WO2007037555A9 - Application therapeutique ou diagnostique du gene dusp15 - Google Patents

Application therapeutique ou diagnostique du gene dusp15

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Publication number
WO2007037555A9
WO2007037555A9 PCT/JP2006/320043 JP2006320043W WO2007037555A9 WO 2007037555 A9 WO2007037555 A9 WO 2007037555A9 JP 2006320043 W JP2006320043 W JP 2006320043W WO 2007037555 A9 WO2007037555 A9 WO 2007037555A9
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WIPO (PCT)
Prior art keywords
cancer
gene
protein
antibody
seq
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PCT/JP2006/320043
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English (en)
Japanese (ja)
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WO2007037555A1 (fr
Inventor
Shinichirou Niwa
Yasutaka Makino
Tomoki Ikuta
Kazuya Arai
Takayuki Shindou
Hiromichi Ogura
Original Assignee
Link Genomics Inc
Shinichirou Niwa
Yasutaka Makino
Tomoki Ikuta
Kazuya Arai
Takayuki Shindou
Hiromichi Ogura
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Application filed by Link Genomics Inc, Shinichirou Niwa, Yasutaka Makino, Tomoki Ikuta, Kazuya Arai, Takayuki Shindou, Hiromichi Ogura filed Critical Link Genomics Inc
Priority to JP2007537785A priority Critical patent/JPWO2007037555A1/ja
Publication of WO2007037555A1 publication Critical patent/WO2007037555A1/fr
Publication of WO2007037555A9 publication Critical patent/WO2007037555A9/fr

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/12—Dual-specificity kinases (2.7.12)
    • C12Y207/12001—Dual-specificity kinase (2.7.12.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a D U S P 15 gene which is a gene specifically amplified in cancer, and its therapeutic nucleotide relates to a diagnostic use.
  • Cancer Malignant tumors. (Cancer) are characterized by growth, invasion and metastasis. Local therapies such as surgical resection or radiotherapy cannot adequately deal with metastatic recurrent cancers, and the development of pharmacotherapy, which is a systemic therapy, will improve future cancer treatment results. Expected.
  • Chemotherapy the current center of cancer drug therapy, is a cell killing agent that acts directly on the DNA and / or RNA of cancer cells to kill them.
  • Other than this for example, bone marrow cells, germ cells, hair matrix cells, and gastrointestinal epithelial cells also acted on normal cells with many divisions, causing strong side effects.
  • recent advances in molecular cell biology have elucidated the mechanisms involved in cancer cell invasion, proliferation, and metastasis, and molecular target drugs that act specifically on the specific mechanisms of cancer cells Development is attracting attention.
  • EGFR epidermal growth factor receptor
  • Yretza genetic name: gefitinib
  • a tyrosine kinase inhibitor WO 9 6 Z3 3 9 80
  • breast cancer Herceptin gene: trastuzumab
  • HE R-2 human epidermal growth factor receptor 2
  • the present inventors have conducted extensive research, and as a result, the gene that is frequently amplified in cancer (especially colorectal cancer) is the DUSP 15 gene. I found out.
  • the present inventors have further found that by inhibiting the expression of DU SP 15 protein in colorectal cancer cell lines and cervical cancer cell lines, the growth of cancer cells can be suppressed,
  • the present invention has been completed. That is, the present invention is directed to a cancer therapeutic agent, a method for screening a candidate substance having a cancer-suppressing action, a cancer diagnostic agent, a kit for cancer diagnosis, and a method for cutting cancer described below. Etc.
  • a therapeutic agent for cancer containing an inhibitor of DU S P 15 gene expression as an active ingredient.
  • the cancer therapeutic agent according to (1) ′ above comprising a substance selected from the group consisting of: (3) D'U S P 1 5.
  • the cancer treatment agent according to (3) above which comprises a substance selected from the group consisting of: .
  • a method for screening a substance that inhibits the expression of DU S P 15 gene comprising:
  • a screening method comprising a step of selecting a compound that reduces the expression level as compared with a case where a test compound is not contacted.
  • a method for screening DU SP 15 protein inhibitory substances ′ (a) a step of contacting p USP 15 protein with a test compound, (b) a step of measuring the binding activity between the DU SP 1 5. protein and the test compound, and
  • a screening method comprising a step of selecting a compound that binds to the U S P 15 protein.
  • a cancer therapeutic agent comprising the antibody according to (9 ') above.
  • a cancer diagnostic agent comprising the antibody according to (9) above.
  • a cancer diagnostic agent comprising a nucleotide sequence that can be hybridized under stringent high-precipitation conditions to a DUSP15 gene or a partial nucleotide sequence thereof.
  • a cancer diagnostic kit comprising a polynucleotide comprising a nucleotide sequence that can be hyper-predated under a stringent hyper-precipitation condition in a D U S P 15 gene or a part of the nucleotide sequence thereof.
  • the biological sample is whole blood, serum, or plasma, (1 9) The method described in 3 ⁇ 4.
  • ⁇ (b) a step of detecting and Z or quantifying the binding between the antibody in the above test and DU S P.1 '5' protein.
  • a method for treating cancer comprising the step of administering to a patient a substance that inhibits the activity of DU S P 15 protein.
  • a cancer depressant containing a DUSP 15 gene expression inhibitor as an active ingredient comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, .. SEQ ID NO: 9, or A therapeutic agent for cancer, comprising a polynucleotide having a nucleotide sequence of SEQ ID NO: 10; INDUSTRIAL APPLICABILITY
  • novel drugs, needles and methods useful for the treatment and diagnosis or diagnosis of cancer for example, colorectal cancer
  • candidate compounds having cancer suppressive action for example, colorectal cancer
  • a screening method is provided. Brief description of the drawings ''
  • Fig. 1 is a histogram showing the frequency of DU S P 15 gene relative to the degree of gene amplification in 2.00 samples from colon cancer patients.
  • Figure 2 shows an optical micrograph (phase contrast image) showing the results of RNA i analysis when the siRNA of the DUS ⁇ 1.5 gene was transcribed into the colon cancer cell lines Caco 2 and RKO ⁇ 6. It is.
  • FIG. 3 is a graph showing the results of quantitative evaluation of the R N Ai effect by R T—P C R, when s R N A of the DU S P 15 gene was transferred to the colon cancer cell line C ao 2. .
  • Figure 4 shows the results of evaluating the RNA i effect by measuring the number of viable cells when siRNA of the DU SP 15 gene was transfected into colon cancer cell lines C ac ⁇ 2 and R KO E 6 It is a graph which shows.
  • Figure 5 is a photograph showing the results of verifying the RNA i effect with an optical microscope when siRNA of the DU SP 15 gene is transferred to the cell line C CD 18 CO derived from normal colon tissue. Phase difference image).
  • Figure 6 shows the RNA i effect when siRNA of the DU SP 15 gene is transfected into the cell line C CD 1 8 CO derived from normal colon tissue. It is a graph which shows the result verified by cell number measurement.
  • ⁇ 7 is a photograph showing the results of Northern Hybridization performed using various normal organ tissues.
  • Fig. 8 is an optical micrograph (fluorescence image) of a portion of the tissue (6 cells) of each sample tissue (A to J) derived from a colon cancer patient analyzed by the FISH method.
  • FIG. 9A and FIG. 9B are graphs showing the results of (A) serum derived from a colon cancer patient and (B) serum derived from a healthy subject analyzed by mass spectrometry, respectively.
  • Figures 10A-C are determined by analysis M S / M S,: shows the correspondence between pic and amino acids (or amino acid sequences) shown in Figure 9,.
  • Figure 1 is a graph showing the results of verifying the RNA i effect by measuring the number of viable cells when siRNA of DUSP 1.5 gene was transfected into a cervical cancer cell line.HeLa cell line. . .
  • Figure 12 is a photomicrograph: True (differential interference image) showing the result of observing the movement of the experiment of Figure 11 in time series under a microscope and in detail.
  • the inventors of the present invention conducted verification of amplified genes by an array GH method using a sample derived from a colon cancer patient, and identified a gene amplification region specific to colorectal cancer.
  • Specimen In the region where amplification is occurring at high frequency, the DU DU SP 15 (Dualspe ci ficityphosphatase— 1 i ke 1 5) gene is frequently found in specimens from patients with colorectal cancer. I found it.
  • DUSP 15 belongs to the non-receptor type protein tyrosine phosphatase family, Protein—tyrosinephosphatasef am i 1 y, and includes protein tyrosine phosphatase activity and serine / threonine-specific phosphatase (S erine / threonine—specificphosphatase) with activity '14' Therefore, it is called a bispecific phosphatase (D ua, le secifieityphosphatase (DSP)). .
