WO2007037533A9 - Application therapeutique ou diagnostique du gene ppp1r3d - Google Patents

Application therapeutique ou diagnostique du gene ppp1r3d

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Publication number
WO2007037533A9
WO2007037533A9 PCT/JP2006/320009 JP2006320009W WO2007037533A9 WO 2007037533 A9 WO2007037533 A9 WO 2007037533A9 JP 2006320009 W JP2006320009 W JP 2006320009W WO 2007037533 A9 WO2007037533 A9 WO 2007037533A9
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WIPO (PCT)
Prior art keywords
cancer
gene
protein
ppp
antibody
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PCT/JP2006/320009
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English (en)
Japanese (ja)
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WO2007037533A1 (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 JP2007537776A priority Critical patent/JPWO2007037533A1/ja
Publication of WO2007037533A1 publication Critical patent/WO2007037533A1/fr
Publication of WO2007037533A9 publication Critical patent/WO2007037533A9/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
    • 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 P P P 1 R 3 D gene that is a gene specifically amplified in cancer, its therapeutic or diagnostic use, and the like.
  • Cancers Malignant tumors (cancers) are characterized by lethality due to generalization through proliferation, invasion, and metastasis. Local therapies such as surgical resection or radiation therapy cannot adequately address metastatic recurrent cancer, and the development of systemic pharmacotherapy is expected to improve the outcome of cancer treatment in the future. Yes.
  • Chemotherapy which is the current center of cancer drug therapy, often uses cell killing agents that directly act on the DNA and / or RNA of cancer cells and cause the cells to die. Bone marrow cells, germ cells, hair matrix
  • 3 ⁇ 4-It acted on normal cells such as cells and gastrointestinal epithelial cells, which had many divisions, and had strong side effects.
  • recent advances in molecular cell biology have elucidated the mechanisms involved in cancer cell invasion, proliferation, and metastasis, and the development of molecular targeted drugs that specifically act on the specific mechanisms of cancer cells. Attention has been paid.
  • EGFR epidermal growth factor receptor
  • Yretza genetic name: gefitinib
  • Herceptin gene: trastuzumab
  • HER-2 human epidermal growth factor receptor 2
  • W ⁇ 94 4 Z 0 0 1 3 6 human epidermal growth factor receptor 2
  • Japanese colon cancer tends to increase year by year, and the number of deaths is lung cancer and stomach It is in 3rd place. By age, 60s is the most common, followed by 50s and 70s.
  • the cause of the increase in colorectal cancer may be due to genetic factors or environmental factors, but it has been pointed out that it may be due to westernization of the diet, especially excessive consumption of animal fat.
  • Development of an effective molecular target drug for colorectal cancer is awaited.
  • about half of the tumor markers used for diagnosis (CEA, CA 19-9) are positive even in advanced colorectal cancer, with no organ specificity, and higher performance diagnostic agents. Development is desired. Disclosure of the invention
  • the present inventors have found that the gene that is frequently amplified in cancer (especially colorectal cancer) is the PPP 1 R 3 D gene. I found out. Furthermore, the present inventors have found that the proliferation of cancer cells can be suppressed by inhibiting the expression of PPP 1 R 3 D protein in colorectal cancer cell lines and cervical cancer cell lines. It came to complete. That is, the present invention provides a cancer therapeutic agent, a screening method for a candidate substance having a cancer-suppressing action, a cancer diagnostic agent, a cancer diagnostic kit, a cancer diagnostic method, and the like described below. provide.
  • a cancer therapeutic agent comprising, as an active ingredient, a substance that inhibits the expression of PPP1R3D gene.
  • the cancer therapeutic agent according to (1) above comprising a substance selected from the group consisting of: (3) A cancer therapeutic agent comprising a substance that inhibits the activity of PPP1R3D protein as an active ingredient.
  • the cancer therapeutic agent according to (3) above comprising a substance selected from the group consisting of:
  • 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 a PPP 1 R 3 D protein activity inhibitor (a) contacting the PPP 1 R 3 D protein with a test compound,
  • a screening method comprising the step of selecting a compound that binds to the PPP 1 R 3 D 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 base sequence that can be hybridized to a PPP 1 R3D gene or a part of the base sequence thereof under stringent high-precipitation conditions.
  • (2 7) A method for treating cancer, comprising a step of administering a PPP 1 R 3 D gene expression inhibitor to a patient.
  • a method for treating cancer comprising the step of administering to a patient an inhibitor of PPP 1 R 3 D protein activity.
