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WO2007037533A1 - Application therapeutique ou diagnostique du gene ppp1r3d - Google Patents

Application therapeutique ou diagnostique du gene ppp1r3d Download PDF

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Publication number
WO2007037533A1
WO2007037533A1 PCT/JP2006/320009 JP2006320009W WO2007037533A1 WO 2007037533 A1 WO2007037533 A1 WO 2007037533A1 JP 2006320009 W JP2006320009 W JP 2006320009W WO 2007037533 A1 WO2007037533 A1 WO 2007037533A1
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Prior art keywords
cancer
ppp
protein
gene
antibody
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English (en)
Japanese (ja)
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WO2007037533A9 (fr
Inventor
Shinichirou Niwa
Yasutaka Makino
Tomoki Ikuta
Kazuya Arai
Takayuki Shindou
Hiromichi Ogura
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Link Genomics Inc
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Link Genomics Inc
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Priority to JP2007537776A priority Critical patent/JPWO2007037533A1/ja
Publication of WO2007037533A1 publication Critical patent/WO2007037533A1/fr
Publication of WO2007037533A9 publication Critical patent/WO2007037533A9/fr
Anticipated expiration legal-status Critical
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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
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • 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 gene R 3 D gene specifically amplified in cancer, its therapeutic or diagnostic use, and the like.
  • cancers Malignant tumors (cancers) are characterized by lethality due to proliferation, invasion, and metastasis. With surgical excision or local radiation therapy, the development of pharmacotherapy that can adequately deal with metastatic recurrent cancer is at the center of improving the outcome of future cancer treatment. Chemotherapy often acts directly on cancer and Z or RNA to kill cells, leading to death, but other than cancer cells, such as bone marrow cells, living cells, and gastrointestinal epithelial cells It had strong side effects on many normal cells. On the other hand, recent molecular cellular organisms, mechanisms related to cancer cell invasion, proliferation, and metastasis, are attracting attention as molecular targets that specifically act on the specific mechanism of cancer cells.
  • non-small cell lung cancer treatments such as GF R (epidermal growth factor receptor), tyrosine kinase inhibitor sa (generic name: gefitinib) (WO 9 6 3 3 9 8 0), It is in 3rd place.
  • GF R epimal growth factor receptor
  • tyrosine kinase inhibitor sa generic name: gefitinib
  • WO 9 6 3 3 9 8 0 WO 9 6 3 3 9 8 0
  • age group 60s are in the order of 50s and 70s.
  • the cause of the increase in colorectal cancer and environmental factors may be considered, but it has been pointed out that this is due to the westernization of eating habits and excessive consumption.
  • the large intestine is waiting for the development of targeted drugs.
  • CEA CA 19-9 used for diagnosis only indicates that it is advanced colorectal cancer, has no organ specificity, and is a higher performance diagnostic agent. Disclosure of the invention
  • the present inventors have found that the P P 1 R 3 D gene is frequently amplified in cancer (especially colorectal cancer).
  • the present inventors have suppressed the growth of cancer cells by inhibiting the expression of PPPP1 in cancer cell lines and cervical cancer cell lines, and have completed the present invention. That is, the present invention relates to a cancer therapeutic agent, a screening agent for a candidate substance having a cancer suppressive effect, a cancer diagnostic kit, a cancer diagnostic method, and the like.
  • PPP 1 R 3D gene expression inhibitor as an active ingredient (c) For PPP 1 R 3 D gene or part thereof
  • a therapeutic agent for cancer which comprises a substance that inhibits the activity of PPP1R3D protein described in (1) above, comprising a substance selected from the group consisting of
  • a PPP 1 R 3 D gene expression inhibitor is a screen
  • a cancer therapeutic agent comprising the antibody according to (9) above.
  • the above (1 therapeutic agent further comprising a vector carrying a radioisotope, a therapeutic protein, a low molecular drug gene.
  • a cancer diagnostic agent comprising the antibody according to (9) above.
  • (2 4) (a) a step of subjecting a biological sample derived from a subject to a polynucleotide comprising a base sequence capable of hybridizing to a base sequence of a PPP 1 R 3 D fragment thereof under a stringent high-pridity; and
  • the cancer is colorectal cancer. (30) Use of a P protein activity inhibitor for the manufacture of a medicament for the treatment of cancer.
  • the present invention provides novel drugs, kits and methods useful for the treatment and treatment of cancer (eg, colorectal cancer), and methods for screening candidate compounds for cancer.
  • cancer eg, colorectal cancer
  • Figure 1 is a histogram showing the frequency of the PPP 1 R 3 D gene relative to the degree of gene amplification in 2 from colorectal cancer patients.
  • Figure 2 shows the colorectal cancer cell lines DLD-1 and RK ⁇ E 6 3 D.
  • FIG. 2 is an optical micrograph (phase contrast image) showing the results of when s 1 RNA of a gene was transfected.
  • Figure 3 is a graph showing the results of R quantitative RT-PCR evaluation of the colon cancer cell line DLD-1 and s 1 RNA of the RKO E 6 D gene.
  • Cancer cell line DLD-1 and RKO E 6 D gene siRNA when transfected Figure 7 A- (: shows the correspondence between quin and amino acid (or amino acid sequence) determined by MS / MS analysis
  • Figure 8 shows PPP 1 R 3 in cervical cancer cell line HeLa cell line It is a graph which shows the result of verifying the RNA i effect when RNA was transfected.
  • FIG. 9 is an optical micrograph (differential interference image) showing the results of detailed observation of the experiment of FIG. 8 in time series, taken under a microscope.
  • BEST MODE FOR CARRYING OUT THE INVENTION Using a patient-derived specimen, we verified the amplified genes and increased the number of genes specific for colorectal cancer. Of the regions where amplification occurs frequently in the specimen, it was found that the R 3 D (P r o t e n p h o hs p h a t a s e 1, a t o r y s u b u n i t 3 D) gene is high in colorectal cancer.
  • PPP1R3D (PPP1R6) (Protein phosphatase 1, regula 3D) is a glycogen-binding protein that binds and glycates with protein phosphatase 1 (PP1).