  • D ua le secifieityphosphatase
  • Protein phosphatase ⁇ f is a serine / threonine specific phosphatase (PP 1; PP 2 etc.) and tyrosine phosphatase (P TP) containing cysteine residues. Divided (T onks
  • P ⁇ P is P T P'I B (C ha r b o n n e au, H. ⁇ e t a 1. (1 9 8 9) P r o c. N at: 1. A c a d. S c i. U
  • DSP DSP
  • M itogen-activate dproteinkinase M itogen-activated protein kinase
  • NA 'phosphatase MA' P kinasephosphat a. Se (MKP)
  • MK P kinase targeting motif In addition to typical D S P like MK P, MK P kinase targeting motif
  • V H 1 derived from ⁇ xinia virus (V a c c i n i a V i r u s) is D S P. (G u a n, K. L., e t a 1.
  • DU SP 15 (VHY) is expressed in testis-specific expression in normal tissues, and in the detailed digit, it is expressed in the pakiten phase (the mitotic stage) spermatocytes, which is the first metaphase of meiosis. It is done.
  • myristate which is required for membrane localization, has a binding site (glycine residue) at the N-terminus, and is localized in the cell membrane and Golgi apparatus. The direct association of DU S P 15 with cancer has not been reported. ⁇
  • the present inventors have also confirmed that cancer cell growth can be suppressed by suppressing the expression of DU S P 15 gene by R.NA i (RNA 1000) ⁇ ). Therefore, by suppressing the expression of the DU S P 15 gene, it becomes possible to treat cancer. It is also possible to diagnose cancer by determining the expression level of DU S P.15 gene.
  • the present invention includes (1) a cancer therapeutic agent containing an OUSP 15 gene expression inhibitor as an active ingredient, and (2) a DU S ⁇ 1 ⁇ protein active or sex inhibitor as an active ingredient.
  • a cancer therapeutic agent is provided. .
  • DUS ⁇ 15 gene refers to NCBI nucleotides on the basis of A ccession No ..: NM_ 8 8 6 1 1 It means the human DU SP 15 gene (SEQ ID NO: 1) consisting of 3 bases (A lonso, A., eta (2 0 0 4) J. B iol. C he m.
  • stringent conditions may be any of f stringent conditions, medium stringent conditions, and high stringent conditions.
  • Low stringent conditions are, for example, the conditions of 5 XSSC, ⁇ X Denhardt's solution, 0. ⁇ % SDS, 50% holmium, 3 2.
  • Medium stringent conditions are, for example, the conditions: 5 XSSC, 5 X Denhardt's solution, 0'. ⁇ % SDS ⁇ 50% formamide, 42 t.
  • “High stringency conditions” are; for example, 5 XSSC 5 X Denhardt's solution; 0.5% SDS, 50% formamide, 50 conditions. Under these conditions, it can be expected that DNA with higher compatibility can be obtained efficiently as the temperature is increased.
  • factors affecting the hybridization stringency may include multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration. For example, it is possible to achieve the same triggency by appropriately selecting these elements. , ⁇ ⁇ ⁇ ' ⁇ '
  • hybridizable polynucleotide the base sequence of SEQ ID NO: 1, for example, 70% or more, when calculated using the default parameters by homology search software such as FAS TA and BLAST, 7 5% or more, 80% or more, 85% or more, 90% or more, 9 1% or more, 9 2% or more, 93% or more, 94% or more, 95% or more, 96% or more, Examples include polynucleotides having 9 7% or more, 98% or more, and 99% or more identity.
  • inhibiting expression of a gene from gene to protein production; the sequence of events (e.g., transcription '(generation of mR NA), translation (generation of protein): including) of By inhibiting any event, it means inhibiting the production of protein encoded by the gene.
  • DU SP 15 protein refers to 2 3 5 amino acid residues registered in the NC Br protein database as A ccession No .: N P — 0 5 4 2 1 7 8
  • the activity of human DUSP 1 5 protein (SEQ ID NO: 2) and substantially the same activity as this protein (eg, protein tyrosine phosphatase activity and serine / threonine specific phosphatase activity)
  • SEQ ID NO: 2 protein tyrosine phosphatase activity and serine / threonine specific phosphatase activity
  • a mutant protein consisting of
  • the amino acid mutation site and number in the above mutant protein are not particularly limited as long as the mutant protein retains substantially the same activity as the original protein, but the number of mutations is, for example, 1 to 50, 1 to 40, ⁇ ! ⁇ 30, 1-2,5! ⁇ 2 0 ,! ⁇ 1 5 pieces, 1 ⁇ ; 1 0 pieces, 1 ⁇ 9 pieces,:! ⁇ 8 pcs! ⁇ 7, 1-6 (1 ⁇ several)! ⁇ 5 pcs! ⁇ Four, .:! ⁇ 3, .1 ⁇ 2, 1 In general, the smaller the number of mutations, the better.
  • such a mutant protein has approximately .70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 9% or more of the amino acid sequence of SEQ ID NO: 2. 2% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more amino acid sequence having identity And proteins having substantially the same activity as the original protein. In general, the larger the homology value, the better.
  • the DU SP 15 protein includes “partial peptides” of the DU SP 15 protein.
  • a partial peptide of DU SP 15 protein a part of the amino acid sequence (SEQ ID NO: 2) of DU SP 15 protein is linked. Any partial peptide consisting of a sequence of X mific acid followed by any of the above may be used as long as it has an activity similar to that of the DU SP 15 protein described above.
  • amino acid sequence represented by SEQ ID NO: 2 at least 20, preferably at least .50, more preferably at least 70, more preferably at least 100, most preferably Preferred examples include polypeptides having an “amino acid sequence” consisting of at least 200 amino acid residues.
  • these polypeptides contain an amino acid sequence corresponding to the portion responsible for the activity of the DU SP 15 protein.
  • the partial peptide used in the present invention is the above-mentioned polypeptide! In ⁇ , one or more amino acid residues in the amino acid sequence (for example, about 1 to 20 pieces, more preferably about 1 to 10 pieces, and more preferably about 1 to 5 pieces) are deleted.
  • the D U S P; l S protein used in the present invention can be prepared from cells or tissues in which the protein is expressed. These proteins can also be synthesized by known peptide synthesizers, or prepared by recombinant methods using appropriate host cells selected from prokaryotes or eukaryotes. Can do.
  • the DU S P 15 protein used in the present invention may be derived from any species. Preferably, it is derived from human.
  • fermentation rate activity protein tyrosine phosphatase 'activity and serine threonine-specific phosphatase activity, etc.
  • fermentation rate activity protein tyrosine phosphatase 'activity and serine threonine-specific phosphatase activity, etc.
  • L 0 to 0 times preferably about 0.5 to 20 times
  • More preferably about 0.5 to 2 times quantitative factors such as the degree of activity and the molecular weight of the protein may be different.
  • Enzyme activity was measured using p-nitrophenyl phosphorate dephosphorylation 3 ⁇ 4 (A lonso, A., Eta 1. (2 04 04) J. B iol. m.
  • cancer therapeutic agent means anticancer agent, cancer metastasis inhibitor, cancer cell apoptosis inducer, cancer cell proliferation inhibitor, cancer cell invasion inhibitor, cancer preventive agent, etc. Used to include.
  • cancer and “tumor” are used as square words having the same meaning.
  • the present invention provides a cancer therapeutic agent containing a substance that inhibits the expression of DU S P 15 gene as an active ingredient.
  • the “DU SP 15 gene expression inhibitor” is not limited as long as it inhibits the expression of DU SP 15 gene.
  • DU SP 15 gene to DU A substance that inhibits transcription to SP 15 mRNA; and
  • RNA i activity against D.U S P 1.5 mRNA or a portion thereof eg, siRNA
  • nucleic acid means R N A or DN A.
  • Nucleic acid as used herein may contain not only purine and pyrimidine bases but also those with other heterocyclic bases that have been modified. 'Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or ethers, amines, etc. It may be converted to a functional group.
  • RNA i is a phenomenon in which the expression of the introduced foreign gene and target endogenous gene is inhibited when a double-stranded RNA having the same or similar sequence as the target gene is introduced into the cell.
  • R ⁇ ⁇ ⁇ ⁇ ⁇ is, for example, a double-stranded RNA that generates RNA interference with a length of 1.9 to 30 bases, such as ds RNA (doublesrand RNA) ⁇ , si RNA (sma 1 1 interfering RNA) Or sh RNA (shorthairpin RA).
  • ds RNA doublesrand RNA
  • si RNA si RNA
  • sh RNA shorthairpin RA
  • -Delivery of RNA, ribosome, etc . The system can be locally delivered to the desired site, and it can be expressed locally using a single vector so that the above double-stranded RNA can be generated. You can.
  • the methods for preparing and using such double-stranded RNA are known from many literatures (Special Table 2).
  • the length of the double-stranded RNA exhibiting the RNA i effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21. ⁇ 25 bases, most preferably 2 1-23 bases.
