient is used.
  • the dose may be set to be administered once a month.
  • a retroviral titers can be selected from the amount range of about l X 10 3 pfu- 1 X 10 1 5 pfu per day of patient body weight LKG.
  • about 1 ⁇ 10 4 cells / body-IX 10 15 cells / body may be administered.
  • the present invention comprises (i) a double-stranded RNA containing a part of the RNA encoding the protein of the present invention and RNA complementary thereto, and (ii) the double-stranded RNA.
  • a drug (iii) a ribozyme containing a part of the RNA encoding the protein of the present invention, (iv) a drug containing the ribozyme, (V) a gene (DNA) encoding the ribozyme.
  • An expression vector or the like is also provided.
  • a double-stranded RNA, ribozyme or the like can also destroy RNA transcribed from the DNA of the present invention or suppress its function.
  • the double-stranded RNA, ribozyme and the like capable of suppressing the function of the protein A or the DNA encoding the protein A of the present invention can be used, for example, as a prophylactic or therapeutic agent for cancer diseases (for example, cancer of the like). Can be used.
  • the double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, vol. 411, p. 494, 2001).
  • the ribozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, vol. 7, p. 221, 2001). For example, it can be produced by substituting a part of a known ribozyme sequence with a part of RNA encoding the protein of the present invention.
  • RNA encoding the protein of the present invention As a part of the RNA encoding the protein of the present invention, a consensus that can be cleaved by a known ribozyme And a sequence near the base sequence NU X (wherein N represents all bases and X represents a base other than G).
  • NU X a sequence near the base sequence
  • the above-mentioned double-stranded RNA or ribozyme is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.
  • the expression vector (V) is used in the same manner as known gene therapy methods and the like, and is used as the above-mentioned prophylactic / therapeutic agent.
  • the present invention relates to a cytotoxic ⁇ cell (hereinafter referred to as CTL) inducer comprising, as an active ingredient, ASK, CKS1, MELK, STK12, TTK or GPR87 (hereinafter sometimes referred to as the tumor antigen protein of the present invention).
  • CTL cytotoxic ⁇ cell
  • the present invention provides a CTL inducer containing an amino acid sequence identical or substantially identical to the amino acid sequence described in any of SEQ ID NOs: 13 to 18.
  • the tumor antigen protein as an active ingredient of the CTL inducer of the present invention may be a protein derived from a natural product (for example, a lung cancer cell line) or a recombinant protein.
  • a protein containing an amino acid sequence identical to the amino acid sequence of any of SEQ ID NOs: 13 to 18 Is a protein comprising the amino acid sequence described in any one of SEQ ID NOs: 13 to 18, or the amino acid sequence described in any one of SEQ ID NOs: 13 to 18; And / or a protein comprising an amino acid sequence having another amino acid sequence added to the C-terminal side.
  • a protein containing an amino acid sequence substantially the same as the amino acid sequence described in any of SEQ ID NOs: 13 to 18 specifically refers to proteins listed in the following (a) to (c): Include:
  • a protein containing an amino acid sequence showing 70% or more sequence identity with the amino acid sequence of any of SEQ ID NOs: 13 to 18, and cells expressing the protein are expressed by CTLs.
  • a protein with recognized characteristics (c) a protein encoded by a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide encoding the amino acid sequence of any of SEQ ID NOs: 13 to 18, and A protein having the characteristic that cells expressing the same are recognized by CTL.
  • a protein comprising an amino acid sequence substantially identical to the amino acid sequence described in any of SEQ ID NOs: 13 to 18 is exemplified.
  • Examples of the protein consisting of an amino acid sequence substantially identical to the amino acid sequence described in any one of SEQ ID NOs: 13 to 18 include the following proteins (a ′;) to (c ′ :) :
  • (a ′) a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted and Z or added in the amino acid sequence of any of SEQ ID NOs: 13 to 18, and A protein whose expressed cells have characteristics recognized by CTLs,
  • (b ') a protein consisting of an amino acid sequence showing 70% or more sequence identity to the amino acid sequence of any one of SEQ ID NOs: 13 to 18, and cells expressing the protein are expressed by CTL.
  • a protein with recognized characteristics
  • (c ′) a protein encoded by a polynucleotide that hybridizes under stringent conditions to a complementary strand of the polynucleotide encoding the amino acid sequence of any of SEQ ID NOs: 13 to 18, and A protein that has the characteristics that cells expressing the protein are recognized and recognized by CTL.
  • the number of amino acid mutations and mutation sites in the protein are not limited as long as the activity of the protein of the present invention is maintained. Indices for determining how and how many amino acid residues should be deleted, substituted and / or added without losing the activity are computer programs well known to those skilled in the art, for example, DNA Star. can be found using software.
  • the number of mutations is typically within 10% of all amino acids, and preferably within 5% of all amino acids.
  • the amino acids to be substituted are protein From the viewpoint of maintaining the structure of the amino acid, the amino acid is preferably an amino acid having properties similar to the amino acid before substitution, such as the polarity, charge, solubility, hydrophobicity, hydrophilicity and amphiphilicity of the residue.
  • Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are amino acids classified as non-polar amino acids, and Gly, Ser, Thr, Cys, Tyr, Asn and Gin are non-charged amino acids.
  • Asp and Glu are amino acids classified as acidic amino acids, and Lys, Arg and His are amino acids classified as basic amino acids. Therefore, amino acids belonging to the same group can be appropriately selected using these as indices.
  • the “protein containing an amino acid sequence showing 70% or more sequence identity with the amino acid sequence according to any one of SEQ ID NOs: 13 to 18” in the above (b) is, for example, a protein of SEQ ID NOs: 13 to 18
  • Specific examples include a protein comprising a partial sequence of the amino acid sequence described in any of SEQ ID NOs: 13 to 18.
  • sequence identity refers to sequence identity and homology between two proteins.
  • sequence identity is determined by comparing two optimally aligned sequences over the region of the sequence to be compared.
  • the protein to be compared may have an addition or deletion (for example, a gap or the like) in the optimal alignment of the two sequences.
  • sequence identity is calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. can do.
  • the sequence identity is measured using sequence analysis software, specifically, analysis tools provided by Vector NTI, GE ETYX-MAC, and public databases.
  • the public database is generally available, for example, at the homepage address http: @ www.ddbj.nig.ac.jp.
  • the “polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding the amino acid sequence according to any one of SEQ ID NOs: 13 to 18” in (c) above includes, for example, SEQ ID NO: : Amy according to any of 13 to 18 And at least about 40%, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, Preferably, a polynucleotide containing a base sequence having about 95% or more sequence identity is used.
  • Hybridization is performed by a method known per se or a method analogous thereto, for example,
  • the method can be performed according to the method described in a basic book such as Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • tumor antigen protein of the present invention has substantially the same activity as the protein having the amino acid sequence of any one of SEQ ID NOs: 13 to 18.
  • substantially the same activity means that cells expressing the tumor antigen protein of the present invention are recognized by CTL, that is, the cells show reactivity to CTL, in other words, the tumor antigen protein of the present invention. Alternatively, it exhibits the property that an antigen peptide derived from the protein activates CTL or induces CTL.
  • the cell is preferably a cell that expresses an HLA antigen. Therefore, the substantially equivalent activity is more specifically defined by expressing the tumor antigen protein of the present invention in a cell that expresses an HLA antigen such as HLA-A24.
  • HLA-A24 an HLA antigen such as HLA-A24. This refers to the property that a complex of a peptide and an HLA antigen is presented on the cell surface, and as a result, the cell is recognized by CTL, that is, CTL is activated (CTL is induced).
  • Such properties of the protein of the present invention is a method known per se or how modifications thereof (e.g. 5 1 Cr release mediation Si (J.ImmunoL, 159: 4753, 1997 ), LDH release mediation Si (LDH Cytotoxicity Detection Kit (Takara The present invention can be easily measured by measuring the amount of cytokines, etc.)