  • PP1 binds to more than 20 different proteins and is dephosphorylated from reversible proteins, allowing various intracellular regulation such as glycogen binding by glycogen binding protein and muscle contraction by myosin binding protein. Or bind by signals from outside the cell GM (PPP1R3) is a skeletal muscle-specific Darikogen binding subuni P, et al (1985) Eur J Biochem 149, 295-303, et al (1991) J Biol Cheni 266, 15782-15789, Ch al (1994) Diabetes 43 , 1234-1241), GUPPP1R4) identified in rats (Doherty, MJ, et al (1995) FE 284-298), PPP1R5 (PP1R MJ, et al (1996) FEBS highly expressed in skeletal muscle and liver Lett 399, 339-343 PPP1R3D (PPP1R6) is highly expressed in skeletal muscle and heart, and is also expressed in medium to low levels in placenta, lung, liver, kidney, and
  • PPP1R3D has a PP1 binding site and (R / K) (V / I) XF-sequence (Egloff, MP, et al (1997) 1876-1887), Mi togen-act lvated protein kinases (M-oxidation-PX (S / T) P-sequence, MAPK-activated pro (MAP-KAP-K2) and Calmodul in-dependent protein kinase-2-LXRXXSL-sequence (Stokoe, D, et al (1993) 296, 843-849)
  • GM does not contain the phosphorylation site of the dependent protein kinase (PKA) and the binding site of GL Phosphorylase ⁇ , and the role of the function is divided.
  • colon cancer-derived cell line SW480 was included in 8,447 genes reported as high genes (01/94629).
  • colon cancer-derived cell lines L0V0, KM12, COL-derived cell lines ( ⁇ 322, PC10, H157, SBC5, ⁇ 209), stomach cancer
  • the present inventors confirmed the growth of cancer cells by suppressing the expression of the PPP 1 R 3 D gene by RN interference. Therefore, cancer can be treated by the expression of the PPP 1 R 3 D gene.
  • the cancer diagnosis is performed by measuring the expression level of PP molecule.
  • the cancer therapeutic agent, screening method, and diagnosis of the present invention will be described in detail.
  • the present invention provides (1) a cancer therapeutic agent containing as a PPP 1 R 3D gene expression component, and (2) a cancer therapeutic agent containing a PPP 1 R activity inhibitor as an active ingredient.
  • PPP 1 R 3 D gene means a human gene (SEQ ID NO: 1) consisting of 3 4 8 1 bases registered in Accession N 0 6 2 4 2 in the database. (Armstrong, CG, FEBS Lett 418, 210-214), but not limited to this, a variant that is altered by having one or more base substitutions, deletions, additions, etc.
  • a gene consisting of a base consisting of a base sequence that can be hybridized under stringent hyphenation conditions to the base sequence or its complementary sequence, such as ⁇ PPP 1 R 3 D '' is also used in this specification. . Therefore it can be done.
  • stringent stringent conditions, medium stringent conditions, or moderate conditions can be used.
  • Low stringent conditions include 5 XSSC, 5 X Denhardt's solution, 0 5% SD.
  • “Middle string is a condition of 5 XSSC, 5 X Denhardt solution, 050% formamide, 42 ° C.”
  • the “trin condition” is, for example, the conditions of 5 XSSC, 5 X Denhardt's solution, 050% formamide, 50 ° C. The higher these conditions, the more efficiently DNA having high homology can be obtained. However, there are several factors that can influence the hybridization pattern, such as temperature, probe concentration, long probe time, and salt concentration. Achieving trigency.
  • the base sequence of SEQ ID NO: 1 when compared with the nucleotide sequence of SEQ ID NO: 1, when calculated using the default by homology search software such as FAST, more than 75%, more than 80%, more than 85% % Or more, 90% or more 92% or more, 93% or more, 94% or more, 95% or more, 97% or more, 98% or more, 99% or more Can do.
  • homology search software such as FAST, more than 75%, more than 80%, more than 85% % Or more, 90% or more 92% or more, 93% or more, 94% or more, 95% or more, 97% or more, 98% or more, 99% or more Can do.
  • “inhibition of gene expression” refers to gene expression.
  • 0 R 6 D from 2 9 9 amino acid residues registered in 0 0 6 2 3 3 (SEQ ID NO: 2) and the activity of this protein (for example, PP1 binding activity, one or more activities of glycogen binding activity)
  • a mutant protein consisting of amino acid sequence of deletion, substitution, insertion, and deletion of one or more amino acid residues of this protein.
  • the amino acid mutation site has substantially the same quality of activity as the original protein, but there is no particular limitation, but the number of mutations is, for example, 1 to 50, 1 to 30, 1 ⁇ 25, 1 ⁇ 20, 1 ⁇ 15, 1 9, 1 ⁇ 8, 1 ⁇ 7, 1 ⁇ 6 (1 ⁇ several), 1, 1 ⁇ 3, One or two, one.
  • the number of mutations is generally good.
  • such a mutant protein has a sequence of SEQ ID NO: 2 and about 70% or more, 75% or more, 80% or more, 85% or more 91% or more, 92% or more, 93% or more, 9% It contains 4% or more, 96% or more, 97% or more, 98% or more, 99% or more of the same amino acid sequence, and contains substantially the same protein as the original protein.
  • PPP 1 R 3 D “partial peptide” is also included in the PPP 1 R 3 D protein as the homology value is generally larger.
  • the PPP 1 R 3 D protein includes a partial peptide consisting of a part of the amino acid sequence of the PPP 1 R 3 D protein, or the same activity as the PPP 1 R 3 D protein described above.
  • the term “do” used in the present invention refers to the amino acid residue of several amino acid sequences (for example, about 1 to 20, more preferably about 1 to 1, more preferably about 1 to 5) in the above polypeptide. The group may be changed by deletion or insertion.
  • the PPP 1 R 3 D protein used in the present invention can be prepared from the cells and tissues.
  • the protein can be synthesized by a known peptide synthesizer, or can be prepared in a combination using appropriate host cells selected from eukaryotes.
  • P P P 1 R 3 used in the present invention may be derived from any species, but preferably human “substantially the same quality activity” indicates that these activities are characteristic. Therefore, it is preferable that the activity (PP1 binding activity, glyco, etc.) is equivalent (for example, about 0 to 1: L 0 0 times, preferably 0 times, more preferably about 0 5 to 2 times). Quantitative factors such as degree and protein molecular weight are good. The measurement of these activities can be applied to known methods described in literatures such as Armstrong, CG, szo Lett 418, 210-214, etc. For example, the screening method described later can be used.