  • the following si RNA (used in Example 3) can be used.
  • antisense nucleic acid or “antisense poly'nucleotide” includes: a polynucleotide that is complementary to at least one PN.A region of interest, and the polynucleotide is A nucleic acid that can hybridize with at least a part of the region.
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • the antisense nucleic acid of the present invention is Ri ⁇ A, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include sulfur derivatives of nucleic acids, thiophosphate derivatives, and those that are resistant to degradation of oligonucleotides, but are not limited to them. Is not to be done.
  • the antisense nucleic acid to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
  • the nucleic acid thus prepared can be transformed into a desired animal using a known method.
  • the sequence of the antisense nucleic acid is determined by the animal being transformed. It is preferable that the sequence is complementary to the endogenous gene or a part thereof, but it may not be completely complementary as long as the gene expression can be effectively suppressed. '
  • Antisense nucleic acid effective for inhibiting gene translation is about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably, of the target gene transcript. Has a complementarity of about 95% or more. ''
  • the length of the antisense nucleic acid is at least about 0 bases (for example, about 10 to 40), preferably about 15 bases More preferably, it is about 10 bases or more, more preferably about 500 bases or more.
  • 'Antisense nucleic acids can be referred to by publicly known literature: Design: can be made' (eg, Hira. Shima and Inoue, New Chemistry Laboratory 2 Nucleic acid IV gene replication and expression, 'Japan Biochemical Society, Tokyo, Japan) Chemistry, 1.9 93, p. .3 1 9-3 4 7), J. Kawak am ieta 1., P harm T e Ja Pan. V o 1 .8, p.
  • a nucleic acid having a ribozyme activity that specifically cleaves a transcript of the DU SP 15 gene can be used as an active ingredient.
  • ribozyme activity refers to a nucleic acid that specifically cleaves mRNA, which is a transcription product of a target gene.
  • Some ribozymes have a size of 400 nucleotides or more, such as group I introns and MRNA contained in RNase P. 40 nucleotides called hammerhead or hepin types Some have a sex. Domain (protein nucleic acid enzyme, 1.990, .35, p.
  • a compound other than a nucleic acid that inhibits the transcription activity of the DU S P 15 gene can be used as an active ingredient.
  • a compound is, for example, a compound that binds to a factor involved in transcription of the expression of the DU S P 15 gene.
  • Such a compound may be a natural product or a synthetic compound.
  • Such compounds can be obtained by the screening method described below. 1. Cancer treatment containing 2 DU S P 15 protein activity inhibitor M
  • the present invention also provides a cancer therapeutic agent containing a DU.S P 15 protein activity inhibitor.
  • D U S P 15 protein activity inhibitor includes, for example,
  • the “antibody” means an antibody that reacts with the full length or fragment of a protein.
  • the form of the antibody of the present invention is not particularly limited, so long as it binds to the D1J SP 15 protein of the present invention, in addition to the above polyclonal antibody, monoclonal antibody, and human antibody, a human antibody, a genetically modified hydride Also included are typed antibodies, as well as antibody fragments and antibody modifications.
  • An antibody that binds to DU S P 15 protein can be prepared by methods known to the abandoned person. Details of the anti-D, U.S P I ⁇ antibody will be described later.
  • DUS ⁇ 15; a DUSP 15 protein variant with dominant negative properties for proteins means that by expressing the gene that encodes it.
  • a protein that has the function of eliminating or reducing the activity of the endogenous wild-type DU SP 15 protein Kermanihiro Tsuchida, Gene Activity Inhibition Experiment, edited by Takuma Yoshikazu, Yodosha. (2 0 0 1) 2 (See 6-3 3). .
  • a compound other than the above antibody or mutant that binds to DU S P 15 protein can be used as an active ingredient.
  • Such compounds are, for example, compounds that bind to DUSP 15 protein and inhibit its activity.
  • Such compounds can be natural or synthetic.
  • Such a compound can be obtained by a screening method described later.
  • the above-described substance capable of inhibiting the activity of the DUPSP15 protein of the present invention can be used as a cancer therapeutic agent. .
  • the present invention relates to a screening method for a candidate compound having a cancer suppressing action. Also provide ... .
  • One preferred embodiment is a method using as an index the binding between DU S P1.5 protein and a test compound.
  • a compound that binds to DU S P 15 protein is expected to have an effect of inhibiting the activity of DU S P 15 protein.
  • the compound preferably binds to the active site of DU S P 1 5. protein.
  • DU S P 15 protein protein is contacted with a test compound.
  • DU SP 15 protein depending on the indicator for detecting binding to the test compound, ⁇ for example, a purified form of the DU SP 15 protein, still intracellular; is expressed extracellularly, or It may be in a form bound to an affinity column.
  • the test compound used in this method can be appropriately labeled as necessary. As examples of knowledge, radiolabels, fluorescent labels, and the like can be given.
  • the binding of DU S P.1 protein and test compound is then detected.
  • test compounds and substances used in this method There are no particular limitations on the test compounds and substances used in this method. For example, natural. Compound, organic compound, inorganic compound, protein, peptide, etc., and compound library., Gene library expression product, cell extract, cell culture supernatant, fermenting microorganism product , Marine organism extracts, extracts, plant extracts, etc., but are not limited to these. .
  • the binding between the DU S P 15 protein and the test compound can be detected by, for example, a label attached to the test compound bound to the PU S P 15 protein.
  • a change in the activity of the DU S P 15 protein caused by the binding of the test compound to the DU S P 15 protein expressed intracellularly or extracellularly can also be detected as an indicator.
  • the binding activity between a protein and a test compound can be measured by a known method (for example, the p-nitrophenyl phosphate dephosphorylation method (A 1 onso, A., eta I. (2 0 0 4) J. B iol. C he m. 2 7 9, 3 2 5 8 6— 3 2 5 9 1)).
  • the compound isolated by this method is expected to have a cancer suppressing action and is useful as a cancer therapeutic agent.
  • Another aspect of the screening method of the present invention is a method using the expression of the DUPSP15 gene as an index.
  • a test compound is brought into contact with cells expressing the DUSP 15 gene.
  • cells expressing the DUSP 15 gene examples include human, mouse, cat, inu, ushi, hidge, and birds, such as cells derived from pets and livestock.
  • Dusp 15 m gene expressing ⁇ “ cell. ”Refers to cells expressing endogenous DUS ⁇ 15 gene, or exogenous DUS; P 15 gene introduced.
  • cells expressing the gene can be used.
  • Cells in which an exogenous DUSP15 gene is expressed can usually be prepared by introducing each into an expression vector into which the DUSP15 gene has been inserted.
  • the expression vector can be prepared by a general genetic engineering technique. ' ⁇
  • test compound used in this method is not particularly limited.
  • a natural compound, an organic compound, an inorganic compound, a single compound such as a protein, a peptide, and the expression product of a compound library or gene library are used.
  • Cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, etc. are used.
  • Contact of a test compound to a cell expressing the DUSP 15 gene is usually performed by adding the test compound to the culture medium of the cell expressing the DUSP 15 gene. Not. When the test compound is a protein or the like, “contact” can be performed by introducing a DNA vector that expresses the protein into the cell.
  • the expression level of the DUSP 15 gene is measured.
  • gene expression includes both transcription and translation. Measurement of gene expression levels should be performed by methods known to those skilled in the art. And can. For example, mRNA is extracted from cells expressing the DU S PI 5 gene according to a conventional method, and the transcription level of the gene is determined by carrying out the Northern hybridization method or RT-PCR method using this mRNA. Measurements can be made.
  • the promoter region of the DU SP 15 gene is isolated according to a conventional method, and a marker gene (for example, luminescence of luciferase, GFP, galactosidase, etc., a gene that can be detected as an indicator such as fluorescence, color development, etc.)
  • the transcription level of the gene can also be measured by observing the activity of the marker gene.
  • by collecting the protein fraction from the cells that express the DU SP 15 gene and detecting the expression of each DU SP 15 protein by electrophoresis such as SDS-PAGE It is also possible to measure the translation level.
  • it is also possible to measure the translation level of the gene by detecting the expression of the protein by performing Western plotting using an antibody against DUS P.15 protein. Is possible.
  • the antibody used for detecting the DU SP 1; 5 protein is not particularly limited as long as it is a detectable antibody. For example, both monoclonal antibodies, '. Or polyclonal antibodies can be used.
  • a compound that lowers the expression level is then selected as compared with the case where the test compound is not contacted (control).
  • Compounds selected in this way become candidate compounds for cancer therapeutics. .
  • the present invention also provides an anti-DU SP 15 antibody, a cancer therapeutic agent containing this antibody, and the like.
  • the cancer therapeutic agent is used for cancer targeted therapy or targeted drug delivery.