  • a cancer therapeutic agent containing a PPP 1 R 3 D gene expression inhibitor as an active ingredient SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, Alternatively, a cancer therapeutic agent comprising a polynucleotide having the base sequence of SEQ ID NO: 8.
  • FIG. 1 is a histogram showing the frequency of PPP 1 R 3D gene versus the degree of gene amplification in 200 samples from colon cancer patients.
  • Figure 2 shows an optical micrograph (phase difference) showing the results of RNA i analysis when s 1 RNA of the PPP 1 R 3 D gene was transfected into the colon cancer cell lines DLD_1 and RKO E6. Image).
  • Figure 3 is a graph showing the results of quantitative RT-PCR evaluation of the RNA i effect when siRNA of the PPP 1 R 3 D gene was transfected into colon cancer cell lines DLD-1 and RKOE 6. is there.
  • Figure 4 is a graph showing the results of evaluating the RNA i effect by measuring the number of living cells when siRNA of the PPP 1 R 3 D gene was transfected into colon cancer cell lines DL D-1 and RKOE 6. It is.
  • Fig. 5 is an optical micrograph (fluorescence image) of a portion (6 cells) of cancer cells in a sample tissue derived from a colon cancer patient analyzed by the FISH method.
  • Figures 6A and B are graphs showing the results of (A) serum derived from a colon cancer patient and (B) serum derived from a healthy subject, respectively, analyzed by mass spectrometry.
  • 7A to C show the correspondence between the peak shown in FIG. 6 and amino acids (or amino acid sequences) determined by MS / MS analysis.
  • FIG. 8 is a graph showing the results of verifying the RNAi effect by measuring the number of viable cells when sciRNA of the PPP1R3D gene was transfected into the cervical cancer cell line HeLa cell line.
  • Fig. 9 is an optical micrograph (differential interference image) showing the result of detailed observation of the dynamics of the experiment in Fig. 8 taken in a time-series and under a microscope.
  • the present inventors used a specimen derived from a colorectal cancer patient to verify the gene amplified by the array CGH method and to identify a gene amplification region specific to colorectal cancer.
  • the human PPP 1 R 3 D (.Proteinphosphatase 1, regu 1 atorysubunit 3 D) gene was found to be frequent in specimens from colon cancer patients. It was.
  • PPP1R3D PPP1R6 (Protein phosphatase 1 and regulatory subunit 3D) is a glycogen-binding protein that binds to protein phosphatase 1 (PP1) and participates in glycogen metabolism.
  • PP1 binds to more than 20 different proteins and is responsible for various intracellular regulation by dephosphorylation from reversibly phosphorylated proteins. For example, it regulates glycogen metabolism by glycogen binding protein, regulates muscle contraction by myosin binding protein, regulates in the nucleus, and changes its binding protein by signals from outside the cell.
  • glycogen binding protein regulates glycogen metabolism by glycogen binding protein
  • myosin binding protein regulates muscle contraction by myosin binding protein
  • PP 1 activates glycogen synthase by dephosphorylation and regulates glycogen synthesis.
  • glycogen binding subunits of PP1 including PPP1R6.
  • GM PPP1R3
  • GM PPP1R3
  • GM PPP1R3
  • PPP1R4 Liver-specific GL
  • PPP1R5 PPP1R3C
  • PPP1R3C PPP1R5
  • PPP1R3D PPP1R6
  • PPP1R6 is highly expressed in skeletal muscles and the heart, and has been reported to be expressed at moderate to low levels in other organs (brain, placenta, lung, liver, kidney, sputum) (Armstrong, CG, et al. (1997) FEBS Lett. 18, 210-214).
  • PPP1R3D PPP1R6
  • PPP1R6 PPP1R6
  • V / I XF-sequence
  • MAPKs Mitogen-act ivated protein kinases
  • S / T P-sequence
  • MAPK-activated protein kinase-2 MAPK-activated protein kinase-2
  • Calmodul in- There is a sequence consistent with the LXRXXSL-sequence (Stokoe, D., et al. (1993) Biochem. J.
  • colon cancer-derived cell lines (LOVO, KM12, COL0320), lung cancer-derived cell lines (H322, PC10, H157, SBC5, ⁇ 209), stomach cancer-derived cell lines ( ⁇ 7, ⁇ 0 ⁇ ), ovarian cancer-derived cell lines ( ⁇ 2780) , 2008) has been reported (Takakura, S., et al. (2001) Int. J. Oncology 18, 817-824)).