  • B LA ST and Gapped B LAS T the default parameters of each program are used.
  • cancer therapeutic agent is used to refer to anticancer agents, cancer cell apoptosis inducers, and cancer cell growth inhibition. Used to include drugs, cancer drugs, cancer preventives, etc. Note that the terms “cancer” (or cancer) and “tumor” in this application have the same meaning.
  • the present invention provides a cancer therapeutic agent containing a PPP1R3D harmful substance as an active ingredient.
  • PPP 1 R 3 D gene expression inhibitor 1 No restriction as long as it inhibits R 3 D gene expression (1) PPP 1 R 3 D gene to PPP 1 R 3 D mR And (1 i) PPP 1 R 3 D mRN R 3 D protein translation inhibitors are included.
  • nucleic acid means RNA or DNA.
  • Nucleic acid includes those having other decorated heterocyclic bases containing purine and pyrimidine bases. May include methylated and pyriminated purines and pyrimidines, acylated purines, or other heterocycles. Leosides and modified nucleotides may also be sugar moieties, for example, one or more hydroxyl groups may be halogenated, substituted, or functionalized such as ether, amine.
  • RNA i is a phenomenon in which when a double-stranded RNA having the same sequence as the target gene sequence is introduced into a cell, both the expression and the expression of the target endogenous gene are inhibited. It is expressed locally using a vector that produces Methods for preparing and using such double-stranded RNA (ds RNA, sl RNA A) are known from many literatures.
  • Double-stranded RN having the R N A1 effect used in the present invention usually 19 to 30 bases, preferably 20 to 27 bases
  • an antisense nucleic acid which is a nucleic acid that has a unique polynucleotide and that can be hybridized with a part of the polynucleotide, is RNA, DNA, or modified NA, DNA).
  • the antisense nucleic acid of the present invention is a modified nucleic acid (RNA, DNA). It may be NA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or NA hybrid.
  • Modified nucleic acid ingredients include, but are not limited to, nucleic acid sulfur derivatives, thiophosphate derivatives, and resistance to degradation of oligonucleotide amides.
  • the antisense nucleic acid to be used is appropriately promoted, and preferably the nucleic acid prepared in such a manner that a sequence containing a transcription termination signal on the 3 ′ side can be transformed into this using a known method.
  • the sequence of the antisense nucleic acid is not a sequence complementary to the endogenous gene of the trait or a part thereof, but may be omitted as long as the gene expression can be effectively suppressed.
  • a complementary antisense sequence is designed near the 5 'end of the mRNA 1 of the PPP 1 R 3 D gene, it is a gene conversion. It can complement the coding region or the 3 'untranslated region. About 70% or more, preferably more preferably about 90% or more, and most preferably about 95% or more of an anti-target gene transcript effective in inhibiting gene translation Hirashima and Inoue, Laboratory for Neonatal Chemistry 2 Nucleic Acid IV Genes, Japanese Biochemical Society, Tokyo Chemistry, 1 9 9 3, p 3 1 9 Kawak am ieta 1, P harm T ec V ol 8, p 2 4 7, 1 9 9 2, Vol 8, p 2; see STC rookeeta 1, ed, se Research and Applicate Press, 1 9 3 3 etc.).
  • a nucleic acid having a liposomal activity that specifically cleaves a PPP 1 R 3 product can be used.
  • the term “ribozyme activity” used here refers to site-specific mRNA transcripts of the gene. Some ribozymes have more than 400 nucleotides, such as Ml RNA contained in the group I intron type, but some have an active domain of a hammerhead type or hairpin type (protein) 9 0, 3 5, p 2 1 9 1).
  • Non-header type liposomes eg FEBSL ett, 1 9 8 8, 2 2 8, p SL ett, 1 9 8 8, 2 3 9, p 2 8 5; tamper 1 9 90, 3 5, p 2 1
  • N ature 1 p 3 4 9, Nuc 1 Ac ids R es, 1 9 9 1, 7 5 1; Kikuchi Hiroshi, Chemistry and Biology, 1 9 9 2, 3 0, p 1 1 It is a compound.
  • Such a compound can be a natural product.
  • Such a compound can be used in the screening method described below. 1. 2 PPP 1 R 3 D protein activity inhibitor Therapeutic
  • the present invention also provides a cancer therapeutic agent containing a PPP1R3 activity inhibitor.
  • antibody means the full length protein or antibody.
  • the antibody form of the present invention is not limited to the above-described monoclonal antibody, as long as it specifically binds to the PPP 1 R 3 D protein. Antibody modifications are also included.
  • An antibody that binds to P protein can be prepared by the following method. Anti-PPP Yodosha (see 2 0 0 1) 2 6-3 2 etc.).
  • a compound that binds to PPP 1 R 3 D protein or uses a compound other than a mutant as an active ingredient is, for example, PPP 1 R 3 D protein. It is a compound that inhibits binding to 3D protein. Such a compound may be a natural product. Such compounds can be obtained by screening described below.
  • the activity of the above-described PPP1R3D protein of the present invention can be used as a cancer therapeutic agent.
  • the present invention also provides a screen for a candidate compound having a cancer suppressing action.
  • One preferred embodiment is a method using PPP 1 R 3 D protein and binding as an index.
  • compounds that produce PPP 1 R 3 D are expected to inhibit the activity of the PPP 1 R 3 D protein.
  • the compound preferably binds to the active site of PPP 1 R.
  • the P 1 R 3 D protein is brought into contact with the test compound.
  • P protein is a purified form of PPP 1 R 3 D protein, a cell that is an indicator for detecting binding to a test compound.
  • the binding between the PPP 1 R 3 D protein and the test compound can be marked on the test compound bound to the 1 R 3 D protein.
  • changes in the activity of PP protein that is expressed intracellularly or extracellularly and is caused by the binding of the test compound to the R 3 D protein can be detected as an index, and the binding activity to the test compound is known.
  • PP 1 binding activity, glycogen binding activity (Arms trong, CG, et al (1997) FEBS Let 418, 2
  • PPP 1 R 3 D protein A test compound that inhibits the activity of is selected.