  • anti-DU S P 15 antibody includes an antibody that specifically binds to DU S P 1.5 protein (including fragments (partial peptides) or salts thereof).
  • the anti-DU SP 15 antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.
  • the antibody glass is not particularly well-defined, I g G,. I gM, I gA, I g
  • Antibodies having any isotype such as D or IgE are also included. I g G or I g M is preferable, and ⁇ g G is more preferable in consideration of ease of purification.
  • the term “antibody” is used to mean any antibody fragment or derivative, such as F ab, F ab '. 2 , CD, humanized antibody, multifunctional Antibodies, single chain antibodies (S c F v): etc.
  • the antibody of the present invention can be produced by a known method. Methods for producing such antibodies are well known in the art (eg, Harlow
  • the protein used as a sensitizing antigen is usually DUSP 15 protein or a salt thereof.
  • the DUSP 15 protein includes partial peptides thereof, which are not limited to force, for example, fragments of the amino acid sequence of SEQ ID NO: 2, for example, 20 or more
  • the partial peptide having a continuous amino acid sequence portion of 4 or more, 60 or more, 80 or more, or 1.00 or more.
  • these fragments for example, amino (N) terminal fragment and carboxy (C) terminal fragment are used.
  • one or more (preferably about 1 to 10, more preferably several (1 to 6)) amino acid residues in the above amino acid sequence are deleted. , Substitutions, insertions and additions or additions.
  • salts of DU SP 15 protein or partial peptides used herein include inorganic acids (eg, hydrochloric acid, sulfuric acid) Or a salt with an organic acid (for example, acetic acid, formic acid, propionic acid).
  • the DU S, P "l 5 protein of the present invention used as a sensitization anti-antibody for antibody acquisition is not limited to the animal species from which it is derived, but is a protein derived from mammals such as mice and humans. Proteins are preferred, and proteins derived from special baboon are preferred.
  • DU SP 15 protein as described above, partial peptide thereof or salt thereof (in the present specification, in the description of the antibody, these are collectively referred to as “DU SP, 15 protein”) as an antigen.
  • Administer to mammals such as rats, mice, and rabbits.
  • the dose per anti-thick animal is 0.1 to 100 mg when no adjuvant is used, and 1 to L.0.00 when the adjuvant is used.
  • adjuvants include bromine complete adjuvant (F..CA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally.
  • the immunity 'interval' is not particularly limited, but it may be several days to several weeks, preferably 2 to 5 weeks.
  • antibody-producing cells are collected 1 to 60 days after the final immunization, preferably 1 to 14 days after.
  • Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells. Spleen cells or local lymph node cells are preferred.
  • myeloma cells to be fused with antibody-producing cells generally available cell lines of animals such as mice can be used.
  • the cell line to be used has drug selectivity and cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) in an unfused state, but can survive only in a state fused with antibody-producing cells.
  • HAT selection medium including hypoxanthine, aminopterin, and thymidine
  • myeloma cells are X 6 3 A g. 8. 6 5 3, MSI myeloma cell lines such as NSI /.1.—A g 4—1, N S.0 Zo 1, and small myeloma cell lines such as Yg 2 ”0.
  • the myeloma cell and the antibody-producing cell are fused.
  • the cell fusion, DM EM serum-free, R.PM I - 1 6 40 culture 3 ⁇ 4 for animal cell culture medium such as, 1 XI .0 6 ⁇ : the LX 1 0 7 or Zm 1 of antibody-producing cells 2 X 1.0 5:. ⁇ 2 X 1 0 6 or Zm 1 myeloma were mixed with cells (antibody producing cells with myeloma cells and the cell ratio 2: 1 to 3: 1 is preferred), cells fusion promoter present Perform the fusion reaction under As the cell fusion promoter, polyethylene glycol having an average molecular weight of 100 to 600 daltons can be used. Also, commercially available cell fusion apparatus electrical stimulation (e.g. elect port Poresho emissions) and for ⁇ li: antibody-producing cells and myeloma Y-, and Ma cells can be fused with.
  • the culture supernatant of the growing hybridoma is screened for the presence of antibodies that react with DU S P 15 protein.
  • the screening of pipridoroma may be performed according to a normal method and is not particularly limited. For example, a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by enzyme immunoassay, radioimmunoassay or the like. Cloning of fused cells is performed by limiting dilution. Finally, a hybridoma that is a cell producing a monoclonal antibody that reacts with the DUSP 15 protein is established.
  • a normal cell culture method or ascites formation method can be employed.
  • mammalian derived Eroma cells hive Li dormer about 1 X 1 0 7 cells were administered intraperitoneally to same species animals are grown hybridomas in a large amount. And.:!
  • the antigen described above is administered to mammals such as rabbits, mice, and rabbits.
  • the dose of the antigen per animal is 0.:! To 100 mg when adjuvant is not used, and 10 to 100 g when adjuvant is used.
  • adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide, and juvant.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Aluminum hydroxide aluminum hydroxide
  • juvant juvant.
  • Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. Further, the immunization interval is not particularly limited, and immunization is performed for several days to several weeks, preferably 2 to 5 weeks, 1 to 10 times, preferably 2 to 5 times.
  • enzyme immunoassay enzyme immunoassay (ELISA (enz ume—11 nkedi mmu nosorbentassy) or EIA (enzyme immunassay)
  • radioimmunoassay RIA; radioi mm unoassay
  • Etc. blood is collected on the day when the maximum antibody titer is shown to obtain antiserum.
  • polyclonal antibodies in the antiserum Collect antibody that reacts with DU SP 15 protein (column adsorption 0 min) over a fixed affinity column.
  • the reactivity of the polyclonal antibody in the antiserum against the DU SP 15 protein can be measured by the ELIS A. method or the like. .:
  • F ab or F ab ' 2 fragments can be prepared by digestion with a protease (eg,' pepsin yet papain) by conventional methods.
  • a protease eg,' pepsin yet papain
  • Humanized antibodies are described, for example, by Riechmann et al. (Riechmann JM o 1 B iol. Oct 5; 2 0 3; (3): 8 2 5-8, 1 9 8 8), 'and J ones et al. (J H. nes et al., Nature .3 2 1: 5 2.2—5 2.5, 1 9 8 6). .
  • the antibody that binds to the DU S P 1,5 protein of the present invention can be used for the purpose of, for example, suppressing the proliferation or metastasis of cancer cells.
  • a human antibody or a human antibody is preferred in order to reduce immunogenicity.
  • the antibody When the antibody is used as a diagnostic agent, it may be labeled with a labeling substance for monitoring (eg, radioisotopes, fluorescent substances, etc.). If necessary, it is labeled with radioactive substances, fluorescent compounds, etc. can do.
  • a labeling substance for monitoring eg, radioisotopes, fluorescent substances, etc.
  • radioactive substances, fluorescent compounds, etc. can do.
  • fluorescent labeling compounds fluorescein isothiocyanate
  • the antibody DU SP 15 antibody can be labeled with a bioluminescent compound. The presence of a bioluminescent protein is measured by detecting the presence of fluorescence.
  • bioluminescent compounds for this purpose are luciferin, luciferase and aequorin.
  • the antibody of the present invention can be used for specifically detecting DU S P 15 protein in a subject such as body fluid or tissue.
  • the anti-DU SP 15 antibody used in the present invention itself is an agent having a neutralizing activity that attenuates the activity of the antigen in the therapeutic agent or diagnostic agent of the present invention. However, if necessary, it can be used in combination with other drugs for producing a therapeutic effect. Therefore, in another aspect, the present invention provides an anti-DUP 15 antibody for use in targeted therapy or targeted imaging of cancer (eg, colorectal cancer) and other drugs. And a composition containing such a complex is also provided. According to such an embodiment, the anti-DU SP 15 antibody used in the present invention is used to express other drugs having a therapeutic effect or a labeling agent for diagnosis, etc., and highly express the DU SP 15 protein. Can be delivered to the target site.
  • cancer eg, colorectal cancer
  • Examples of the “other drug” used in the present invention include a viral vector or a non-viral vector for introducing a gene into a target, such as a radioisotope, a therapeutic protein, or a small molecule drug. .
  • examples of the “radioisotope” include fluorine 1 18, Examples include radioactive halogen elements such as iodine—1 2 5 ( 1 2 )) and iodine—1 3 1. Like these radioactive halogen element also radioactive metallic element described above: a labeled antibody or peptide may be wide immediately utilized as radioactive Osamu therapeutic agent or a radioactive diagnostic agent. For example, it can be bound to an antibody or antibody fragment by a known method such as odorization with 1 2 5 I. or 1 3 1 I, or chloramine sputum method.
  • technetium 99 m for diagnosis, indium—11 1 1 and gallium 6 7 ( 6 7 Ga), etc., and for treatment, yttrium 1 90 ( 9 0 Y), rhenium—1 8 6 ( -1 8 6 Re) or rhenium 1 8 8. ( 1 88 Re) can be used.