  • uterine cancer cell line HeLa cells there is a report that the induction of apoptosis by RNAi was 2.1 times that of the control (McKeigan, JP, et al. (2005) Nature Cell Biology). 7 (6), 591-600).
  • RNA i RNA NA interference
  • cancer can be treated by suppressing the expression of the PPP 1 R 3 D gene. It is also possible to diagnose cancer by measuring the expression level of the PPP 1 R 3 D gene.
  • the cancer therapeutic agent, screening method, diagnostic agent and the like of the present invention will be described in detail.
  • the present invention includes (1) a cancer therapeutic agent containing a PPP 1 R 3 D gene expression inhibitor as an active ingredient, and (2) a PPP 1 R 3 D protein activity inhibitor as an active ingredient. Provide cancer treatment.
  • PPP 1 R 3 D gene refers to a 3 4 8 1 base sequence registered under the Accession No .: N M — 0 0 6 2 4 2 on the NCBI nucleotide base.
  • G means PPP 1 R 3 D gene (SEQ ID NO: 1) (Armstrong, CG, et al. (1997) FEBS Lett. 418, 210-214), but is not limited to this.
  • the base of the gene A stringent hyperidase such as a variant that is altered by having one or more base substitutions, deletions, additions, or insertions in the sequence, or a complementary sequence thereof.
  • a gene consisting of a polynucleotide having a base sequence that can be highly pre-synthesized under a running condition is also included in the “PPP 1 R 3 D gene” used in this specification.
  • stringent conditions may be low stringent conditions, medium stringent conditions, or high stringent conditions.
  • Low stringency conditions are, for example, conditions at 5 XSSC, 5 X Denhal solution, 0.5% SDS, 50% formamide, 32.
  • “medium stringent conditions” are, for example, conditions of 5 XSSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 42.
  • “High stringent conditions” are, for example, conditions of 5 XSSC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide, 50. Under these conditions, it can be expected that DNA having high homology can be obtained efficiently as the temperature is increased. However, there are several factors that can influence the stringency of the hybridization, such as temperature, probe concentration, probe length, intensity, time, and salt concentration. The same stringency can be achieved by selecting it as appropriate.
  • nucleotide sequence of SEQ ID NO: 1 for example, 70 0 when calculated using the default parameters by homology search software such as FASTA and BLAST. % Or more, 75% 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 Polynucleotides having% or more, 97% or more, 98% or more, 99% or more identity can be mentioned.
  • inhibition of gene expression means any event in a series of events from gene to protein production (for example, transcription (production of mRNA), translation (production of protein)) By inhibiting the production of the protein encoded by the gene.
  • PPP 1 R 3 D protein refers to the “Accession No .: NP” in the NC BI protein database.
  • a human PPP 1 R 3 D protein (SEQ ID NO: 2) consisting of 2 9 9 amino acid residues registered under 0 0 6 2 3 3 and an activity substantially equivalent to this protein (eg, PP1 binding activity, One or more activities selected from glycogen binding activity), and from amino acid sequences in which deletion, substitution, insertion, and / or addition of one or more amino acid residues to the amino acid sequence of this protein occurs.
  • a mutant protein consisting of 2 9 9 amino acid residues registered under 0 0 6 2 3 3 and an activity substantially equivalent to this protein (eg, PP1 binding activity, One or more activities selected from glycogen binding activity), and from amino acid sequences in which deletion, substitution, insertion, and / or addition of one or more amino acid residues to the amino acid sequence of this protein occurs.
  • 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.
  • the smaller the number of mutations the better.
  • such a mutant protein has an amino acid sequence of SEQ ID NO: 2 and about 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 9 2 % Or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of amino acid sequences having identity
  • a protein having substantially the same activity as the original protein In general, the larger the homology value, the better.
  • the PPP 1 R 3 D protein includes a “partial peptide” of the PPP 1 R 3 D protein.
  • a partial peptide of the PPP 1 R 3 D protein a partial peptide consisting of a partial amino acid sequence of the amino acid sequence of the PPP 1 R 3 D protein (SEQ ID NO: 2) is preferable. Any one having the same activity as that of the aforementioned PPP 1 R 3 D protein may be used.
  • the 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 at least And a polypeptide having an amino acid sequence consisting of 20 amino acid residues.
  • these polypeptides are paired with a portion involved in the activity of the PPP 1 R 3 D protein. Contains the corresponding amino acid sequence.