  • the compound isolated by this method has a cancer suppressing action and is useful as a cancer therapeutic agent.
  • Another embodiment of the screening method of the present invention is a method using PPP 1 R expression as an index.
  • a PPP 1 R 3 D gene is contacted with a test compound.
  • a test compound examples include, but are not limited to, cells such as mice, cats, dogs, ushi, hidge, and birds. Can be made.
  • Test compounds used in this method are not particularly limited, but compounds, organic compounds, inorganic compounds, proteins, pep compounds, compound libraries, gene libraries, cell extracts, cell culture supernatants, fermented microorganism products, Marine raw extracts are used.
  • Test compound to cells expressing PPP 1 R 3 D gene Usually, each compound is added by adding a compound of cells expressing the PPP 1 R 3 D gene, but this method is limited. In this case, “contact” is performed by introducing the protein into the cell. In this method, the PPP 1 R 3 D gene is then measured.
  • expression of gene is transcribed and reversed. A person skilled in the art can measure the expression level of a gene. For example, NA that expresses PPP 1 R 3D gene is extracted according to a conventional method, and the transcription level of the gene is measured by carrying out the normalization method or RT-PCR method using this mRNA as a saddle type. Can do.
  • the promoter region of the D gene isolates the promoter region of the D gene according to a conventional method, connect the gene (for example, a gene that can be detected by using luciferase, GFP, galactosyl fluorescence, color development, etc. as an index), and connect the marker gene Seeing the activity
  • the transcription level of the gene can be measured.
  • the antibody used for detecting D protein but for example, both a monoclonal antibody and an antibody can be used.
  • the compound selected as the compound that decreases the expression level as compared to the roll that is not contacted with the test compound is then used for the cancer therapeutic agent.
  • the present invention also provides an anti-PPPP1R3D antibody, a composition containing this antibody, and the like.
  • an anti-PPPP1R3D antibody a composition containing this antibody, and the like.
  • an “anti-PPP 1 R 3 D antibody” includes an antibody that binds a PPP 1 protein (including fragments (partial peptides) or salts thereof).
  • the anti-PPP used in the present invention may be a polyclonal antibody or a monochrome antibody.
  • the class of the antibody is not particularly limited, and may have any isotype such as IgG, IIgD, or IgE. Preferred is IgG or IgM, and more preferably IgG after purification. Also here 9 8 8))).
  • the protein P P 1 R 3 D protein or a salt thereof used as a sensitizing antigen also includes its partial peptide, which includes, but is not limited to, a fragment of the amino acid sequence of SEQ ID NO: 2, 0 or more, 40 or more, 60 or more, 80 As described above, it is a partial peptide having 10 consecutive amino acid sequence portions. For example, amino (N) terminal fragment or carboxy (C).
  • the partial peptide used in the present invention one or two or more (preferably about 1 to 10, more (1 to 6)) amino acid residues are deleted, substituted or inserted. It may be a thing.
  • PPP 1 R 3D protein used as an antigen is preferably a protein whose origin is a mammal such as a mouse or a protein derived from a human.
  • the interval between immunizations mainly by intravenous, subcutaneous or intraperitoneal injection is not particularly limited, and is several days to several weeks, -5 weeks, 1 to 10 times, preferably 2 to 5
  • the immunization is collected 1 to 60 days after the last immunization, preferably 1 to 14 days.
  • antibody-producing cells include spleen cells, phosphocytic cells, etc., but spleen cells or local lymph nodes (11) cell fusion
  • fusion with antibody-producing cells is performed.
  • Cell lines that use generally available cell lines of animals such as myeloma cells to be fused with antibody-producing cells are drug-selective, unfused medium (hypoxanthine, aminopterin, thymidine) It is preferable that it can survive only when fused with antibody-producing cells
  • myeloma cells for example, a mouse mye such as X 6 3 Ag NSIZ 1—A g 4 _l, NS 0-1
  • cell fusion between the above myeloma cells and antibody-producing cells is carried out by adding 1 X 10 6 to 1 X 1 in DMEM, RPI-1640 cell culture medium without serum.
  • the cell ratio between the production cell and the myeloma cell is 2.1 to 3 1 in the presence of the cell fusion promoter. 0 7 cells Zm 1 and 2 X 10 5 to 2 X 10 6 cells Cell fusion promotion Average molecular weight 1 0 0 0-6 0 0 0 Dalton polyethylene grease After appropriately diluting with 0 medium, etc., sprinkle about 1 microweed plate and add selective medium to each well. As a result, cells that grow after the start of culture in a selective medium can be obtained as a hybridoma. Next, it is determined whether or not antibodies that react with the protein are present in the culture supernatant of the proliferated hybridoma. Screening for a screened riviera is not done according to normal practice.
  • a normal cell culture method or ascites formation method can be used as a method for monochromating from the hybridoma obtained as described above.
  • 10% of hybridoma is contained in RPMI-1640 medium, MEM medium, or serum-free medium.
  • Normal culture conditions for example, 37 ° C, 5% 7-14 days
  • cultured is obtained from the culture supernatant antibody. If the re dormer myeloma cells from mammalian the same species animal about 1 X 1 0 7 or administered, and large quantities of High Priestess dormer, 1-2 weeks Later, ascites is collected. To be administered.
  • the dose of the antigen per animal is 0 1 to 10 Omg when adjudicated, and 1 to 100 zg with adjuvant.
  • adjuvants include Freuvant (F CA), Freund's incomplete adjuvant (FI aluminum adjuvant, etc.
  • Immunization is performed by injection into the main subcutaneous or intraperitoneal cavity. Immunization is performed every few days to several weeks, preferably 2 to 5 1 to 10 times, preferably 2 to 5 times, and after 6 to 60 days, an enzyme immunoassay (ELISA (enznkedi mmu nosorbentassy) or Blood is collected on the day when the antibody titer is shown by radioimmune A, radlol mmunoassay), etc., to obtain antiserum.
  • ELISA enzyme immunoassay
  • the polyclonal antibody in the antiserum is applied to an affinity column fixed with P protein, and the antibody that reacts with the P protein (column adsorbed fraction) is collected.
  • Polyclonal in the antiserum against the 3D protein It can be measured by antibody LISA method.