  • 5 1 Cr release mediation Si J.ImmunoL, 159: 4753, 1997
  • LDH release mediation Si LDH Cytotoxicity Detection Kit (Takara
  • the present invention can be easily measured by measuring the amount of cytokines, etc.
  • the specific Atsey method is exemplified below:
  • the protein of the present invention is applied to host cells such as 293-EBNA cells (Invitrogen).
  • DNA encoding the HLA antigen used herein for example, DNA encoding the HLA-A24 antigen HLA-A24 antigen
  • the DNA to encode includes the cDNA of HLA-A2402 (Cancer Res., 55: 4248-4252 (1995), Genbank Accession No. M64740).
  • the transfection can be performed, for example, by a lipofectin method using a ribophenatamine reagent (manufactured by GfflCO BRL). Thereafter, restricted CTL is added to the HLA antigen to be used and allowed to act, and the amount of various cytokines, for example, IFN- ⁇ produced by the reaction (activation) of the CTL is measured by, for example, ELISA. Can be checked.
  • CTL CTL prepared by stimulating human peripheral blood lymphocytes with the protein of the present invention described in any of SEQ ID NOs: 13 to 18 and Int. J. Cancer, 39, 390- 396, 1987, N. Eng. J. Med, 333, 1038-1044, 1995 and the like can be used.
  • the tumor antigen protein of the present invention is capable of inducing CTL-inducing activity in vivo by subjecting it to, for example, Atsusei (WO 02/47474, Int J. Cancer: 100,565-570 (2002)) using a human model animal. You can find out.
  • the tumor antigen protein of the present invention can be produced from a natural product (eg, an osteosarcoma cell line, a renal cancer cell line, etc.) by a method known per se for purifying a protein. It can also be produced by culturing a transformant containing a nucleic acid containing the encoding polynucleotide.
  • a natural product eg, an osteosarcoma cell line, a renal cancer cell line, etc.
  • the present invention provides, as an active ingredient, a partial peptide of the above-described tumor antigen protein of the present invention, which is a tumor antigen peptide that is bound to an HLA antigen and recognized by CTL (hereinafter, may be referred to as “peptide of the present invention”).
  • peptide of the present invention Provided is an agent for inducing CTL.
  • the peptide which is an active ingredient of the CTL inducer of the present invention is a peptide consisting of a part of the amino acid sequence of the tumor antigen protein of the present invention, and further comprises the following:
  • the binding complex is a peptide that is recognized by CTL, it may be a peptide of any length at any position in the amino acid sequence of the tumor antigen protein of the present invention.
  • Such a peptide of the present invention synthesizes a candidate peptide consisting of a part of the JB tumor antigen protein of the present invention, and is a complex of the candidate peptide and the HLA antigen recognized and recognized by CTL? No, that is, whether the candidate peptide has activity as a tumor antigen peptide Identification can be performed by ascertaining
  • the peptide can be synthesized according to a method used in ordinary peptide chemistry. Such known methods are described in the literature (Peptide Synthesis
  • sequence regularity (motif) of the antigenic peptide presented by binding to the HLA is known (see, for example, Immunogenetics, 41: pl78, 1995).
  • the amino acid at the second position in the peptide consisting of 8 to 11 amino acids is tyrosine, phenylalanine, methionine or tryptophan, and the amino acid at the C-terminus is phenylalanine. It is known to be leucine, isoleucine, tryptophan or methionine (J. Immunol., 152, p3913, 1994; Immunogenetics, 41: p178, 1995; J. Immunol, 155: p4307, 1994). As for the motif of HLA-A2, the motifs shown in Table 1 below are known (Immunogenetics, 41, pl78, 1995; J. Immunol., 155: p4749, 1995).
  • peptide moieties related to these motifs from the amino acid sequence of the tumor antigen protein of the present invention.
  • a sequence predicted to be capable of binding to the HLA antigen can be easily selected by a search using the BIAS software.
  • the selected candidate peptides are synthesized by the method described above, and whether or not the candidate peptides bind to the HLA antigen and are recognized by CTL, that is, the activity of the candidate peptide as a tumor antigen peptide
  • the peptide of the present invention can be identified by measuring whether or not it has the same.
  • Specific examples of the method for identifying the tumor antigen peptide of the present invention include the method described in J. Immunol., 154, p2257, 1995. That is, when peripheral blood lymphocytes are isolated from humans positive for the type of HLA antigen which is considered to present a candidate peptide, and stimulated in vitro with the candidate peptide added thereto, the candidate peptide is expressed as a candidate peptide. Induction of CTL that specifically recognizes the pulsed HLA antigen-positive cells indicates that the candidate peptide can be a tumor antigen peptide.
  • the presence or absence of CTL induction is determined by, for example, measuring the amount of various cytokins (eg, IFN- ⁇ ) produced by CTL in response to antigen peptide-presenting cells, for example, by ELISA, ELISP0T, etc. Can be checked. It can also be examined by a method for measuring the toxicity of CTLs to 51 Cr-labeled antigen peptide-presenting cells ( 5l Cr release atsey, Int. J. Cancer, 58: p317, 1994).
  • cytokins eg, IFN- ⁇
  • a candidate peptide was pulsed into cells that had been transfected into an expression plasmid that expresses a cDNA encoding an HLA antigen of the type that is thought to present the candidate peptide, for example, into 293-EBNA cells (Invitrogen).
  • the cells are allowed to react with a restricted CTL against an HLA antigen of a type that is thought to present the candidate peptide, and the amount of various site-in (eg, IFN- ⁇ ) produced by the CTL is measured.
  • the HLA antigen includes HLA-A24 antigen.
  • HLA-A24-restricted tumor antigen peptide As the cDNA encoding the HLA antigen, the cDNA of HLA-A2402 (Cancer Res., 55: 4248-4252 (1995), Genbank Accession No. M6474 0) can be used.
  • the CTL may be prepared by stimulating human peripheral blood lymphocytes with peptides, or may be prepared from Int. J. Cancer, 39, 390-396, 1987, N. Eng. J. Med, 333, 1038-. CTLs established by the method described in 104, 1995, etc. can be used.
  • the peptide of the present invention is tested for in vivo activity by subjecting it to, for example, Atsusei (W002 / 7474, Int J. Cancer: 100, 565-570 (2002)) using a human model animal. be able to.
  • the peptide of the present invention as described above include a partial peptide of the tumor antigen protein of the present invention comprising the amino acid sequence of any one of SEQ ID NOs: 13 to 18, and which binds to the HLA antigen. And peptides recognized by CTL. From the viewpoint of the HLA antigen to which the peptide of the present invention binds, the peptide of the present invention that binds to the HLA-A24 antigen can be mentioned.
  • HLA-A24 binding tumor antigen peptides include the following Table 2 (partial peptide of ASK), Table 3 (partial peptide of CKS1), Table 4 (partial peptide of MELK), A peptide consisting of any of the amino acid sequences described in Table 5 (partial peptide of STK12), Table 6 (partial peptide of TTK) and Table 7 (partial peptide of GPR87) (SEQ ID NO: 37 to 225) A peptide comprising an amino acid sequence), which binds to the HLA-A24 antigen and is recognized by CTL.