  • a metal chelator is usually used.
  • EDTA, DTPA, diaminodithio compounds, cyclam, and DOT A are known as gold genus chelating agents. These chelating agents can be pre-bound to the antibody and then labeled with a radioactive metal, or they can be labeled after binding to the antibody after forming a radioactive metal gilt. ;
  • a cytokine that activates cells responsible for immunity is suitable, for example, a hydrointerleukin 2, a human granulocyte, a macrophage, and a colony stimulating factor Examples include human macrophage colony-stimulating factor, human interleukin 12 and the like.
  • toxins such as ricin and diphtheria toxin can be used to directly kill colon cancer cells.
  • the cDNA encoding the therapeutic protein is linked to the cDNA encoding the antibody or antibody fragment, and the DN A encoding the fusion antibody is constructed.
  • a fusion antibody can be produced by inserting the expression vector into a eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • “Small molecule drug” is used herein to mean a diagnostic or therapeutic compound other than “radioisotope” or “therapeutic protein”.
  • small molecule drugs include Nitrogen 'Mustard, Alkylating agents such as cyclophosphamide, antimetabolites such as 5-fluorouracil and mesotrexate, daunomycin, bleomycin, mitomycin C, antibiotics such as daunorubicin and doxorubicin, vincristine, vinblastine, vindesine
  • Anti-cancer agents such as hormonal agents such as plant algiroids, evening moxifen, dexamethasone (clinical oncology (Japan Clinical Oncology Society 1 9 9 6 Cancer and Chemotherapy)), or Hyde mouth Cortisone, Steroids such as prednisone, non-sterolides such as 7spirin, Indomethacin, immunoregulators such as gold zomarate and penicillamine, immunosuppressants such as cycl
  • clemacytin ' Antihistamines such as anti-inflammatory agents (inflammation and anti-inflammatory therapy in 1982 years Biomedical drug Publishing Co., Ltd.) and the like and the like.
  • daunomycin and antibody can be bound by binding between daunomycin and the amino group of the antibody via dartal aldehyde, or the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carpositimide. And the like.
  • virus vector a virus vector modified so as to bind to the anti-DU SP 15 antibody of the present invention can be used (for example, adenovirus vector (W ang, P., Eta (1 9 9 5) S oma tie C e 1 1 and Mol e'c. G enet. '2 1, 4 2 9-44 1), retroviral vectors (N aviaux RK, et. A 1. (1 9 9 6) J. Virol 7 0, 5 7 0 1-5 7 0 5), Lentiviral vector (N aldini, L. (1 9 9 8) C urr. O pin. B iotechno 1 9, 4 5 7-4 6 3)).
  • adenovirus vector W ang, P., Eta (1 9 9 5) S oma tie C e 1 1 and Mol e'c. G enet. '2 1, 4 2 9-44
  • retroviral vectors N aviaux RK, et. A 1. (1 9 9 6) J. Virol 7
  • virus vectors for example, induce apoptosis of cancer cells at target sites (eg, colon cancer) such as cell proliferation-related genes, apoptosis-related genes, and immune regulatory genes.
  • a gene (therapeutic gene) that produces a therapeutic effect such as Viral vectors that bind to anti-DUSP 15 antibodies are When administered to a patient in need of gene therapy with an anti-DUSP 15 antibody, it can be targeted to a site where an antigen recognized by the anti-DUSP 15 antibody (ie, DUSP 1.5) is present.
  • the anti-D U S P 15 antibody and the other drug can be combined chemically or genetically.
  • “chemical bond” includes, for example, ison bond, hydrogen bond, covalent bond, bond by intermolecular force, bond by hydrophobic interaction, etc.
  • ⁇ nativity binding '' includes the mode of binding between the antibody and therapeutic protein when a fusion protein consisting of the antibody and therapeutic protein is produced using a technique such as genetic recombination. To do.
  • Cancer treatment agent containing IS harmful substance Cancer treatment agent containing DUSP 15 protein activity inhibitor
  • anti-D SP15 antibody used in the present invention carries a radioisotope, a therapeutic protein, a small molecule drug, and a therapeutic gene.
  • a therapeutic agent chemically or genetically engineered with any one or any combination of these can be formulated based on a known acupuncture method.
  • a pharmaceutically acceptable carrier can be added as necessary according to a conventional method.
  • surfactants for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity promoters, taste masking
  • the present invention is not limited to these, and other commonly used carriers can be appropriately used.
  • the types of dosage forms of the therapeutic agent of the present invention include, for example, oral tablets,. Powders, pills, powders, granules, fine granules, soft, hard capsules, film coating agents , Pellets, sublinguals, pastes, etc.
  • the optimal dosage form can be selected according to the target.
  • Inhibitors of DU S P 1.5 protein activity (or expression of DU S P 15 gene) as an active ingredient may be included in the preparation in an amount of 0.1 to 99.9% by weight.
  • the dose of the active ingredient of the drug of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc.
  • the daily dose is about 0.1 mg to l, 100 mg, preferably about 1.0 to: I 00 mg, more preferably about 1.0 to 50 mg.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc.
  • patients 60 kg About 0.1 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to: about 1 O mg is conveniently administered by intravenous injection. It is.
  • the type of dosage form, method of administration, age and weight of the patient, patient symptoms, etc. can be taken into account, and can be determined as appropriate by the physician or veterinarian.
  • the preparation thus obtained can be administered, for example, to humans or other mammals (eg, rabbits, magpies, hidges, buyu, ushi, cats, nu, monkeys, etc.). it can. In the case of animals other than humans, the amount converted per 60 kg can be administered.
  • mammals eg, rabbits, magpies, hidges, buyu, ushi, cats, nu, monkeys, etc.
  • the amount converted per 60 kg can be administered.
  • the therapeutic agent of the present invention is cancer (for example, colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, Prevention of bladder cancer,.
  • Uterine cancer eg cervical cancer, endometrial cancer
  • testicular cancer thyroid cancer
  • knee cancer ovarian cancer
  • brain tumor blood tumor, etc.
  • blood tumor used for treatment, preferably 'prophylaxis of colorectal cancer' treatment.
  • the drug of the present invention contains DUSP 15 protein activity inhibitor IV or DU.
  • S 15 gene expression inhibitor as an active ingredient, anti-cancer agent, cancer.metastasis inhibitor, cancer cell apoptosis It can be used as an inducer or the like.
  • the target cell, tissue, organ, or cancer type is not limited to a specific type.
  • the agent of the present invention may contain both a D U S P 15 protein poor activity inhibitor and a D y S P 15 gene expression inhibitor.
  • an antisense nucleic acid is used in the therapeutic agent of the present invention, the antisense nucleic acid is inserted alone or into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associate virus vector, etc. Thereafter, it can be administered according to known means.
  • Antisense nucleic acids can be formulated alone or with a physiologically acceptable carrier and administered via a catheter such as a gene gun or a hide-mouth gel catheter.
  • a virus vector such as a recombinant adenovirus particle and an anti-DUSP 1 ⁇ antibody
  • these may be used alone, but in general, pharmaceuticals are used.
  • a commercially acceptable carrier used with a commercially acceptable carrier.
  • the above-described carrier and aqueous isotonic solutions such as water, physiological saline, glucose, and human albumin are preferable.
  • additives, preservatives, preservatives, balance, etc. that are usually used in pharmaceutics can also be added.
  • the pharmaceutical composition thus prepared can be administered by an appropriate administration form and administration route depending on the disease to be treated.
  • dosage forms include emulsions, syrups, capsules, tablets, granules, injections, ointments and the like.
  • administering usually from 10 3 to 10 15 virus particles at a time per adult.
  • the administration frequency may be once to several times a day.
  • the administration period may be from 1 day to several months or more, with 1 to several doses as one set. May be administered.
  • the virus vector particles or virus vector nucleic acid molecules used in the present invention can be used for detection of specific cells and Z or tissues, or diagnosis of disease states.
  • a viral vector particle obtained by incorporating a detectable marker gene into a nucleic acid molecule of a virus vector and transfection into an appropriate host cell is anti-D 'USP 1 It can be used to detect and diagnose tumor cells in combination with 5 antibodies.
  • a detectable label can be bound to the anti-DU SP 15 antibody and used to excise and diagnose tumor cells.
  • the cancer diagnostic agent of the present invention comprises: (a) an antibody against DU SP 15 protein; or (b) a DUS P 15 gene or a partial nucleotide sequence thereof.
  • ⁇ ⁇ Contains a polynucleotide consisting of a base sequence that can be hybridized under high hybridization conditions.
  • the diagnostic method using the anti-DU SP 15 antibody of the present invention includes, for example, (a) a step of bringing a subject-derived biological sample into contact with an antibody against DU SP 15 protein, and (b) A step of detecting and Z or quantifying the binding between the antibody in the sample and the DU SP 15 protein or a partial peptide or a salt thereof.