  • F ab or F ab ' 2 fragments can be prepared by digestion with pepsin or papain) by conventional methods.
  • Human antibodies such as Riec hma nn et al. (R in JMol Biol Oct 5, 2 0 3 (3) 1 5, p 1 4 6-1 5 6, 1 9 9 7 ”,“ Naturlcs, Vol 7, p 1 3— 2 1, 1 9 94 ”, JP 3 65 5, International Application Publication W ⁇ 9 4-2 5 5 8 No. 5 public, June issue, pages 40 to 50, 1 9 95 years, Vo l 3 6 8, p 8 5 6-8 5 9, 1 9 94 ”, Respectively, can be produced with reference to Japanese Patent Publication No. 2 33.
  • An antibody that binds to the P 1 R 3 D protein can be used for the purpose of, for example, suppressing cancer metastasis.
  • human antibodies and human antibodies are preferred for immunogens.
  • the antibody When used as a diagnostic agent, the antibody is labeled with a monitoring substance (eg, radioisotope, fluorescent substance, etc.). If necessary, it is labeled with a radioactive substance, fluorescent compound, or the like.
  • a monitoring substance eg, radioisotope, fluorescent substance, etc.
  • the antibody PPP 1 R 3 can also be obtained using a bioluminescent compound as well as fluoresceinate, rhodamine, phycoerythrin and fluoride. The presence of a bioluminescent protein is measured by fluorescence.
  • the antibody of the present invention is used for specifically detecting 1 R 3 D protein or the like in a sample such as a body fluid or tissue. Used to purify PPP 1 R 3 D protein, etc. PPP 1 R 3 D protein in each fraction during purification The ability to be an agent having a neutralizing activity used in combination with other agents for exerting a therapeutic effect Accordingly, in another aspect, the present invention is directed to a targeted therapy or target for cancer We also provide complexes of PPP 1 R 3 D antibodies with other drugs, such compositions, etc. used for synthetic imaging. According to such an embodiment, the anti-PPP 1 R 3 D antibody to be used can be used to deliver a therapeutic agent or a labeling agent for diagnosis to the target site of the PPP 1 R 3 D tamper. it can.
  • radioisotope is iodine - 1 2 5 (1 25 1), and iodine - include 1 3 1 such as emission element.
  • radiohalogen elements can be widely used as radiotherapeutic diagnostic agents by labeling them with antibodies and peptides in the same manner as the above elements.
  • 1 25 I or 1- merization can be bound to an antibody by a known method such as the chloramine T method.
  • indium 1 1 1 1 and gallium 1 6 7 for diagnosis 6 7 Ga for treatment, yttrium 1 90 ( 90 Y), rhenium 6 R e) or rhenium 1 1 8 8 ( 1 8 8 Re)
  • examples of the “therapeutic protein” include site-in which activates, for example, human 2, human granulocyte-macrophage-one colony-stimulating factor, phage colony-stimulating factor, human Tointerleukin 1 2 etc.
  • ricin and diph toxins can be used to directly kill colon cancer cells.
  • a cDNA encoding is linked to a cDNA encoding an antibody or antibody fragment, the fusion antibody is encoded, and this DNA is then expressed in a prokaryotic or eukaryotic expression vector. It can be introduced into prokaryotes or eukaryotes to produce fusion antibodies.
  • “Small molecule drug” as used herein may mean a diagnostic or therapeutic compound other than “radioisotope” and the like.
  • small molecule drugs include alkylating agents such as nitrogen cyclophosphamide, antimetabolites such as 5—flumesotrexate, daunomycin, mitomycin C, daunorubicin, doxorubicin vincristine, Hormone agents such as vinblastine, vindesine, hormonal agents such as evening moxifen, dexamethasone (Japan Clinical Oncology Society 1 9 9 6 years) Cancer and morphogenesis or steroids such as cortisone, prednisone, indomethacin, etc.
  • Non-sterolide agents, immunomodulators such as gold thomalamin, cyclophosphamide, Etc.
  • a viral vector modified so as to be able to bind to the anti-PP of the present invention is used.
  • Adenoviral vector Wang, P, et 5) Somatic Celland Molec 2 1, 4 2 9-44 1
  • retrovirus vector 1 RK, eta 1 (1 9 9 6) JV ir 5 7 0 1-5 7 0 5
  • lentiviral vector N a L (1 9 9 8) C urr Op ⁇ n B ⁇ ot 9, 45 7-46 3).
  • genes that have therapeutic effects such as induction of apoptosis at target sites (eg, colon cancer) such as cell proliferation-related genes and apoptosis-related genes are incorporated.
  • the anti-PPP 1 R 3D antibody and the other drug can be chemically coupled.
  • “chemical bond” includes a bond, covalent bond, bond by intermolecular force, and hydrophobic interaction
  • “genetic engineered bond” consists of a therapeutic protein.
  • Anti-PPP 1 R 3 D antibody to be used is chemically or genetically engineered with any of the following radioisotopes, therapeutic molecular drugs, and viral vectors carrying therapeutic genes: Therapeutic agents can be formulated on the basis.
  • a chemically acceptable carrier can be added according to a conventional method.
  • excipients for example, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, binders, disintegrants, lubricants, fluidity promoters, flavoring agents, etc.
  • the usual carrier should be used as appropriate.
  • Lightly anhydrous anhydrous, lactose, crystalline cellulose, mumps, carmellose calcium, carmellose sodium propylcellulose, hydroxypropylmethyl cell mouth dilute rutile Examples include amino acetate, polyvinyl latin, medium-chain fatty acid tridalylide, polyoxyethylene oil 60, sucrose, carboxymethylcellulose, cornstar and the like.
  • Examples of the dosage form of the therapeutic agent of the present invention include oral powders, pills, powders, granules, fine granules, soft and hard capsules, pellets, sublinguals, and pastes.
  • non-propellants, suppositories, transdermal agents, ointments, plasters, and liquids for external use have optimal dosage forms according to the route of administration and subjects of administration.
  • the single dose varies depending on organs, symptoms, administration method, etc. For example, is usually about 30 mg per day for patients (for 60 kg) However, it is preferable to administer about 0 1 to 20 mg or about 0 1 to 10 mg by intravenous injection.