  • VVDDIVSKL (SEQ ID NO: 52)
  • EFEYRHVML (SEQ ID NO: 68) 18-26 EYRHV LPK (SEQ ID NO: 81)
  • VYKRLVEDI (SEQ ID NO: 100) 568-576 LVNPDQLLN (SEQ ID NO: 118)
  • RLVNPDQLL SEQ ID NO: 1034 130-138 HRDLKPENL (SEQ ID NO: 122)
  • KLEKNSVPL (SEQ ID NO: 166) 501-509 TPLQNLQVL (SEQ ID NO: 184) 201-209 KNLSASTVL (SEQ ID NO: 167) 588-596 LYDYEITDQ (SEQ ID NO: 185) 467-475 RTPWKNDF (SEQ ID NO: 168) 172-180 RGAV PLEML (SEQ ID NO: 186) 102-110 KYGQNESFA (SEQ ID NO: 169) 645-653 HSDLKPANF (SEQ ID NO: 187) 19-27 KVRDIKNKF (SEQ ID NO: 170) 76-84 SVPLSDALL (SEQ ID NO: 188) 777-785 KQRIS1PEL (SEQ ID NO: 171) 712-720 SPKSDVWSL (SEQ ID NO: 189) 75-83 NSVPLSDAL (SEQ ID NO: 172) 810-818 KYVLGQLVG (SEQ ID NO: 190) 605-613 GNIDLNSWL (S
  • LYYCKEITL (SEQ ID NO: 193) 208-216 TYVNSGLFV (SEQ ID NO: 208) 225-233 CYIAISRYK SEQ ID NO: 194) 78-86 FYLKNIVVA (SEQ ID NO: 209)
  • GFNLTLAKL (SEQ ID NO: 199) 299-307 1 "shi 5 01 ⁇ (SEQ ID NO: 214) 276-284 TFSHLDRLL (SEQ ID NO: 200) 300-308 1 ⁇ shi 5 01 ⁇ 0 (SEQ ID NO: 215) 272-280 RIPFTFSHL (SEQ ID NO: 201) 260 -268 1 "0" shi (sequence number: 216) 115-123 RYTSVLFYA (sequence number: 202) 127-135 cout 51 shi 01_1 (sequence number: 217) 103-111 GFGPWYFKF (sequence number: 203) 228-236 AISRY1HKS (SEQ ID NO: 218) 48-56 LYUIFVAS (SEQ ID NO: 204) 72-80 ⁇ c5 ⁇ ⁇ (SEQ ID NO: 219) 267-275 PYHLCRIPF (SEQ ID NO: 205) 125-133 MYTSIVFLG (
  • one or more amino acid sequences are compared with the amino acid sequence of the protein of the present invention, more specifically, the amino acid sequence of the peptide of the present invention consisting of a part of the amino acid sequence described in any of SEQ ID NOs: 13 to 18.
  • a modified peptide obtained by modifying an amino acid residue and having an activity as a tumor antigen peptide that is recognized by CTL by binding to an HLA antigen is also included in the scope of the peptide of the present invention. .
  • modification of an amino acid residue means substitution, deletion, and Z or addition of an amino acid residue (including addition of an amino acid to the N-terminal or C-terminal of a peptide), and preferably an amino acid residue.
  • group substitution In the case of modification involving substitution of amino acid residues, the number and position of amino acid residues to be substituted are arbitrary, as long as the activity as a tumor antigen peptide is maintained, but as described above, tumor antigen peptides are usually used. Since the length is about 8 to 14 amino acids, the number is preferably one to several.
  • the length of the modified peptide of the present invention is preferably about 8 to 14 amino acids (however, HLA-DR, -DP, and _DQ may have a length of 14 amino acids or more.)
  • HLA types such as -Cw0401 and -Cw0602
  • sequence regularity (motif) of the antigenic peptide presented by binding to the HLA is known.
  • a peptide sequence expected to be capable of binding to the HLA antigen can be searched on the Internet (http: @bimas, dcrt. Nih. Gov / molbio / hla-bind /). Therefore, it is possible to prepare the modified peptide based on the motif and the like.
  • the second amino acid of the peptide consisting of 8 to 11 amino acids is tyrosine, phenylalanine, methionine, or tryptophan.
  • the C-terminal amino acid is phenylalanine, leucine, isoleucine, tryptophan or methionine (J. Immunol., 152: p3913, 1994; I: unogenetics, 41: pl78, 1995; J. Immunol., 155: p4307, 1994).
  • HLA-A2 the motifs shown in Table 1 above are known.
  • a peptide sequence that is expected to be able to bind to the HLA antigen is shown on the Internet (http: ⁇ bimas.dcrt.nih.gov/tnolbio/hla_bind/).
  • Amino acids with are acceptable. Therefore, the amino acid at the position where amino acid substitution on these motifs is possible (in HLA-A24 and HLA-A2, the 2nd position and the C-terminal of the peptide) can be combined with another amino acid (preferably on the Internet).
  • Predicted amino acids and modified peptides having an activity of binding to HLA antigen and being recognized by CTL.
  • a modified peptide having the above-mentioned activity which is a peptide containing an amino acid sequence obtained by substituting any of the amino acid residues known on the motif at the position. That is, in the case of an HLA-A24 binding peptide as shown in SEQ ID NOs: 37 to 225, the second amino acid is substituted with tyrosine, fenilalanine, methionine, or tributophan, and the Z- or C-terminal amino acid is substituted. Modified peptides substituted with phenylalanine, leucine, isoleucine, tryptophan or methionine, and having the above-mentioned activity are mentioned. Of these, a peptide in which the amino acid at the second position is substituted with cysteine is more preferable.
  • Examples of the modifying group for the amino group of the N-terminal amino acid include, for example, 1 to 3 anoalkyl groups having 1 to 6 carbon atoms, pheninole groups, cycloalkyl groups, and acyl groups.
  • Examples are alkanoyl groups having 1 to 6 carbon atoms, alkanoyl groups having 1 to 6 carbon atoms substituted by phenyl group, carboyl groups substituted by cycloalkyl group having 5 to 7 carbon atoms, and 1 to 6 carbon atoms.
  • Examples of peptides in which the carboxyl group of the C-terminal amino acid is modified include ester forms and amide forms. Specific examples of the ester form are alkyl esters having 1 to 6 carbon atoms and substituted with phenyl groups. Examples thereof include an alkyl ester having 0 to 6 carbon atoms and a cycloalkyl ester having 5 to 7 carbon atoms.
  • the amide form include one or two of an amide and an alkyl group having 1 to 6 carbon atoms.
  • a substituted amide, an amide substituted with one or two alkyl groups having 0 to 6 carbon atoms substituted with a phenyl group, a 5- to 7-membered ring containing a nitrogen atom of the amide group Amides that form azacycloalkanes are exemplified.
  • the nucleic acid contained in the CTL inducer of the present invention is a polynucleotide encoding the tumor antigen protein of the present invention described in (6) above (hereinafter, referred to as the nucleic acid of the present invention). (Sometimes referred to as a polynucleotide).
  • the polynucleotide of the present invention may be any of cDNA, mRNA, cRNA, genomic DNA, or synthetic DNA of various cells and tissues, for example, cells and tissues derived from osteosarcoma and renal cancer. Further, it may be a single-stranded or double-stranded form.
  • the nucleic acid containing the polynucleotide of the present invention can be in any of single-stranded and double-stranded forms.
  • a recombinant expression vector for expressing the protein of the present invention can be prepared by inserting the polynucleotide of the present invention into an expression vector. That is, the category of the nucleic acid of the present invention also includes a recombinant expression vector prepared by inserting the double-stranded polynucleotide of the present invention into an expression vector.
  • the expression vector used here can be appropriately selected depending on the host used, the purpose, and the like, and examples thereof include a plasmid, a phage vector, and a virus vector.
  • examples of the vector include a plasmid vector such as pUC118, pUC119, pBR322, and pCR3, and a phage vector such as ⁇ and gtll.
  • yeast the vector includes , PYES2, pYEUra3, etc.
  • the host is an insect cell, pAcSGHisNT-A, etc.
  • viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors.
  • the vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, and a terminator.
  • a sequence expressed as a fusion protein with thioredoxin, His tag, GST (daltathione S-transferase), or the like may be added.
  • a GST fusion protein such as pGEX4T
  • an appropriate promoter such as lac, tac, trc, trp, CMV, or SV early promoter
  • a tag sequence such as Myc or His.