  • a step of bringing a subject-derived biological sample into contact with an antibody against DU SP 15 protein includes, for example, (a) a step of bringing a subject-derived biological sample into contact with an antibody against DU SP 15 protein, and (b) A step of detecting and Z or quantifying the binding between the antibody in the sample and the DU SP 15 protein or a partial peptide or a salt thereof.
  • the detection and / or quantification step using the labeled anti-DU SP 15 antibody, DU SP 15 protein or The binding of the fragment to the anti-DUSP 1 ⁇ antibody is detected and / or quantified. : '"
  • biological sample derived from a test refers to a tissue, cell, or body fluid derived from a subject (eg, blood (including whole blood, plasma, serum, etc.), urine, lymph, saliva, sweat, semen Etc.)
  • a “subject” is a human subject who usually receives or desires to undergo a cancer screening, and is a human subject who has or is suspected of having cancer. Etc. are included. Examples of such cancers are colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer. , Uterine cancer (eg cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, knee cancer., Ovarian cancer, brain tumor, blood. Colorectal cancer is preferred.
  • Immunoassays to detect the expression of DUS ⁇ 15 in a biological sample from a subject as described above may be suspected of having cancer (eg, colorectal cancer) or risk of cancer. Including contacting a biological sample taken from a subject with anti-DUS ⁇ 15 antibody under conditions that produce specific anti-antigen-antibody binding, and then measuring the amount of immunospecific binding by the antibody. To do. Such antibody binding is used to detect the presence and increased expression of the DUS 15 protein. In this case, detection of increased D U S 15 protein expression is an indicator of disease state. If necessary, the level of DUS 15 protein in a biological sample may be compared with the level of a healthy person who does not have cancer. ..
  • a biological sample such as a serum sample is contacted with a solid support or carrier such as nitrocellulose for the purpose of immobilizing all proteins present in the sample.
  • the support is then washed with buffer and subsequently treated with detectably labeled anti-DUSP 15 antibody.
  • the solid support is then washed twice with buffer to remove unbound antibody.
  • the amount of bound antibody on the solid support is measured according to well-known methods. Detection conditions suitable for each measurement are determined using conventional test methods. It can be appropriately determined by those skilled in the art.
  • the antibody is conjugated to an enzyme; for example the enzyme used in the enzyme immunoassay (EIA): [V oi 1 er, A. “The enzyme-labeled immunosorbent assay” (ELISA)., 1 9 7 8 ,; Diagnostic Horizons, 2: 1-7 , Microbiological A ssociates Quarterly Pub 1 ication, W a 1 kersvi 1 1 'e .. MD; V oi 1 er, A. by J' ⁇ CI in. Pathol., 3 1: 5 0 7-5 2 0: 1 9 7 8: B tier, J. E. by M eth.
  • EIA enzyme immunoassay
  • the photometric reaction produces a chemical molecule that can be detected by fluorescence measurement with visible means, and is reacted with an appropriate substrate, preferably a chromogenic substrate. Used for Enzymes which can and Turkey, it encompasses Peruokishidaze and alkaline phosphatase, and the like.
  • the 'Detection may also be accomplished by colorimetric methods using a chromogenic substrate for the enzyme.
  • Radioimsoatase RIA
  • sandwich immunoassay immunometric method
  • FIA fluorescence immunoassay
  • TRFIA 'time-resolved fluorescence immunoassay
  • EIA Enzyme immunoassay
  • ⁇ IA luminescence immunoassay
  • ELIA electrochemiluminescence immunoassay
  • latex agglutination method immunoprecipitation atse
  • precipitin reaction method gel diffusion precipitin reaction method
  • an immunoassay selected from the group consisting of an assay, an agglutination assay, a complement binding assay, an immunoradiometric assay, a fluorescence immunoassay, and a protein A immunoassay (WO 0).
  • each of the species associated with DU SP ⁇ 5 protein dysfunction Diagnosis of disease can be made. For example, if an increase in the concentration of DU SP 1.5 protein is detected, it may be caused by, for example, a disease caused by overexpression of DU SP 15 'protein (eg, cancer (eg, colon cancer)). Can be diagnosed as being high or likely to be affected in the future. .
  • DU SP 15 'protein eg, cancer (eg, colon cancer
  • the anti-DU SP 15 antibody of the present invention can also be used for diagnosis with i n v i V o.
  • Antibody preparations, product preparations and methods of use that can be used here are well known in the art.
  • antibody-chelating agents are described in Nuc1, Med. Biol. 1 990 017: 24 7-254.
  • an antibody having a label and a paramagnetic ion used in magnetic resonance imaging is described in, for example, M agne tic 'Re e n a c e e N e c i c e e 1 9 9 1 2 2: 3 3 9 ⁇ 34 2.
  • a probe or primer designed based on the nucleotide sequence of DUSP 15 gene can be used.
  • a diagnostic method includes, for example, (a) a biological sample derived from a subject, and a hybridization process that is based on the nucleotide sequence of the DU S 15 gene or a fragment thereof. And (b) contacting the polynucleotide in the sample with the DU SP 15 gene or a fragment thereof. Includes the process of detecting and Z or quantifying the session.
  • DNA of the DUSP 15 gene (or gene fragment thereof) in a biological sample derived from a subject is detected using the probe. And / or quantify.
  • the length of the base sequence used as a probe is, for example, 12 bases or more, 15 bases or more, .18 bases or more, 21 bases or more, 24 bases or more, 27 bases or more, 30 bases or more, or It may be a longer length polynucleotide fragment.
  • the low, high or high stringency conditions described above may be used.
  • a nucleotide sequence that can be hybridized under hybridization conditions that are stringent to the sequence” includes “DU SP 15 gene or fragment thereof”.
  • ⁇ a base sequence complementary to the base sequence (antisense polynucleotide): ⁇ .
  • Methods of hybridization of probes and nucleic acids are known to those skilled in the art. For example, International Publication No. 8 9/0 6 6 9 8, ⁇ ⁇ — A 0 2 0 0 3 6 2, US Pat. No. 2 9 1 5, 0 8 2, EP—A 0 0 6 3 8 7 9, E P.—A
  • the target sequence can be detected or quantified by using a known polynucleotide probe or primer for the DUPSP15 gene;
  • known methods for example, Southern hyperpla- dization, 'Northern hybridization, RT-PCR method, PCR ⁇ SSCP method (Genomics, Sections 5, IV, 8) 7 4-8 7 9 (1 9 8 9)), Proceedingsofthe Natio ⁇ a 1 A cademyof Sc
  • the array C GH method is a method that applies the chromosomal C GH method (K allioniemi, A. eta 1. (1 9 9 2) Science 2 5 8, 8 1 8-8 2 1). Genome D covering the region Using DNA chips with highly densely spotted NA fragments (BAC, PAC, YAC, etc.), cancer-derived DNA and normal DNA labeled with different dyes are converted into 'genomic DA fragments on the slide. In this method, DNA and copy number abnormalities in cancer are detected with high resolution by simultaneously performing hybridization and detecting the binding state (P nkel, D. et a. 9 9 8) N at. G enet. 2 0, 2 0 7-2 1 1).
  • the mR 1ST A level of DU SP 15 in the cell is expressed as a standard gene '(' housekeeping gene.
  • a standard gene ' housekeeping gene.
  • rnRNA levels can also be compared, preferably by RT-PCR.
  • the target sequence (DNA, mRNA, etc.) is detected and quantified by the method described above and overexpression of the DU SP 15 gene is confirmed, for example, it is caused by overexpression of DUS P 15 It can be diagnosed that the disease is likely (eg, cancer (eg, colorectal cancer)) or is likely to be affected in the future.
  • cancer eg, colorectal cancer
  • the presence of a target protein or a fragment thereof in a test sample can be identified using a mass spectrometer (MS). That is, by using a mass spectrometer, it is possible to determine the amino acid sequence of a target protein or a fragment thereof, and determine whether DU SP 15 protein is present in a biological sample derived from a subject. be able to.
  • Mass spectrometry uses MS to ionize samples such as proteins and peptides, and separates them according to the resulting mass charge (mZ z) It is a method to determine the mass of a sample by measuring its intensity. From the results of mass spectrometry, individual amiso acids' constituting the amino acid sequences of proteins and peptides can be identified.
  • MAL DI matrix assisted laser deposition
  • EI electrospray ionization
  • EI gas phase
  • FD field desorption
  • ion separation an ion separation method that is compatible with the ionization method is used.
  • TI me off 1 ight: TOF time-of-flight mass spectrometer
  • QM S quadrupole type
  • ion trap type ion trap type
  • magnetic field type ion trap type
  • Mass spectrometers are sometimes used in evening and in demem.