  • the type of dosage form, administration method, patient's symptoms, etc. This can be done at the discretion of a doctor or veterinarian.
  • the preparation thus obtained can be administered to, for example, humans and their (eg, rats, rabbits, hidges, buyu, ushi, cats, etc.). Of animals other than ⁇
  • the therapeutic agent of the present invention is cancer (for example, colorectal cancer, stomach cancer, cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen bladder cancer, uterine cancer (eg cervical cancer) Uterine body cancer), adenocarcinoma, knee cancer, ovarian cancer, brain tumor, blood tumor, etc.)
  • cancer for example, colorectal cancer, stomach cancer, cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen bladder cancer, uterine cancer (eg cervical cancer) Uterine body cancer), adenocarcinoma, knee cancer, ovarian cancer, brain tumor, blood tumor, etc.
  • it is used for the prevention / treatment of colorectal cancer.
  • the agent of the present invention inhibits the activity of P P P 1 R 3 D protein.
  • PP 1 R 3D gene expression inhibitor is used as an active ingredient and is used as an anti-cancer agent, cancer metastasis inhibitor, cancer cell apoptosis inducer, etc.
  • the cell, tissue, organ, or cancer type of interest is not specified.
  • the agent of the present invention includes both a PPP 1 R 3 D tamper harmful substance and a PPP 1 R 3 D gene expression inhibitor. They can also be formulated and administered via a gene gun or a hydrogel catheter.
  • a combination of a recombinant adenovirus particle vector and an anti-PPP 1 R 3D antibody when used for cancer treatment, these may be used alone, but generally used together with a carrier to be produced.
  • the Such carriers are preferably pre-existing carriers, as well as water, saline, glucose, and human isotonic solutions. Furthermore, preservatives and weights that are commonly used in pharmaceutics can be added.
  • the preparation can be administered in an appropriate dosage form, depending on the disease to be treated. Examples of the dosage form include emulsion, capsule, tablet, granule, injection, ointment and the like.
  • PP 1 R 3D antibody when administering viral vector particles or for therapeutic purposes, it is usually preferable to administer 10 15 viral particles per adult, depending on the nature of the disease cell or tissue. You may change it.
  • the administration frequency may be 1, the administration period may be from 1 day to several months or more, and it may be intermittently set over a long period of time with 1 set as the input.
  • the viral vector vector nucleic acid molecule used in the present invention can also be used for diagnosis of specific cells and Z or disease states. For example, a viral vector obtained by integrating a detectable marker gene into one nucleic acid molecule and transfecting this cell.
  • the cancer diagnostic agent of the present invention comprises: (a) a PPP 1 R 3 D antibody, or (b) a PPP 1 R 3 D gene or one of its stringent conditions under a highly pre-precipitation condition.
  • the diagnostic method using the present R 3 D antibody includes, for example, (a) contacting a subject with an antibody against PPP 1 R 3 D protein, and (b) the antibody in the sample, PPP 1 R 3 D data includes the step of detecting and binding to the partial peptide or salt thereof.
  • the labeled P protein or a fragment thereof and the anti-PPPP1R3D antibody are quantified or quantified using a labeled anti-PPPP1R3D antibody.
  • subject-derived biological sample includes subject-derived or body fluid (for example, blood (including whole blood, plasma, serum, etc.), saliva, sweat, semen, etc.).
  • a “subject” is a human subject who is, or is suspected to be, a human subject who is to receive, or wants to receive, an example of such a cancer is the large intestine. , Stomach cancer, lungs Or an anti-PPP 1 R 3 D collected from a subject at risk of cancer under conditions that produce antigen-antibody binding, and then measuring the amount of immunospecific binding by the antibody. Binding is used to detect PPP 1 R 3 D protein Z or increased expression. In this case, detection of D protein expression is an indicator of disease state. Required The level of PPP 1 R 3 D protein in the sample may be compared to the level of cancer.
  • a serum sample is contacted with a solid support or carrier such as a target loose that immobilizes all proteins present in the sample.
  • a solid support or carrier such as a target loose that immobilizes all proteins present in the sample.
  • wash with buffer followed by treatment with detectably labeled anti-PPPP1.
  • the solid support is then removed twice with buffer.
  • the amount of bound antibody on the solid support is then measured. Detection conditions suitable for each measurement can be appropriately determined by a person skilled in the art through routine trials.
  • One method for detectably labeling anti-PPP 1 R 3D antibodies is to bind the antibody to an enzyme, such as the enzyme Imnoassay (EIA) [Immunoadsorption assay by V o 1 1 er, A ("T he Enzyme Limmunosorbent A ssay) (ELISA) D lagnostic Horizons, 2 1-7, lological A ssociates Quart React with chromogenic substrate.
  • EIA Imnoassay
  • Enzymes capable of attaching a detectable label to the antibody include, but are not limited to, peroxidase and alvease. This can be achieved by a colorimetric method using a chromogenic substrate for detection.
  • RIA race
  • sandwich immunoassay immunometry
  • fluorescence immunoassay FIA
  • time-resolved fluorescence RFIA enzyme immunoassay
  • EIA enzyme immunoassay
  • luminescence immunoassay luminescence immunoassay
  • electrochemiluminescence immunoassay ELIA
  • various diseases associated with PPPP1R3D dysfunction can be diagnosed by utilizing the in vivo P P protein quantification method using the antibody of the present invention. If an increase in the concentration of P 1 R 3 D protein is detected, it is likely that the disease is due to overexpression of 1 R 3 D protein (eg, intestinal cancer) or may be affected in the future. it can.
  • 1 R 3 D protein eg, intestinal cancer
  • the anti-PPP 1 R 3 D antibody of the present invention is in vivo -It is described in 34-2.
  • a diagnostic method using a probe or primer designed based on the PPP 1 R 3 D gene, such a diagnostic method can be performed by, for example, (a) subject and PPP 1 R 3 D gene. Or a polynucleotide (probe) that can be hybridized under high hybridization conditions suitable for the nucleotide sequence of the fragment (b) the polynucleotide in the sample and PPP 1 or A step of detecting hyperpredation with the fragment.
  • the PP gene DNA (or gene fragment thereof) in a biological sample derived from a subject is probed and Z or quantified.