  • the partial peptide used in the present invention is one or more of the above-mentioned polypeptides in the amino acid sequence (for example, about 1 to 20 and more preferably about 1 to 10). Further preferably, about 1 to 5 amino acid residues may be changed by deletion, addition, substitution, or insertion.
  • the PPP1R3D protein used in the present invention can be prepared from cells or tissues expressing the protein. These proteins can also be synthesized by a known peptide synthesizer, or prepared by a recombinant method using an appropriate host cell selected from prokaryotic organisms or eukaryotic organisms. Can do.
  • the P P.1 R 3 D protein used in the present invention may be derived from any species, but is preferably derived from human.
  • “Substantially the same activity” indicates that these activities are qualitatively equivalent. Therefore, the activities (PP1 binding activity, glycogen binding activity, etc.) are equivalent (for example, about 0.01 to: 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to However, quantitative factors such as the degree of activity and the molecular weight of the protein may be different. These activities can be measured according to known methods described in the literature such as Armstrong, CG, et al. (1997) FEBS Lett. 418, 210-214. For example, screening methods described later are used. Can be measured according to
  • B LA ST and Gapped B LAS T programs the default parameters of each program are used.
  • cancer therapeutic agent includes anticancer agents, cancer metastasis inhibitors, cancer cell apoptosis inducers, cancer cell proliferation inhibitors, cancer cell infiltration inhibitors, cancer preventive agents, and the like. Used in meaning.
  • cancer (or cancer) and “tumor” are used as terms having the same meaning.
  • the present invention provides a cancer therapeutic agent containing a PPP 1 R 3 D gene expression inhibitor as an active ingredient.
  • PPP 1 R 3 D gene expression inhibitor is not limited as long as it inhibits the expression of the PPP 1 R 3 D gene.
  • PPP 1 R 3 D A substance that inhibits transcription from a gene to PPP 1 R 3 D mRNA
  • PPP 1 R 3 D mRNA a substance that inhibits translation from PPP 1 R 3 D mRNA to PPP 1 R 3 D protein.
  • substances that inhibit transcription from P P P 1 R 3 D gene to P P P 1 R 3 D mRNA include:
  • substances that inhibit translation from PPP 1 R 3 D mRNA to PPP 1 R 3 D protein include: (e) a polynucleotide having an RNA i action on PPP 1 R 3 D mRNA or a part thereof (for example, siRNA),
  • nucleic acid means R N A or DN A.
  • nucleic acid 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 are both inhibited when a double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into the cell.
  • Examples of RNA used here include double-stranded RNA that causes RNA interference of 19 to 30 bases in length, such as ds RNA (doublestrand RNA), si RNA (small interfering RNA), or sh RNA ( shorthairpin RNA).
  • dsRNA doublestrand RNA
  • siRNA small interfering RNA
  • sh RNA shorthairpin RNA
  • the length of the double-stranded RNA that exhibits the RNAi effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21.
  • ⁇ 25 salt most preferably 21-23 bases.
  • si R N A used in Example 3
  • antisense nucleic acid or “antisense polynucleotide” is complementary to at least a portion of a DNA region of interest.
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). They may be double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA, or even a DNA: RNA hybrid.
  • modified nucleic acids include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those that are resistant to degradation of polynucleotide amides and oligonucleotide amides. is not.
  • 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 preferably a sequence complementary to the endogenous gene of the animal to be transformed or a part thereof, but is completely complementary as long as the gene expression can be effectively suppressed. It doesn't have to be.
  • an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of the PPPP1R3D gene is effective in inhibiting gene translation.
  • a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
  • Antisense nucleic acid effective for inhibiting gene translation is about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more of the transcript of the target gene. % Complementarity.
  • the length of the antisense nucleic acid is at least about 10 bases (for example, about 10 to 40 bases), preferably about 15 bases or more. More preferably about 100 bases or more, and still more preferably about 500 bases or more.
  • Antisense nucleic acids can be designed with reference to known literature (for example, Hirashima and Inoue, Laboratory for Neonatal Chemistry 2 Replication and Expression of Nucleic Acid ⁇ V Gene, edited by The Japanese Biochemical Society, Tokyo Chemical Dojin, 1 9 9 3, p. 3 1 9 ⁇ 3 4 7), J. Kawakamieta 1, V ol. 8, p. 2 4 7, 1 9 9 2; V o 1.8, p. 3 9 5, 1 9 9 2; S. T. C rookeetal., Ed , Antisense Research and Applications, CRCP ress, 1 9 9 3 etc.).