  • Other amino acid sequencing methods may be used, for example, sequencing using a sequencer (eg, gas phase sequencer);
  • the present invention also provides a kit for detecting and Z or determinating and quantifying a DU SP 15 protein or fragment thereof in a body fluid sample of a subject containing an anti-DU SP 15 antibody as a cancer marker.
  • the DU SP 15 gene or a part of its nucleotide sequence contains a stringent high-prididase nucleotide sequence that can be hybridized under the conditions of the DU SP 15 gene in a biological sample derived from a subject.
  • It also provides a kit for detecting and determining or quantifying the fragment as a cancer marker. These kits are used to detect a cancer marker by the above-mentioned immunological technique or hybridization method.
  • cancers examples include colon cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, and uterus.
  • cervical cancer, endometrial cancer testicular cancer, thyroid cancer, spleen cancer, Ovarian cancer, brain tumor, blood tumor, etc. are included, but colorectal cancer is particularly preferable.
  • the term “'cancer marker” refers to a subject's body fluid (eg, blood, urine, lymph fluid, saliva, sweat, semen, etc.) or cells or tissues that are not derived from normal tissue. Or a molecule that is selectively up-regulated in cancer cells or tissues, and the presence of the molecule in the body fluid or cell or tissue of the subject indicates or suggests the presence of cancer. Say things.
  • the kit according to the first aspect includes a component for detecting and / or quantifying a D U S P 15 antigen (including D U 'S P .15 protein and its partial peptide) in a body fluid sample from a subject.
  • a D U S P 15 antigen including D U 'S P .15 protein and its partial peptide
  • a D U S P 15 antigen including D U 'S P .15 protein and its partial peptide
  • a D U S P 15 antigen including D U 'S P .15 protein and its partial peptide
  • a body fluid sample from a subject.
  • a component for detecting and / or quantifying a D U S P 15 antigen including D U 'S P .15 protein and its partial peptide
  • a body fluid sample from a subject.
  • DUSP 15 protein is detected and / or quantified by ELISA
  • Such components can be expressed in, for example, tissue sections or levels of D 'USP 15 in body fluid samples such
  • the kit according to the second embodiment contains a polynucleotide having a base sequence that can be hyper-precipitated under a high-pridition condition stringent to the base sequence of the DUPSP 15 'gene or a part thereof.
  • the kit of the present invention may contain the above-mentioned polynucleotide immobilized on a DNA chip.
  • the kit of the present invention can be used for a high-pridition condition under high-precipitation conditions.
  • Containers and labels may be included.
  • the label on or associated with the container may indicate that the drug is used to detect a colorectal cancer marker.
  • other items such as instructions for use may be further included.
  • Example 1 Identification of colon cancer specific amplified gene by array 'C GH method
  • sample preparation of 200 colorectal cancer specimens and verification based on the array CGH method were performed.
  • the gene information in the public DB (NCBI: http: ZZwww. Ncbi. N 1 m. Nih. Gov /) is used in the region where amplification occurs frequently in colorectal cancer specimens.
  • the used B AC C 1 one.
  • the gene was found to be frequently and highly elevated in colorectal cancer patients (Fig. 1, Table 2).
  • Figure 1 shows a histogram showing the frequency of amplification of DU S P 15 gene in 200 colorectal cancer patients.
  • Table 2 shows the degree of amplification (GZR value) and frequency of 200 samples of DUSP 15 gene in colorectal cancer patients.
  • the average value shows the average value for samples with a .'GZR value of 1.2 or more.
  • the 'DUSP 15 gene was amplified in 56.0% of 20 0 colon cancer patients, with an average amplification of 1.7. .
  • the maximum value was 2.4, and remarkable amplification occurred very frequently. ..
  • Example 2 Verification of gene amplification in a colon cell culture
  • the degree of amplification in a colon cancer-derived cultured cell line was examined for a high-frequency and high-value gene region in colorectal cancer patients.
  • the cultured cell lines used were C a c o 2 and R KO. E 6 which are cell lines derived from colorectal cancer. Genomic DNA was extracted from the cultured cells using B 1 ood & CellCu1ture D.NA Kit (Q I AGEN) according to the protocol attached to the kit.
  • Table 3 shows the degree of amplification (G / R value) of DU.
  • S ⁇ 15 gene in a cell line derived from colorectal cancer. As shown, in the colon cancer-derived cell line, it was demonstrated that amplification occurred in the DUSP I 5 gene located in B AC Clone RP 1 1 — 2 4 3 J 16. -
  • Quantitative PCR was performed to confirm the amplification of the DU SP 15 gene region. Quantitative PCR is performed using S YB RG reen RT—PCRR eagents, Applied B iosys te rn s) according to the attached protocol. iosystems) did. Primers were synthesized using the following sequences (commissioned to OPE RON).
  • Table 4 shows the degree of amplification of the DU.SP15 gene in a colon cancer-derived cell line. Values are relative to control DNA (normal). As shown, it was found that amplification occurred in the DU S P 15 gene region also in the colon cancer-derived cell line. (Table 4)
  • DU SP 15 gene was also amplified in a colon cancer-derived cell line (C aco 2, RKOE 6). Therefore, cultured cells that could be used for functional analysis of DUSP 1'5 gene in cancer were selected.
  • Example 3 Functional analysis by RNA 1 analysis using colorectal cancer cell lines
  • the DU SP 15 gene which was frequently amplified in 20 colorectal cancer patients, was a colon cancer-derived cell line, and gene amplification was observed at the genomic level.
  • Cell line (C aco 2, R Using KOE 6), RNAi analysis was performed and the phenotype was observed.
  • RNA i analysis was performed and the phenotype was observed.
  • s i RN A was selected as a specific 21 m er within the gene and s i RN A was synthesized with the sequence as the target (commissioned to Q I AGEN).
  • siRNA When siRNA was introduced into Caco 2 cells, O 1 I gofeet amine (Invitrogen) was used, and si and RNA of ⁇ ⁇ ⁇ ⁇ ⁇ were introduced into the cells according to the attached protocol.
  • siRNA was introduced into kKO E 6 cells using R NA i FECT (QIAGEN), and 1 0 0 111 ⁇ 3 i RN A was introduced into the cells according to the attached pro-collet.
  • N e g. Ativ e. Controlsi RNA (QI AG EN) was used. The cells were observed under an inverted microscope for 4 days after introduction into the cells.
  • siRNA was verified at the mRNA level using a quantitative RT-PCR method. From the cells 24 hours after siRNA introduction, total RNA was extracted according to the attached protocol using Micro-to-Midi Total RNA Purification System (Invitrogen). Then S superer S cript III CDNA was synthesized according to the attached protocol using FIrst—Strand Synthesis Systemfr; RT-PCR (Invitrogen).
  • the standard gene for calculating the relative ratio is G: lycer a.1 dehyde— 3-phosphatedehydrogenase (GAP DH) Control Reagents (A plied B iosyst em s). The ratio was calculated. Measurement of living cell count>
  • Fig. 2 A and Fig. 2 B show that after transfeetion, siRNA of DU SP 15 gene was transferred to Caco 2 cells and RKOE 6 cells, respectively.
  • the observation images on the 4th day are shown (in each figure, the upper row: ⁇ ⁇ 40; the lower row: ⁇ 2 0 0).
  • a, b, and c are 3 types of siRNA of DU SP 15 gene, and N C ′ represents a negative control.
  • N C ′ represents a negative control.
  • the number of cells was significantly reduced for both a, b, and c siRNAs compared to negative control (NC) (Fig. 2A,. B).
  • Fig. 3 shows the results of quantitative RT-PCCR after cells were harvested 24 hours after siRNA of DU S P15 gene was transformed into C. a o 2 cells.
  • the relative amount of N C was shown using the contrast of GAPDH.
  • the effect at the RNA level was confirmed by quantitative RT-PCR.
  • the amount of all three siRNAs was significantly reduced (Fig. 3).
  • Figures 4A and B show that after transfeetion of DUSP 15 gene iRNA to Caco 2 cells and RK0 E 6 cells, respectively, the number of viable cells was measured with the measurement reagent using the cells on day 4. The measurement results are shown. The graph shows the relative amount to N C. As shown, the number of viable cells was measured. As a result, for Caco 2 cells, one of the DU SP 15 gene siRNAs (c), for R KO E 6 cells, DUSP 1 In all three types of siRNA of 5 genes (a, b, c), the number of cells was significantly decreased compared to Negatube contfo 1 (NC), similar to the phenotype (Fig. 4 ⁇ ). , ⁇ ). All siRNAs of the above DU S ⁇ 15 genes in which a small number of cells were observed were significant in the t test ( ⁇ 0. 0 1).