  • pET32a that expresses a fusion protein with thioredoxin and His tag.
  • a transformed cell containing the expression vector By transforming a host with the expression vector prepared above, a transformed cell containing the expression vector can be prepared.
  • the host used herein includes Escherichia coli, yeast, insect cells, animal cells, and the like.
  • Escherichia coli include E. coli K-12 strain HB101, C600, JM109, DH5a, and AD494 (DE3).
  • yeast include Saccharomyces cerevisiae.
  • Animal cells include L929 cells, BALB 3T3 cells, C127 cells, CH0 cells, COS cells, Vero cells, Hela cells, 293-EBNA cells and the like. Insect cells include sf9.
  • a normal introduction method suitable for the host cell may be used. Specific examples include a calcium phosphate method, a DEAE-dextran method, an electoral poration method, and a method using a lipid for gene transfer (Lipofectamine, Lipofectin; Gibco-BRL).
  • the cells are cultured in a usual medium containing a selectable marker, whereby transformed cells in which the expression vector has been introduced into host cells can be selected.
  • the protein of the present invention can be produced by continuously culturing the transformed cells obtained as described above under suitable conditions.
  • the obtained protein can be further isolated and purified by general biochemical purification means.
  • Purification means here include salting out, ion exchange chromatography, adsorption chromatography, and And affinity chromatography, gel filtration chromatography and the like.
  • the protein of the present invention when expressed as a fusion protein with the aforementioned thioredoxin, His tag, GST, etc., it can be isolated and purified by a purification method utilizing the properties of these fusion proteins and tags. it can.
  • the category of the nucleic acid of the present invention also includes a nucleic acid containing a polynucleotide encoding the peptide of the present invention described in (7).
  • the polynucleotide encoding the peptide of the present invention may be in the form of DNA or RNA. These polynucleotides of the present invention can be easily produced based on the amino acid sequence information of the peptide of the present invention and the sequence information of the DNA encoded thereby. Specifically, it can be produced by ordinary DNA synthesis or amplification by PCR.
  • polynucleotide encoding the peptide of the present invention include the polynucleotide encoding the above-mentioned peptide.
  • the nucleic acid containing the polynucleotide encoding the peptide of the present invention can take any of single-stranded and double-stranded forms.
  • a recombinant expression vector for expressing the peptide (epitope peptide) of the present invention is produced by inserting the polynucleotide of the present invention into an expression vector. be able to.
  • the expression vector, the host cell, the method of transforming the host cell, and the like are the same as those described above.
  • CTL inducer comprising the tumor antigen protein of the present invention as an active ingredient
  • the present invention provides a CTL inducer containing the tumor antigen protein of the present invention as an active ingredient.
  • Cells containing the tumor antigen protein of the present invention are characterized by being recognized by CTL. That is, the tumor antigen protein of the present invention is an inducer of CTL.
  • the induced CTL can exert an antitumor effect through the production of cytotoxic action lymphoforce. Therefore, the tumor antigen protein of the present invention can be used as an active ingredient of a medicine (cancer vaccine) for treating or preventing a tumor.
  • the CTL inducer containing the tumor antigen protein of the present invention as an active ingredient is useful for the tumor antigen protein of the present invention in tumor patients.
  • the administration can treat or prevent a tumor.
  • the protein When the protein is administered to a tumor patient, it is taken up into antigen-presenting cells, and then the tumor antigen peptide generated by intracellular degradation binds to the HLA antigen to form a complex. CTLs, which are presented on the surface of the presenting cell and specific for this complex, proliferate efficiently in the body and destroy tumor cells. As described above, the treatment or prevention of a tumor is achieved.
  • the CTL inducer comprising the tumor antigen protein of the present invention as an active ingredient is used for any tumor patient positive for ASK, CKS1, MELK, STK12, TTK or GPR87 described in SEQ ID NOs: 13 to 18. be able to. Specifically, for example, it can be used for the prevention or treatment of cancer (tumor) such as lung cancer, breast cancer, prostate cancer, and colon cancer.
  • cancer tumor antigen
  • the CTL inducer containing the tumor antigen protein of the present invention as an active ingredient is administered in combination with a pharmaceutically acceptable carrier, for example, an appropriate adjuvant, or used in combination or in combination, so that cellular immunity can be effectively established. Can be administered.
  • a pharmaceutically acceptable carrier for example, an appropriate adjuvant, or used in combination or in combination, so that cellular immunity can be effectively established. Can be administered.
  • the adjuvant those described in the literature (Clin. Microbiol. Rev., 7: 277-289, 1994) can be applied, and specifically, components derived from bacterial cells or derivatives thereof, cytokines, plants, and the like.
  • surfactants such as lysolecithin, pull-mouth nic polyols, polyanion, peptides, or oil emulsions (emulsion preparations)
  • ribosome preparations, granular preparations bonded to beads having a diameter of several ⁇ preparations bonded to lipids, microsphere preparations, and microcapsule preparations are also conceivable.
  • the cell-derived components or derivatives thereof include, for example, (i) dead bacteria of bacteria, (ii) cell wall skeleton (abbreviated as Cell Wall Skeleton, CWS) derived from bacteria, (iii) cells It is classified into a specific component derived from or a derivative thereof.
  • CWS cell wall Skeleton
  • killed bacteria include, for example, streptococcus powder (for example, Picibanil; Chugai Pharmaceutical Co., Ltd.), dead bacterial suspension cocktail (for example, Broncasma Berna; Sanwa Chemical Laboratory), or Mycobacterium tuberculosis And the like.
  • streptococcus powder for example, Picibanil; Chugai Pharmaceutical Co., Ltd.
  • dead bacterial suspension cocktail for example, Broncasma Berna; Sanwa Chemical Laboratory
  • CWS derived from bacteria include CWS derived from Mycobacteria (for example, CWS of BCG strain that is Mycobacterium spp.), CTS derived from Nocardia (for example, CWS of Nocardia.Nobra), Or CWS derived from Corynebacterium ——
  • Specific components derived from bacterial cells or derivatives thereof include, for example, polysaccharide components derived from Mycobacterium tuberculosis, which are polysaccharides derived from bacterial cells (eg, Answer; Zeria Shinyaku Kogyo Co., Ltd.) and polysaccharides derived from Basidiomycetes ( For example, lentinan; Ajinomoto, Krestin; Sankyo Co., Ltd., Basidiomycetes kawatake mushroom), muramyl dipeptide (MDP) -related compound, lipopolysaccharide (LPS), lipid A-related compound (MPL), glycolipid trehalose dimycolate (TDM) And bacterial DNA (eg, CpG oligonucleotide), or derivatives thereof.
  • polysaccharide components derived from Mycobacterium tuberculosis which are polysaccharides derived from bacterial cells (eg, Answer; Zeria Shinyaku Kogyo Co., Ltd
  • cell-derived components and their derivatives may be obtained from commercially available sources, or may be obtained from known literature (eg, Cancer Res., 33, 2187-2195 (1973), J. Natl. Cancer Inst. ., 48, 831-835 (1972), J. Bacterid., 94, 1736-1745 (1967), Gann, 69, 619-626 (1978), J. Bacterid., 92, 869-879 (1966) , J. Natl. Cancer Inst., 52, 95-101 (1974)) and the like.
  • known literature eg, Cancer Res., 33, 2187-2195 (1973), J. Natl. Cancer Inst. ., 48, 831-835 (1972), J. Bacterid., 94, 1736-1745 (1967), Gann, 69, 619-626 (1978), J. Bacterid., 92, 869-879 (1966) , J. Natl. Cancer Inst., 52, 95-101 (1974)
  • the “cytokine” includes, for example, IFN- ⁇ , -12, GM-CSF, IL-2, IF ⁇ - ⁇ , IL-18, IL-15, and the like. These sites may be natural products or genetically modified products. These site power-ins can be obtained and used if they are already commercially available.