  • Bases used as probes For example, it may be 12 bases or more, 15 bases or more, 18 bases or more, 224 bases or more, 27 bases or more, 30 bases or more, or a polynucleotide fragment. Hybridized low, medium or high stringency conditions may be used.
  • “Hypridly sequence under the base sequence of PPP 1 R 3 D gene or fragment thereof” indicates the base of PPP 1 R 3 D gene or fragment thereof.
  • Polynucleotide probes or primers can be used to detect or quantify the target sequence.
  • the array C GH method is a method that applies the chromosomal C GH method (K allionieta 1 (1 9 9 2) Science 2 5 8, 1).
  • the chromosomal region on the slide is covered with a NA fragment (BAC, PAC, YAC, etc.) with a high density of A chip, and cancer-derived DN labeled with different dyes are simultaneously hyonated on genomic DNA fragments on the slide to detect their binding state
  • This is a method to detect cancer copy number abnormalities with high resolution (P 1 neta 1 (1 9 98) N at Genet 2 0, 1).
  • the PPP 1 R 3 D gene is generated. Can also be compared.
  • Target sequence DNA, mR output and quantification, and overexpression of PPP 1 R 3 D gene is confirmed by the above method.
  • Disease caused by overexpression of PPP 1 R 3 D eg colorectal cancer
  • a mass spectrometer can be used for the presence of or a fragment thereof in a test sample. That is, PPP 1 R 3 D protein can be present in a biological sample from which the amino acid sequence of a protein or a fragment thereof can be determined by using a mass spectrometer.
  • mass spectrometry a sample such as protein is ionized using MS, the mass charge (mZz) obtained is determined, and the intensity of the sample is determined to determine the mass of the sample. Individual amino acids of the peptide can be identified.
  • MALDI matrix assisted drainage desonization
  • EI, CI electrospray ionization phase method
  • FD field desorption
  • the present invention also provides a kit for developing and / or quantifying a cancerous PPP1R3D protein or fragment thereof containing an anti-PPPP1R3D antibody.
  • R 3 D gene or a part of its base sequence that can be hybridized under stringing conditions PPP 1 R 3 D gene in a biological sample from an investigator or detected as Z Kits for quantification are used to detect cancer markers by the above-described immunological technique or Hypri method. Examples include colon cancer, stomach cancer, lung cancer, breast esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder (eg cervical cancer, endometrial cancer) ), Testicular cancer, thyroid ovarian cancer, brain tumor, blood tumor, etc. are included, but it is particularly preferable.
  • cancer marker refers to a subject's body fluid (urine, lymph fluid, saliva, sweat, semen, etc.) or cells or normal tissue, or cancer cells or alternatively A molecule whose expression is accelerating, and whose presence in the subject or tissue suggests the presence of cancer. obtain.
  • Such antibodies may be radioactive, fluorescent, colorimetric, or fermented.
  • the kit of the present invention may be a labeled secondary antibody.
  • the kit according to the second aspect includes a polynucleotide having a base sequence that can be highly prehydidized and re-synthesized to a PPP 1 R 3D gene. It may contain the above-mentioned pores fixed on the chip.
  • the kit of the present invention contains a container in addition to an anti-PPP 1 R 3D antibody, a base sequence that can be hybridized under high-purity conditions stringent to PPP 1 or a part of the base sequence, and the like. Also good. Items that are indicated to be used for detection of cancer markers on the label on the container or accompanying the container, for example, instructions for use, etc. are further included. The range described more specifically is not limited by these examples.
  • Example 1 Colorectal cancer-specific amplification by array CGH method
  • sample preparation and array C verification of colon cancer specimens were conducted for 20 cases of colon cancer-specific gene amplification regions.
  • Figure 1 shows the frequency of amplification of PP children in 200 specimens of colorectal cancer.
  • Table 2 shows the degree of amplification (GZR value) and frequency in 200 PPP 1 R 3 D gene samples. The average value for specimens with an R value of 12 or more is shown.
  • the P P P 1 R 3 D gene was amplified in 645% of 20 cancer patients, an increase of 18%. The maximum value was 27, which occurred very frequently.
  • Example 2 Verification of gene amplification in a colon-derived cultured cell line
  • the amplification in a colon cancer-derived cultured cell line was verified with a high frequency in colon cancer patients.
  • the cultured cell line used was DLD-1E6, a colorectal cancer-derived cell line.
  • Genomic DNA was extracted from the cultured cells using Blood & Cell DN DNAK (QI AGE N) according to the kit call. Also found that amplification occurred at the P 1 R 3 D gene at BAC C lone RP 1 1-2 8 G 1 8.
  • the PPP 1 R 3 D gene was also amplified in a colon cancer cell line (D LD 6). Therefore, cultured cells with a functional PPP 1 R 3 D gene in cancer were selected.
  • Example 3 RNA 1 analysis using colorectal cancer cell lines In this example, the PPP 1 R 3 D gene, which was frequently found in 200 colorectal cancer patients, was derived from the colorectal cancer genome. RNA i analysis using a cell line R KO E 6) where gene amplification was observed at the level ⁇ RNA 1 analysis>
  • s 1 RN A synthesized s l RNA that selectively selects specific 2 1 me r in the gene (Q I AGEN
  • sl RNA DLD-1 and i FECT (QI AG EN) into RKOE6 cells
  • cells were introduced into cells according to a protocol of 100 nM si.
  • N egontrolsi RNA (QI AGEN) was used as a control. Observation was performed under an inverted microscope for 4 days.
  • the number of viable cells after introduction of s i RN A was measured using A 1 a m a r B l u u r c e) according to the attached protocol.
  • Figures 2A and 2B show the observed images of the s l RNAs of D L D-1 and R D ⁇ R 1 R 3 D gene on the day of Tran sfeet, respectively (upper row. X 40, lower row X 2 0 0). And c, respectively, s i RN A 3 of the PPP 1 R 3 D gene represents a negative control. As shown, it was evident in all cells of the table compared to the three types of a, b, and c s l e ga t i v e C o n t r o l (NC) (Fig. 2).