  • Example 4 Functional analysis by RN A 1 analysis using a colon-derived normal cell line
  • RNA 1 analysis was performed using a cell line derived from normal tissue of the large intestine. By conducting this, we verified that the suppressive effect of the target gene is cancer-specific.
  • Example 3 For cell lines, use C CD 1 8 Co purchased from AT C. The culture conditions were in accordance with the attached protocol. As the siRNA used, the c sequence of Example 3 was used. For introduction of siRNA into the cells, .'Lipofect am ine 2200 (Invitrogen) was used, and 25 nM of siRNA was introduced into the cells according to the attached protocol. As a control, N e ga tiv e C o n t r o l. S i R N A (Q ⁇ A G EN) was used. The cells were observed under an inverted microscope for 5 days after introduction.
  • FIG. 5 shows the observation image obtained on the 5th day after Transfection of siRNA of DU SP 15 gene on CCD 18 Co cells (upper: X 40; lower); X 2,0 0 ) D U S P 15 c represents one of the S i shakuhachi of the D U S P 15 gene (c sequence of Example 3), and N C represents a negative control. As shown, phenotypic observations were similar to NC and no effect was observed (Figure 5).
  • FIG. 6 shows the results of measuring the number of viable cells with the measurement reagent using the cells on the 5th day after transfection of siRNA of the DUPSP15 gene into CCD18CO cells by Transfectioion.
  • the graph shows the relative amount to N C.
  • N C NegativyCorntrol
  • the organ-specific expression of the DU S P 15 gene in normal tissues was evaluated by the Northern hybridization method known in the art.
  • RNA levels in normal organ tissues 23 organs (Heart, Brain, Placenta: (Pl'acenta), Lung, Liver) (Liver)., Skeletal muscle (S'keleta ⁇ Muscle), kidney (Kidney), spleen (Pancreas), spleen (Spleen), thymus (Thymus), prostatic gland (Prostate), testis, ovary ( Ovary, 'Small Intestine, Colon, Peripheral Blood, Peripheral Blood Leukocyte, Stomach, Thyroid, Spinal Cord, Lymph Node, Trachea (Trachea), Adrenal Gland, and Bone Marrow were analyzed by the Northern hybridization method.
  • DU SP 15 gene ⁇ ⁇ ⁇ 1 J is the NCBI (http://www.ncbi.nliii.nih.gov), Accession No.NM_080611, and the 1,383 bp sequence is mRNA. It is registered as.
  • Figure 7 shows the results. As shown, Alonso A. et al. (Alonso A. et al. (2004) J. Biol. Chem. 279 (31), 32586- 3259.1) As in the report, a band around 1.5 kb was observed in the testis, confirming significant testis-specific expression. . In this analysis, a new band was observed around 1.3 kb in the heart and skeletal muscle, suggesting the appearance of alternative splicing variants. For other organs, the expression level was confirmed to be low. The specific expression in the testis and the limited expression of organs in the testis are likely to be limited to normal tissues due to the effects of drugs targeting this gene. Example 6 Evaluation of gene amplification
  • the gene amplification degree (G / R) was 1.2 by the P- array CGH method, which was evaluated by the FISH method known in the art that gene amplification occurred in the DUSP 15 gene region in cancer cells in the specimen tissue.
  • Hybridization was carried out using the BAC Clone RP11-243J16 DNA Probe in which the DUSP 15 gene is located using the specimen tissue (derived from a colon cancer patient).
  • Figure 8 shows optical micrographs (fluorescence images) of some of the specimen tissues (A ⁇ : a portion of cancer cells (6 cells) observed with sputum. As shown, there are signals in the cancer cells. .3 Spots or more were found It was confirmed that the DU SP 15 gene region was amplified in 10 specimens with a gene amplification level of 1.2 or more by the array CGH method. It was shown that gene amplification occurred during the latter period of time, indicating that the DU SP 15 gene region is not only used as a molecular target for cancer therapeutics, but also for cancer diagnosis by FISH.
  • Example 7 Detection of DU SP 15 protein in blood by mass spectrometry
  • Serum specimens from colorectal cancer patients and healthy specimens 10 / zL Dilute with 500 L of a solution (10 mM Tris HC1 ph 7.4 + 150 mM NaCl), and then use a ProteomeLab IgY-12 SC proteome partitioning kit (BECKMAN COULTER: A24618) to obtain a large amount of protein in serum such as albumin and globulin. Was removed. Dithiothreitol (Wako: 049-08972) was added to the obtained fraction to a final concentration of 10 mM, and the reduction reaction was performed at 60 for 30 minutes.
  • a solution 10 mM Tris HC1 ph 7.4 + 150 mM NaCl
  • odoacetate and amide (SIGMA: 144-48-9) were added to a final concentration of 10 mM, and the alkylation reaction was carried out at room temperature for 30 minutes in the dark. After completion of the reaction, add 4 times the amount of cold acetone (with Wako: 014-08681, —20) to the reaction solution, let stand at 80 for 1 hour, and then centrifuge at 15,000 xg for 30 minutes to precipitate the protein. It was collected. The recovered protein was dissolved in 80 L of 2 M urea + lOOmM ammonium bicarbonate solution. After dissolution, a portion was subjected to protein concentration measurement by the BCA method. ⁇ Lipsin (Promega: V511C) was added to the sample protein to 1/50 (w / w), and digestion was performed at 37 for 16 hours.
  • Nano-column (C18 PepMap 3 m, 100 A, 75 m) directly bound to 0.7 g of peptide fragment with X-Precoluinn cartridge (C18 PepMap300, 5 m, 300A, 300 M m id x 5 mm LC PACKINGS: 163589) It was separated by id ⁇ ⁇ 150 mm LC PACKINGS: 160321). As the HPLC apparatus, Ultimate Plus (LC PACKINGS) was used.
  • the flow rate is 200nL / min, and the concentration gradient of 0.formic acid (Wako: 062-02901) -containing ⁇ acetonitril (MERCK: 1287229) and 0.formic acid-containing 90% acetonitrile is 0.573 ⁇ 4 / min.
  • 0.formic acid Wako: 062-02901
  • MERCK ⁇ acetonitril
  • 0.formic acid-containing 90% acetonitrile is 0.573 ⁇ 4 / min.
  • the sample was ionized under the conditions of a capillary voltage of 1500 V, an end plate offset value of 500 V, a dry gas flow rate of 12 L / min, and a dry gas temperature of 250.
  • the ion trap is set to capture 1 Da before and after the target m / z.
  • MS / MS analysis was performed on a mobile phone.
  • Fig. 9A and Fig. 9B show the results of analyzing the above methods for (A) serum derived from colorectal cancer patients and (B) serum derived from healthy subjects, respectively.
  • FIGS. 10A to C show the correspondence between the peaks shown in FIG. 9 and amino acids (or amino acid sequences) determined by MS / MS analysis.
  • si RN A was introduced into cells using O 1 igofec .t am ine (Invitr Q gen, and 100 nM si RN A was introduced into the cells according to the attached protocol.
  • egative Controlsi RNA Q. I AGE N was used ...
  • 01 1 shows the results of measuring the number of viable cells (MTT assay) with the measurement reagent using the cells on the 4th day after transfection of siRNA of DU S P 15 gene into HeLa cells.
  • the graph shows the relative amount with respect to N C (Negative control siRNA (Qiagen)).
  • N C Negative control siRNA (Qiagen)
  • 3 types of siRNA of DUSP 15 gene (a, b, c). Among them, 26% growth inhibitory effect was observed in siRNA a (p ⁇ 0.01 in t-test).
  • differential interference images were taken under a microscope, and the dynamics were observed in detail, specifically, after siRNA (a, b, c) was transferred to HeLa cells, 1,,, A differential interference image of the same visual field was observed after 2, 3, and 4 days, and the results are shown in Fig. 12.
  • Negative control siRNA Qiagen was used for NC. Suppression and induction of cell death (partially indicated by arrows) were confirmed, where a, b, and c correspond to si RNA a, si RNAb, and si RNA c in Figure 11 respectively. Induction of cell death was observed in a in which growth suppression was observed.
  • the present invention provides cancer therapeutic agents, diagnostic agents, diagnostic methods, therapeutic methods, kits used therefor, and the like. Therefore, the present invention is useful in fields such as cancer diagnosis or targeted therapy.

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Abstract

L'invention concerne un agent thérapeutique du cancer contenant un inhibiteur d'expression ou un inhibiteur d'activité de la protéine DUSP15. L'invention concerne également un procédé de criblage d'un composé pouvant servir d'ingrédient actif d'un tel agent thérapeutique; un anticorps contre la protéine DUSP15; un agent diagnostique du cancer et un procédé de diagnostic du cancer utilisant ledit anticorps et analogue.
PCT/JP2006/320043 2005-09-30 2006-09-29 Application therapeutique ou diagnostique du gene dusp15 WO2007037555A1 (fr)

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