  • a genetically modified product the desired gene is cloned by a conventional method based on each base sequence registered in a database such as GenBank, EMBL, or DDBJ, and ligated to an appropriate expression vector. Expression and production can be achieved by transforming host cells with the produced recombinant expression vector.
  • the “plant-derived component or derivative thereof” refers to, for example, Qui.l A (Accurate Chemical & Scientific Corp., QS-21 (Aquila Biopharmac euticals inc.)) Or glycyrrhizin (SIG A-ALDRICH) which is a saponin-derived component And so on.
  • the “marine organism-derived component or derivative thereof” includes, for example, ⁇ -galactosylceramide, which is a saccharide and fat derived from sponge.
  • the oil emulsion includes, for example, a water-in-oil (w / o) emulsion formulation, an oil-in-water (o Zw) emulsion formulation, and a water-in-oil-in-water (wZoZ) formulation.
  • w / o water-in-oil
  • o Zw oil-in-water
  • wZoZ water-in-oil-in-water
  • the water-in-oil (wZo) emulsion preparation takes a form in which an active ingredient is dispersed in a water dispersed phase.
  • Oil-in-water (o Zw) emulsion preparations take the form in which the active ingredient is dispersed in a water dispersion medium.
  • the water-in-oil-in-water (wZoW) emulsion formulation takes a form in which the active ingredient is dispersed in the innermost water dispersion phase. Preparation of such an emulsion preparation can be carried out with reference to, for example, JP-A-8-9885, JP-A-9-122476, and the like.
  • the liposome preparation is a fine particle in which an active ingredient is wrapped in an aqueous phase or a membrane by ribosomes having a lipid bilayer structure.
  • Principal lipids for producing liposomes include phosphatidylcholine, sphingomyelin, and the like. Dicetyl phosphate, phosphatidic acid, phosphatidylserine, etc. are added to the liposome to stabilize it by charging.
  • Examples of the method for preparing ribosome include an ultrasonic method, an ethanol injection method, an ether injection method, a reverse phase evaporation method, a French pre-extraction method and the like.
  • the microsphere preparation is composed of a homogeneous polymer matrix, and is a fine particle in which an active ingredient is dispersed in the matrix.
  • Materials for the matrix include biodegradable polymers such as albumin, gelatin, chitin, chitosan, starch, polylactic acid, and polyalkylcyanoacrylate.
  • biodegradable polymers such as albumin, gelatin, chitin, chitosan, starch, polylactic acid, and polyalkylcyanoacrylate.
  • Known methods for preparing microsphere preparations (Eur. J. Pharm. Biopharm. 50: 129-146, 2000, Dev. Biol. Stand. 92: 63-78, 1998, Pharm. Biotechnol. 10: 1). —43, 199 7), etc., and there is no particular limitation.
  • the microcapsule preparation is fine particles in a form in which the active ingredient is a core substance and is covered with a coating substance.
  • coating materials used for the coating substance include carboxy simethinoresenolylose, cenorellose acetate phthalate, ethynolecellulose, gelatin, gelatin 'Gum arabic, nitrosenorelose, polyvinyl alcohol resin, and hydroxypropylcellulose.
  • Film-forming polymers such as cellulose.
  • the method for preparing the microcapsule preparation include a coacervation method and an interfacial polymerization method.
  • Administration methods include intradermal, subcutaneous, intramuscular, and intravenous administration.
  • the dosage of the tumor antigen protein of the present invention in the formulation depends on the disease to be treated, It can be adjusted appropriately depending on age, weight, etc., but is usually 0.0001 mg to 1000 mg , preferably 0.001 mg to 100 mg , more preferably 0.01 mg to 10 mg , Preferably, it is administered once.
  • the present invention provides a CTL inducer comprising the peptide of the present invention as an active ingredient.
  • the peptide of the present invention is a CTL inducer having CTL inducing activity.
  • the induced CTL can exert an antitumor effect through the production of cytotoxic action lymphoforce. Therefore, the peptide of the present invention can be used as an active ingredient of a medicament for treating or preventing a tumor.
  • a CTL inducer containing the peptide of the present invention as an active ingredient is administered to a tumor patient, the peptide of the present invention is displayed on the HLA antigen of antigen presenting cells, and the binding complex between the displayed HLA antigen and the peptide is expressed.
  • Body-specific CTLs can proliferate and destroy tumor cells, thus treating or preventing a patient's tumor.
  • the CTL inducer comprising the peptide of the present invention as an active ingredient is used for any tumor patient positive for ASK, CKS1, MELK, STK12, TTK or GPR87 protein described in any of SEQ ID NOs: 13 to 18. be able to. Specifically, it can be used for prevention or treatment of cancer (tumor) such as lung cancer, breast cancer, prostate cancer, and colon cancer.
  • the CTL inducer comprising the peptide of the present invention as an active ingredient is administered in a mixture with a pharmaceutically acceptable carrier, for example, an appropriate adjuvant, or in combination with an appropriate adjuvant so that cellular immunity is effectively established. can do.
  • a pharmaceutically acceptable carrier for example, an appropriate adjuvant, or in combination with an appropriate adjuvant so that cellular immunity is effectively established. can do.
  • the adjuvant those described in the literature (Clin. Microbiol. Rev., 7: 277-289, 1994) and the like can be applied, and specifically, components derived from bacterial cells or derivatives thereof, cytokines, plants, and the like.
  • ribosome preparations In addition, ribosome preparations, granular preparations bonded to beads having a diameter of a few meters / zm, preparations bonded to lipids, microsphere preparations, and microcapsule preparations are also conceivable.
  • these adjuvants see the section of “(9) CTL inducer containing tumor antigen protein of the present invention as active ingredient”. 'No.
  • Administration methods include intradermal, subcutaneous, intramuscular, and intravenous administration.
  • the dosage of the peptide of the present invention in the formulation can be appropriately adjusted depending on the disease to be treated, the age of the patient, body weight, etc., but is usually 0.0001 mg to 1000 mg, preferably 0.0001 mg to 1000 mg, more preferably Is 0.1 mg to 10 mg, and is preferably administered once every several days to several months.
  • CTL inducer comprising the nucleic acid of the present invention as an active ingredient
  • Cells expressing the nucleic acid of the present invention are characterized by being recognized by CTL. That is, the nucleic acid of the present invention is an inducer of CTL.
  • the induced CTL can exert an antitumor effect through cytotoxicity and lymphokine production. Therefore, the nucleic acid of the present invention can be used as an active ingredient of a medicament for treating or preventing a tumor.
  • the CTL inducer containing the nucleic acid of the present invention as an active ingredient is capable of treating or preventing a tumor by, for example, administering and expressing the nucleic acid of the present invention to a tumor patient.
  • a tumor antigen protein is highly expressed in antigen-presenting cells.
  • the tumor antigen peptide produced by the intracellular degradation binds to the HLA antigen to form a complex, and the complex is presented at a high density on the surface of the antigen-presenting cell, whereby tumor-specific CTLs are expressed in the body. Proliferates efficiently and destroys tumor cells. In this way, the treatment or prevention of tumors is achieved.
  • the CTL inducer containing the nucleic acid of the present invention as an active ingredient includes an ASK gene represented by SEQ ID NO: 1 and any ASK-positive tumor patient as an expression product of the gene; a CKS1 gene represented by SEQ ID NO: 2; Any CKS1-positive tumor patient, which is an expression product of the gene; the MELK gene described in SEQ ID NO: 3, and any MELK-positive tumor patient, which is an expression product of the gene; Any tumor patient positive for STK12 which is an expression product, any TTK gene described in SEQ ID NO: 5 and any tumor patient positive for TTK which is an expression product of the gene, or any GPR87 gene described in SEQ ID NO: 6 and any It can be used for any GPR87-positive tumor patients that are gene expression products.
  • nucleic acid of the present invention can be used for prevention or treatment of cancer (malignant tumor) such as lung cancer or kidney cancer.