  • Fig. 3 A and Fig. 3B show that DLD-1 and PPP 1 R 3 D gene sl RNA were collected in Transfec 2 4 hours later, and the cells were collected and subjected to quantitative RT-PCR as an endogenous control. Relative amounts using relative ratios of GAP DH are shown. As shown, T-PCR at the RNA level confirmed that Compared to C), the number of cells was significantly reduced (Fig. 4). However, the three s 1 RNAs of the PPP 1 R 3 D gene (a, are significant in the t test (p 0 0 1), so the result of the PPP 1 R 3 D gene due to the RNA 1 effect is DLD-1 and RK ⁇ E 6 were found to be significantly suppressed Example 4. In RNA 1 analysis using colon-derived normal cell lines By using a cell line, the suppression effect of the target gene can be verified in a cancer-specific manner.
  • Example 3 uses the c sequence.
  • siRNA introduction into cells use Lt am lne 2 00 (Invitrogen) to introduce siRNA into cells according to the attached protocol egative Controls RNA (QI AG). Observe for one day under an inverted microscope.
  • Figure 5 shows some of the cancer cells observed in each specimen tissue (A ⁇ ; ⁇ ) (Analysis micrograph (fluorescence image). As shown, more than 3 spots of cancer signal were found. It was confirmed that R ⁇ ⁇ ⁇ 1 R 3 was amplified in 10 samples (G / R) of 11 or higher by array CGH method, and gene amplification occurred throughout the pathological stage This shows that this 1 R 3 D gene region can be used as a molecular target for cancer drugs in cancer diagnosis by the method
  • Example 6 Blood PPP 1 R 3 D by mass spectrometry Protein 1) Pretreatment of blood sample
  • a peptide fragment of 0 1 was separated by a precolumn cartridge (C 5 m, 300 A, 30 ⁇ d x 5 mm LC PACKINGS 163589) Nano column (C18 PepMap 3 m, 100 A, 75 m i PACKINGS 160321).
  • a precolumn cartridge C 5 m, 300 A, 30 ⁇ d x 5 mm LC PACKINGS 163589
  • Nano column C18 PepMap 3 m, 100 A, 75 m i PACKINGS 160321.
  • Ulti PACKINGS was used as the HPLC apparatus.
  • the flow rate was 200 nL / min, and a linear gradient with a concentration gradient between 0 1% formic acid (Wak-containing 2% acetonitrile (MERCK 1287229) and 0 1% formic acid-containing tolyl was 0 57% / min.
  • the separated sample was introduced into ion trap mass spectrometry directly connected through PicoTip (New Object 10-D-20)
  • the mass spectrometer was ionized with HCT Plus (Bruker Dal tonics) material with a capillary voltage of 1500 V, an end plate of 500 V, and a dry plate. Gas flow rate 12 L / min, dry gas temperature 250 ° C
  • MS / MS analysis was conducted with Da uptake before and after the target m / z.
  • the target peptide fragment at amino acid position 1 94 (SEQ ID NO: 1 1) did not show an ion peak corresponding to such a partial sequence in healthy blood (see FIG. 7C). See). Therefore, it was strongly suggested that the PPP 1 R 3 D protein (PPP 1 R 3 D protein) may exist in the blood in a cancer-specific manner.
  • PPP 1 R 3 D protein PPP 1 R 3 D protein
  • Example 7 RNAi effect in uterine cervical cancer-derived strain HeLa cell line In the colon cancer cell line, a knock growth inhibition effect of PPP 1 R 3 D gene was observed. RNAi analysis was performed using the above-mentioned 3 using the HeLa cell line of cervical cancer-derived cells.
  • s 1 RNA A gofect amine I nvitrogen) was introduced into the cells according to the attached protocol with nM siRNA, using eg Negative Control RNA (QI).
  • PPP 1 R 3 D gene s 1 R NA 3 species (9% in a, b, c siRNA a, 17 in siRNA b, s lRNA c showed growth inhibitory effect (p ⁇ 001 in t test) Significant)
  • differential interference images were taken under a microscope and observed in detail, specifically, differential interference in the same visual field 1, 2, 3, and 4 days after s lRNA Transfection in HeLa cells.
  • Fig. 9 shows that NC showed negative control s lRNA (Q was used.
  • the present invention provides a therapeutic agent for cancer, a diagnostic agent, a diagnostic method, a kit used for the therapeutic method, and the like. Therefore, it is useful in the field of this invention 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 PPP1R3D. 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 PPP1R3D; un agent diagnostique du cancer et un procédé de diagnostic du cancer utilisant ledit anticorps et analogue.
PCT/JP2006/320009 2005-09-30 2006-09-29 Application therapeutique ou diagnostique du gene ppp1r3d Ceased WO2007037533A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
WO2004039412A2 (fr) * 2002-10-29 2004-05-13 Engene, Inc. Traitement de cancer par des modulations metaboliques
WO2005002414A2 (fr) * 2003-07-01 2005-01-13 Mor Research Applications Ltd. Determination d'un pronostic chez des patients atteints du sarcome d'ewing au moyen d'un profilage genetique
WO2005098041A2 (fr) * 2004-03-26 2005-10-20 University Of Florida Research Foundation, Inc. Detection et traitement de troubles fibrotiques
WO2005118869A2 (fr) * 2004-05-28 2005-12-15 Dana-Farber Cancer Institute, Inc. Compositions, kits et procedes pour identifier, evaluer, prevenir et traiter un cancer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004039412A2 (fr) * 2002-10-29 2004-05-13 Engene, Inc. Traitement de cancer par des modulations metaboliques
WO2005002414A2 (fr) * 2003-07-01 2005-01-13 Mor Research Applications Ltd. Determination d'un pronostic chez des patients atteints du sarcome d'ewing au moyen d'un profilage genetique
WO2005098041A2 (fr) * 2004-03-26 2005-10-20 University Of Florida Research Foundation, Inc. Detection et traitement de troubles fibrotiques
WO2005118869A2 (fr) * 2004-05-28 2005-12-15 Dana-Farber Cancer Institute, Inc. Compositions, kits et procedes pour identifier, evaluer, prevenir et traiter un cancer

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Title
ARMSTRONG C.G. ET AL.: "PPP1R6, a novel member of the family of glycogen-targetting subunits of protein phosphatase 1", FEBS LETTERS, vol. 418, 1997, pages 210 - 214, XP004261543 *

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