  • a nucleic acid having a lipozyme activity that specifically cleaves a transcription product of a PPP 1 R 3D gene can be used as an active ingredient.
  • ribozyme activity refers to a nucleic acid that cleaves site-specifically mRNA, which is a transcription product of a target gene. Ribozymes have a strength of 40 nucleotides or more, such as the group I intron type and MRNA contained in RNase P. The hammerhead type is about 40 nucleotides called the hairpin type. Some have an active domain (protein nucleic acid enzyme, 1990, 35, p. 2 191).
  • FEBSL ett 1 9 8 8, 2 2 8, p. 2 2 8; FEBSL ett, 1 9 8 8, 2 3 9, p. 2 8 5; protein Reference can be made to nucleic acid enzymes, 1 990, 3 5, p. 2 1 9 1; Nucl Acids Res, 1 9 8 9, 1 7, p.
  • hairpin ribozymes see, for example, Nature, 1 986, 3 2 3, p. 3 4 9; Nuc 1 A cids R es, 1 9 9 1, 1 9, p. 6 7 5 1; You can refer to Hiroshi Kikuchi, Chemistry and Biology, 1 992, 30, p.
  • a compound other than a nucleic acid that inhibits the transcriptional activity of the PPP 1 R 3 D gene can be used as an active ingredient.
  • Such compounds bind to factors involved in the expression and transcription of the PPP 1 R 3 D gene, for example. It is a compound.
  • Such a compound may be a natural product or a synthetic compound.
  • Such a compound can be obtained by the screening method described below. 1. 2 Cancer treatments containing inhibitors of PPP 1 R 3 D protein activity
  • the present invention also provides a cancer therapeutic agent containing a PPP 1 R 3 D protein activity inhibitor.
  • inhibitors of P P P 1 R 3 D protein activity include, for example,
  • 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, as long as it binds to the PPP 1 R 3 D protein of the present invention, in addition to the polyclonal antibody and the monoclonal antibody described above, a human antibody, a human recombinant human form Antibodies, antibody fragments thereof, and modified antibodies are also included.
  • Antibodies that bind to PPP1R3D protein can be prepared by methods known to those skilled in the art. Details of the anti-PPPP1R3D antibody will be described later.
  • a PPP 1 R 3 D protein mutant having a dominant negative property with respect to the PPP 1 R 3 D protein refers to an endogenous wild-type PPP by expressing a gene encoding the same. 1 Refers to a protein that has the function of eliminating or reducing the activity of R 3 D protein (Kunihiro Tsuchida, Gene Activity Inhibition Experiment, edited by Yoshikazu Tahira, Yodosha (see 2 0 0 1) 2 6-3 2 etc.).
  • a compound other than the above antibody or mutant that binds to the PPP 1 R 3 D protein can be used as an active ingredient.
  • a compound is, for example, a compound that binds to the PPP 1 R 3 D protein and inhibits its activity.
  • Such compounds may be natural products or synthetic compounds.
  • Such a compound can be obtained by the screening method described below.
  • the above-mentioned substance capable of inhibiting the activity of the PPP1R3D protein of the present invention can be used as a cancer therapeutic agent.
  • the present invention also provides a method for screening a candidate compound having a cancer suppressing action.
  • One preferred embodiment is a method using as an index the binding between the PPP 1 R 3 D protein and the test compound.
  • a compound that binds to PPP1R3D protein is expected to have an effect of inhibiting the activity of PPPP1R3D protein.
  • P P P 1 R 3 D protein is contacted with a test compound.
  • the PPP 1 R 3 D protein may be a purified form of the PPP 1 R 3 D protein, for example, a form expressed intracellularly or extracellularly, depending on the indicator for detecting binding to the test compound. Or it can be in the form of an affinity ram.
  • the test compound used in this method can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.
  • test compound used for this method.
  • natural compounds, organic compounds, inorganic compounds, proteins, peptides, etc. as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, oceans Examples include, but are not limited to, biological extracts and plant extracts.
  • the binding between the PPP1R3D protein and the test compound can be detected by, for example, a label attached to the test compound bound to the PPP1R3D protein.
  • a change in the activity of the PPP1R3D protein caused by the binding of the test compound to the PPP1R3D protein expressed inside or outside the cell can also be detected as an index.
  • the binding activity between a protein and a test compound can be measured by a known method (for example, measurement such as PP1 binding activity, glycogen binding activity (Armstrong, CG, et al. (1997) FEBS Lett 418, 210-214)).