  • Methods for administering the nucleic acid of the present invention and introducing it into cells include a method using a viral vector and other methods (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, (1), 23-48 ( 1994), Experimental Medicine Special Edition, 12 (15), (1994), and references cited therein) can be applied.
  • Methods using viral vectors include, for example, retrovirus, adenovirus
  • Adeno-associated virus herpes virus, vaccinia virus, box virus, polio virus, simvis virus, and other DNA viruses or RNA viruses, and the method of incorporating and introducing the DNA of the present invention.
  • a method using retrovirus, adenovirus, adeno-associated virus, vaccinia virus and the like is particularly preferable.
  • DNA vaccine and liposomal methods are preferred.
  • nucleic acid of the present invention In order for the nucleic acid of the present invention to actually act as a medicine, an in vivo method in which the nucleic acid is directly introduced into the body, a method in which certain cells are collected from humans, and the nucleic acid is introduced into the cells outside the body, and There is an ex vivo method of returning cells to the body (Nikkei Science, April 1994, pp. 20-45, Monthly Pharmaceutical Affairs, ⁇ (1), 23-48 (1994), Special Issue on Experimental Medicine, 1 ⁇ (15), (1994), and references cited therein). In vivo methods are more preferred.
  • the in vitro method When administered by the in vitro method, it can be administered by an appropriate route depending on the disease, symptom and the like to be treated. For example, it can be administered intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, and the like. When administered by the in vivo method, for example, it can be in the form of a liquid preparation or the like. However, it is generally an injection containing the nucleic acid of the present invention as an active ingredient. An acceptable carrier (carrier) may be added. In addition, a ribosome or a membrane fusion ribosome containing the nucleic acid of the present invention (Sendai virus (HVJ
  • One ribosome, etc. can be in the form of a liposome preparation such as a suspending agent, a cryogen, or a centrifugal concentrated cryogen.
  • the content of the nucleic acid of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient. -It is preferred to administer from 0.01 mg to 100 mg, preferably from 0.001 mg to 10 mg, of the nucleic acid according to the invention once every few days to several months.
  • a polynucleotide encoding an epitope peptide linked to a plurality of CTL epitopes tumor antigen peptides
  • a polynucleotide encoding an epitope peptide linked to a CTL epitope and a helper peptide It has been shown to have CTL-inducing activity efficiently in vivo.
  • Journal of Immunology 1999, 162: 3915-3925 includes six HBV-derived HLA-A2-restricted antigen peptides, three HLA-A11-restricted antigen peptides, and a helper epitope-linked epitope peptide. It is described that the CTL against each epitope was effectively induced in vivo (minigene). .
  • a polynucleotide produced by linking one or more kinds of polynucleotides encoding the peptide of the present invention, and in some cases, also linking polynucleotides encoding other peptides By incorporating it into an appropriate expression vector, it can be used as an active ingredient of a CTL inducer.
  • Such an agent for inducing CTL can also take the same administration method and administration form as described above.
  • antigen-presenting cells can be prepared by bringing any of the proteins, peptides and nucleic acids of the present invention into contact with cells capable of presenting antigen in vitro.
  • the present invention provides a cell of the cell by contacting an isolated antigen-presenting cell derived from a tumor patient with any of the proteins, peptides and nucleic acids of the present invention in vitro. It is intended to provide an antigen-presenting cell having a complex of an HLA antigen and the peptide of the present invention presented on the surface, and a method for producing the same.
  • the term "cells having antigen-presenting ability” is not particularly limited as long as they express an HLA antigen capable of presenting the peptide of the present invention on the cell surface. Dendritic cells are preferred.
  • the substance to be added for preparing the antigen-presenting cell of the present invention from the cell having the antigen-presenting ability includes any of the protein, peptide and nucleic acid of the present invention. 'You can.
  • the antigen-presenting cell of the present invention is obtained by isolating cells having an antigen-presenting ability from a tumor patient, pulsing the cell with the protein or peptide of the present invention in vitro, and allowing the HLA antigen to bind to the peptide of the present invention. Obtained by displaying the complex (Cancer Immunol. I ugly unother., 46:82, 1998, J. Immunol., 158, pl796, 1997, Cancer Res., 59, pi 184, 1999).
  • dendritic cells for example, lymphocytes are separated from peripheral blood of tumor patients by the Ficoll method, non-adherent cells are removed, and adherent cells are cultured in the presence of GM-CSF and IL-4.
  • the antigen-presenting cells of the present invention can be prepared by inducing dendritic cells, culturing the dendritic cells with the protein or peptide of the present invention, and pulsing the cells.
  • the nucleic acid may be in the form of DNA or RNA. good.
  • DNA can be used with reference to Cancer Res., 56: p5672, 1996 and J. Immunol., 161: p5607, 1998.
  • RNA J. Exp. Med. , 184: p465, 1996.
  • the antigen presenting cell can be used as an active ingredient of a CTL inducer.
  • the CTL inducer containing the antigen presenting cell as an active ingredient preferably contains physiological saline, phosphate buffered saline (PBS), a medium, etc., in order to stably maintain the antigen presenting cell. Les ,.
  • Administration methods include intravenous administration, subcutaneous administration, and intradermal administration.
  • the proteins, peptides and nucleic acids of the present invention can be used in vitro in the treatment of tumor patients as follows. That is, CTLs can be induced by bringing any of the proteins, peptides and nucleic acids of the present invention into contact with peripheral blood lymphocytes in vitro. Specifically, the present invention provides CTLs induced by bringing a peripheral blood lymphocyte derived from a tumor patient into contact with any of the proteins, peptides and nucleic acids of the present invention in vivo, and a method for inducing the same. What to do It is.
  • the CTL can be used as an active ingredient of a therapeutic or prophylactic agent for tumor.
  • the therapeutic or prophylactic agent preferably contains physiological saline, phosphate buffered saline (PBS), a medium, and the like, in order to stably maintain CTL.
  • Administration methods include intravenous administration, subcutaneous administration, and intradermal administration.
  • the present invention provides an antibody that specifically binds to the peptide of the present invention.
  • the form of the antibody of the present invention is not particularly limited, and may be a polyclonal antibody using the peptide of the present invention as an immunogen or a monoclonal antibody.
  • the antibody of the present invention is not particularly limited as long as it specifically binds to the peptide of the present invention as described above. Antibodies specifically binding to tumor antigen peptides. Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced by these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons.Section 11.12-1L 13, Antibodies; A Laboratory Manual, Lane, H, D. et al., Cold Spring Harber Labora tory Press, New York 1989).
  • the peptide of the present invention (for example, a tumor antigen peptide consisting of the amino acid sequence of any of SEQ ID NOs: 37 to 225) is used as an immunogen to immunize a non-human animal such as a rabbit, It can be obtained from serum of the immunized animal according to a conventional method.
  • a non-human animal such as a mouse is immunized with the peptide of the present invention (for example, a tumor antigen peptide having an amino acid sequence described in any of SEQ ID NOs: 37 to 225).
  • a hybrid prepared by fusing the obtained spleen cells with myeloma cells is a force obtained from the neutral force of Ima Makoto Itoda (Current protocols in Molecular Biology e dit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11. 4 ⁇ 11. 11).
  • an antibody against the peptide of the present invention can also be carried out by increasing the immunological reaction using various adjuvants depending on the host.
  • adjuvants include Freund's adjuvant, mineral gels such as aluminum hydroxide, as well as lysolecithin, pull-mouthed polyols, polyanions, peptides, oil emulsions, and the emulsions of quinocyanine and dinitrophenol.
  • surface active substances include BCG (Bacillus Calmette-Guerin) and human adjuvants such as Corynebacterium-Parvum.
  • an antibody that recognizes a peptide and an antibody that neutralizes the activity can be easily prepared by appropriately immunizing an animal using the peptide of the present invention in a conventional manner.
  • Uses of the antibody include affinity chromatography, immunological diagnosis and the like. Immunological diagnosis can be appropriately selected from an immunoblot method, a radioimmunoassay method (RIA), an enzyme immunoassay method (ELISA), a fluorescence or luminescence measurement method, or the like.
  • a cancer expressing the ASK gene, CKS1 gene, MELK gene, STK12 gene, TTK gene or GPR87 gene of the present invention for example, a cancer such as a lung cancer or a renal cancer (malignant tumor).
  • Example 1 Example 1
  • DNA chip analysis was performed using total RNA prepared from the sample shown in Example 1.
  • the DNA chip analysis was performed using Affytnetrix's Gene Chip Human Genome U133 set. Specifically, the analysis includes (1) preparation of cDNA from total RNA, (2) preparation of labeled cRNA from the cDNA, (3) fragmentation of labeled cRA, (4) fragmentation of cRNA and probe Hybridization with the array, (5) probe array staining, (6) probe array scanning, and (7) gene expression analysis were performed.
  • DEPC-treated water Sterilized distilled water 91 L, 5X Second Strand Reaction Buffer 30 ⁇ L included in the kit, 10 mM dNTP Mix 3 / iL, E. coli DNA Ligase l included in the kit / L (10 U), 4 ⁇ L of E. coli DNA Polymerase I (40 U) X E. coli RNaseH 1 U2U contained in the kit) were added, and reacted at 16 ° C. for 2 hours. Next, T4 DNA Polymerase 2 / L (10 U) included in the kit was added, and the mixture was reacted at 16 ° C. for 5 minutes, and then 0.5 M EDTA 10 / L was added.
  • DEPC-treated water 17 // L, 10XHY Reaction Buffer 4juL included in BioArray High Yield RNA Transcript Labeling Kit (manufactured by ENZ0), lOXBiotin Labeled Ribonucleotides 4 / iL included in the kit, 10XDTT ⁇ L included in the kit 4 ⁇ L of lOXRNase Inhibitor Mix included in the kit and 20 ⁇ 7 RNA Polymerase included in the kit were mixed, and reacted at 37 ° C for 5 hours to prepare labeled cRNA. After the reaction, 60 L of DEPC-treated water was added to the reaction solution, and the prepared labeled cRNA was purified using the RNeasy Mini Kit (GIAGEN) according to the attached protocol.
  • RNeasy Mini Kit GAAGEN
  • a Human genome U133 probe array (Affymetrix) filled with 1X MES hybridization buffer was rotated in a hybridization oven at 45 ° C and 60 rpm for 10 minutes, and then 1X MES hybridization was performed. The buffer was removed to prepare the probe array. 200 L of the Hyplicocktail supernatant obtained above was added to each of the probe arrays, and rotated at 45 ° C and 60 rpm for 16 hours in a Hyprio oven to obtain a probe array hybridized with the fragmented cRNA.
  • Non-Stringent Wash Buffer (6 X SSPE (diluted 20 X SSPE (manufactured by Nacalai Tesque)), 0.01% Tween 20, 0 005 AntifoamO-30 (Sigma)).
  • the fragmented cRNA was hybridized to a predetermined position of GeneChip Fluidics Station 400 (manufactured by Affymetrix) in which Non-Stringent Wash Buffer and Stringent Wash Buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) were set.
  • the probe array was attached.
  • the primary staining solution (10 / xg / tnL Strepta vidin Phycoerythrin (SAPE) (MolecuLar Probe), 2 mg / mL Acetylated BSA, 10 Om MES, 1M NaCl (Ambion), .05% Tween 20, 0.005% AntifoamO-30), secondary staining solution (100 / ig / mL Goat IgG (Sigma), 3 ⁇ g / mL Biotinylated Anti-Streptavidin antibody (Vector Laboratories), 2mg / mL Acetylated BSA, 100 mM MES, 1M NaCl, 0.05% Tween 20, 0.05% Antifoam 0-30).
  • SAPE Strepta vidin Phycoerythrin
  • secondary staining solution 100 / ig / mL Goat IgG (Sigma), 3 ⁇ g / mL Biotinylated
  • the Human U133 probe name indicates the probe name in Human Genome U133 Chi
  • the Accession No indicates the accession number in the GenBank database.
  • the numerical values indicate the variability in the expression of various cancers with respect to the corresponding normal tissues, and the cancer tissues derived from the same tissues when the gene expression level of the various normal tissues analyzed with the Human Genome U133 Chip is 1 The expression level of the gene was shown.
  • Table 9 show the expression frequency (%) of each gene in various cancer and normal tissues.
  • the six genes selected were genes whose expression levels and expression frequencies were specifically increased in the corresponding cancer tissues as compared with normal tissues derived from various major organs. Therefore, these six genes and their expression products (proteins) were considered to be applicable as disease markers for cancer. In addition, it was considered that by using these genes or their expression products (proteins), it was possible to screen candidate therapeutic agents for alleviating or suppressing cancer cell growth.
  • cancer-specific expressed genes include tumor antigen proteins as active ingredients. It was thought that it could be used as an inducer of CTL.
  • siRNA having a duplex formed by a sense strand and an antisense strand was purchased from PR0LIG0.
  • Table 10 shows the sequences of the prepared siRNAs. Each siRNA name is indicated by using a gene abbreviation.
  • siRNA name sequence (sequence number)
  • MELK ant isense: 5 '' -AUCCUCCAUUGUUUGCCUGUU-3 '(SEQ ID NO: 26)
  • TTK sense 5 '' -GACACCAAGCAGCAAUACCUU-3 '(SEQ ID NO: 27)
  • STK12_C sense: 5 '' -CAUCCUGCGUCUCUACAACUA-3 '(SEQ ID NO: 29)
  • CKS1B sense: 5 '' -UGAAGCUGGCAAGCUACUUUU-3 '(SEQ ID NO: 33)
  • siRNA As a negative control, one of three types of siRNA, Scramble II (Dharmacon Research soil), Control (non-silencing siRNA (Qiagen-Xeragon3 ⁇ 4n and Silencer Negative Control # 1 siRNA (Ambion)) used.
  • Example 8 After transfection of each gene-specific siRNA shown in Example 8 into each human lung cancer cell line, the medium was replaced with the corresponding medium of each cell line containing 10% alamar venore (TREK Diagnostic Systems) after 45 minutes. -Incubated for 2 hours at 37 ° C, and measured the fluorescence intensity with a fluorescent plate reader Fluoroskan Asent (Labsystems) to evaluate the cell proliferation ability.
  • Example 3 For each gene identified in Example 3, the proliferative capacity of a lung cancer cell line transfected with the specific siRNA was calculated assuming that the proliferative capacity of cells not treated with siRNA was 100%.
  • Table 11 shows the names of cell lines that highly express the siRNA into which each gene was introduced, and the fold change in the expression of each gene in normal lung tissue in that cell line.
  • the effect of the negative control siRNA shown in Example 8 on the growth of each cancer cell line was similarly shown in terms of the expression change factor. 'Table 1 1
  • siRNA SEQ ID NOs: 25 to 36
  • the inhibitor obtained by the inhibitor screening for the identified six genes may be a cancer cell-selective growth inhibitor because the expression of this gene shows cancer-specific expression.
  • the method of inducing CTL with peptides and evaluating the activity of the CTL can be performed according to the Atsey method using known human model animals (W002 / 47474, Int. Cancer: 100, 565-570 (2002)).
  • a peptide derived from a cancer antigen is administered to a transgenic mouse expressing human HLA in an appropriate dosage form, and spleen cells or other lymphoid tissues are obtained about one week later.
  • the obtained cells are subjected to antigen stimulation in vitro with the same peptide as that to which the cells were administered, and further cultured for about 50 minutes.
  • the resulting et cells and CTL fraction expressed 5 1 Cr radioactive material such as, or other non-radioactive substance-labeled target cells (administered peptide binds HLA,, further code administration peptide cancer Cells expressing the antigen or the peptide to be administered or cells to which the peptide has been added in advance), and the destruction of target cells by CTL is measured by the amount of radioactive and non-radioactive substances in the culture supernatant. Evaluate activity.
  • the activity of CTL can also be evaluated by measuring the IFN produced in the culture supernatant as a result of the reaction of CTL cells with target cells by ELISA or ELISPOT. .
  • the sequence of the antigenic peptide presented by binding to the HLA molecule has a regularity (motif).
  • the second amino acid of the peptide consisting of 8 to 11 amino acids is tyrosine and phenylalanine. It is known that the C-terminal amino acid is phenylalanine, tryptophan, leucine, isoleucine or methionine (I Oki unogenetics, 41: 178, 1995; J. Immunol., 152: 391). 3, 1994, J. Immunol., 155: 4307, 1994).
  • a peptide portion consisting of 8 to 11 amino acids having an HL ⁇ - ⁇ 24 binding motif is selected from the six amino acid sequences (SEQ ID NO: 13 to SEQ ID NO: 18) of the tumor antigen protein of the present invention. did.
  • the amino acid sequences of these peptides are shown in SEQ ID NO: 37 to SEQ ID NO: 225.
  • one or more peptides for each of the six tumor antigen proteins ie, SEQ ID NOs: 39, 75, 95, 131, 157, 161, 165 , 192, 194, 195, 210, 222-225 were synthesized by the Fmoc method.
  • the peptide represented by SEQ ID NO: 39 is a tumor antigen peptide derived from ASK
  • the peptide represented by SEQ ID NO: 75 is the tumor antigen peptide derived from CKS1
  • the peptide represented by SEQ ID NO: 95, 222-225 Is a tumor antigen peptide derived from MELK
  • the peptide represented by SEQ ID NO: 131 is a tumor antigen peptide derived from STK12
  • SEQ ID NOs: 157, 161 and 165 the peptide is a tumor antigen peptide derived from TTK
  • SEQ ID NO: 192 , 194, 195, 210 are tumor antigen peptides derived from GPR87.
  • HLA-A240 This was performed using Kb transgenic mice.
  • the production of the transgenic mouse and the measurement of in vivo immunogenicity are described in detail in WO 02/47474 and Int J. Cancer: 100, 565-570 (2002). It was carried out with reference to the method described. That is, the above peptide was administered to an HLA-A2402Kb transgenic mouse in an appropriate dosage form, and one week later, spleen cells were obtained.
  • Antigen stimulation was performed in vitro with the same peptide as that to which the obtained cells were administered. The cells obtained about 16 hours later were used as the CTL fraction, and the number of IFNv-producing cells was measured by the ELISPOT method to evaluate the presence or absence of antigen-specific CTL inducing activity.
  • Each synthetic peptide (SEQ ID NO: 39, 75, 95, 131, 157, 161, 165, 192, 194, 195, 210, 222-225) was adjusted to 4 Omg / m1 with DMS O, Each was further diluted to 2.4 mg / ml with physiological saline. Then, using a glass syringe, a water-in-oil emulsion was prepared by mixing with 1.27 times the amount of incomplete Freund's adjuvant (ISA51), and 200 ⁇ l of the drug was added to the solution.
  • ISA51 incomplete Freund's adjuvant
  • spleen was excised, rubbed and broken with a frosted part of a slide glass, and spleen cells were collected and prepared.
  • a CK buffer (0.15 NH 4 C1, lOm KHC0 3, 0. ImM EDTA, H7.2-7.4) a 2 X10 7 ce lls / ml concentration of splenocytes hemolysis treatment with, a plate of ELISPOT was prepared in advance Lic ⁇ Trip / well (2 X 10 6 cells) was added by Triplicate.
  • peptide-added group add the same peptide as the administered peptide so that the peptide concentration becomes 5 g / ml.
  • For the non-added group add only the medium, and add 37 ° C, 5% CO2.
  • the cells were cultured at 2 for about 16 hours.
  • the medium used was RPMI-1640 medium supplemented with 10% FCS and antibiotics.
  • the measurement was performed using a commercially available Mouse IFNy EL I SPOT kit (Mouse IFN gamma ELISPOT Set, Catalog No. BD-551083, BD Pharmingen). The measurement was performed according to the attached manual, and the number of spots / well in the results was measured using KS Elispot Reader Compact (Carl Zeiss). In order to examine peptide specificity, peptides were added during the CTL reaction. The number of IFNy-producing cells was evaluated in the group (Peptide (+)) and the group without peptide (Peptide (-)).
  • the results are shown in FIGS.
  • the vertical axis indicates the number of spots (the number of cells producing IFNy) / 2 ⁇ 10 6 cells, and the horizontal axis indicates the tumor antigen peptide used.
  • the tumor antigen peptide is described by the name of the tumor antigen protein from which it is derived, and the number in the square indicates the amino acid sequence number of the first residue of the peptide sequence. Therefore, for example, the ASK-derived tumor antigen peptide represented by SEQ ID NO: 39 is represented as ASK (228).
  • one or more partial peptides for each of the six proteins (ASK, CKS1, MELK, STK12, TTK, and GPR87), and a total of 15 peptides
  • the induction of targeted CTL revealed that these peptides were tumor antigen peptides.
  • genes whose expression level and / or expression frequency are specifically increased in cancer diseases have been revealed. These genes are useful as marker genes (probes and primers) used for gene diagnosis of cancer diseases.
  • suppression of the expression of the above genes suppresses cancer cell growth, thereby suppressing the expression of these genes, or It is possible to screen and select candidate drugs that can be used as therapeutic agents for cancer diseases by using the expression and suppression of the function (activity) of the protein encoded by the gene as an index.
  • proteins can be used as novel tumor antigens, and tumor antigen peptides derived from these tumor antigen proteins are also provided. These are used as CTL inducers in the field of cancer immunity be able to.
  • the base sequence described in SEQ ID NO: 25 is MELK sense.
  • the base sequence described in SEQ ID NO: 26 is MELK antisense.
  • the base sequence described in SEQ ID NO: 27 is TTK sense.
  • the base sequence described in SEQ ID NO: 28 is TTK antisense.
  • the base sequence described in SEQ ID NO: 29 is STK12 sense.
  • the nucleotide sequence set forth in SEQ ID NO: 30 is STK12 antisense.
  • the base sequence described in SEQ ID NO: 31 is ASK sense.
  • the base sequence described in SEQ ID NO: 32 is ASK antisense.
  • the base sequence set forth in SEQ ID NO: 33 is CKSl sense.
  • the nucleotide sequence set forth in SEQ ID NO: 34 is CKSl antisense.
  • the base sequence described in SEQ ID NO: 35 is GPR87 sense.
  • the nucleotide sequence set forth in SEQ ID NO: 36 is GPR87 antisense.

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Abstract

Le but de la présente invention est de fournir un marqueur de maladies révélant une pathologie cancéreuse ; un procédé de détection d’une pathologie cancéreuse grâce à l’utilisation du marqueur de maladies ; un procédé pour évaluer l’efficacité d’un médicament pour améliorer la maladie, et un agent déclanchant l’action des lymphocytes T cytotoxiques. A savoir qu’un polynucléotide possédant au moins 15 bases consécutives dans la séquence de base du gène codant l’ASK, du gène codant la CKS1, du gène codant la MELK, du gène codant la STK12, le gène codant la TTK ou le gène codant la GPR87 et/ou un polynucléotide y étant complémentaire sont utilisés comme marqueur de maladies pour détecter les pathologies cancéreuses. L’ASK, la CKS1, la MELK, la STK12, la TKK ou la GPR87 ou un peptide en résultant est utilisé comme agent déclanchant l’action des lymphocytes T cytotoxiques.
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