  • test compound that binds to PPP 1 R 3 D protein and inhibits its activity is then selected.
  • the compound isolated by this method is expected to have a cancer suppressing action and is useful as a cancer therapeutic agent.
  • Another embodiment of the screening method of the present invention is a method using the expression of the PPP 1 R 3 D gene as an index.
  • a test compound is brought into contact with a cell expressing the PPP 1 R 3 D gene.
  • the origin of the “cell” used include, but are not limited to, cells derived from pets, livestock, etc. such as humans, mice, cats, cats, dogs, lions, hedgehogs, and birds.
  • PPP 1 R 3 D gene-expressing cell refers to a cell expressing an endogenous PPP 1 R 3 D gene or an exogenous PPP 1 R 3 D gene introduced and the gene is expressed. Cells can be used. Cells in which an exogenous PPP 1 R 3 D gene is expressed can usually be prepared by introducing an expression vector into which a PPP 1 R 3 D gene has been inserted into a host cell. The expression vector is produced by general genetic engineering techniques. Can be made.
  • test compound used in this method is not particularly limited.
  • natural compounds, organic compounds, inorganic compounds, single compounds such as proteins and peptides, compound libraries, expression products of gene libraries, Cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, etc. are used.
  • Contacting a test compound with cells expressing the PPP 1 R 3 D gene is usually performed by adding the test compound to the culture medium of cells expressing the PPP 1 R 3 D gene, respectively. I'm not limited to this method.
  • 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 PPP 1 R 3 D gene is then measured.
  • gene expression includes both transcription and translation.
  • the expression level of the gene can be measured by methods known to those skilled in the art. For example, mRNA is extracted from cells expressing the PPP 1 R 3D gene according to a conventional method, and transcription of the gene is carried out by performing Northern hybridization or RT-PCR using this mRNA as a cocoon. Level measurements can be made.
  • the promoter region of the PPP 1 R 3 D gene is isolated according to a conventional method, and a gene that can be detected downstream using a labeled gene (for example, luminescence, fluorescence, color development, etc.
  • the transcription level of the gene can also be measured by observing the activity of the marker gene.
  • protein fractions are collected from cells expressing the PPP 1 R 3 D gene, and the expression level of the PPP 1 R 3 D protein is detected by electrophoresis such as SDS-PAGE. Can also be done.
  • PPP 1 R 3 The antibody used for detection of D protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.
  • a compound that decreases 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-PPPP1R3D antibody, a cancer therapeutic agent containing the antibody, and the like.
  • the cancer therapeutic agent is used for targeted therapy or targeted drug delivery of cancer.
  • anti-PPP 1 R 3D antibody includes an antibody that specifically binds to a PPP 1 R 3 D protein (including fragments (partial peptides) or salts thereof).
  • the anti-PPP 1 R 3D antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.
  • the class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.
  • antibody used herein is used to include any antibody fragment or derivative.
  • F ab, F ab ' 2 , CDR humanized antibody, multifunctional antibody, single chain antibody (Sc FV).
  • the antibody of the present invention can be produced by a known method. Methods for producing such antibodies are well known in the art (see, for example, Harrowow E. & Lane D., Antibody, Cold Spring Harbor Laboratory Pres (1 9 8 8))).
  • the protein used as the sensitizing antigen is usually P P P 1 R 3 D protein or a salt thereof.
  • the PPP 1 R 3 D protein includes a partial peptide thereof, which is not limited to, for example, a fragment of the amino acid sequence of SEQ ID NO: 2, for example, 20 These are partial peptides having 40 or more, 60 or more, 80 or more, 100 or more consecutive amino acid sequence portions. As 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 amino acid sequence are deleted. It may be a substitution, insertion and / or addition.
  • salts of the PPP 1 R 3 D protein or partial peptide thereof used herein include salts with inorganic acids (eg, hydrochloric acid, sulfuric acid), or salts with organic acids (eg, acetic acid, formic acid, propionic acid). Etc. are used.
  • the PPP 1 R 3 D protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or human, particularly from human. Protein is preferred.
  • PPP 1 R 3 D protein a partial peptide thereof or a salt thereof (in the present specification, these are collectively referred to as “PPP 1 R 3 D protein”) as an antigen.
  • Administer to animals such as rats, mice, and rabbits.
  • the antigen dose per animal is 0.1 to 1 when no adjuvant is used! OO mg, l to 100 g when adjuvant is used.
  • adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Immunity is It is performed mainly by injecting intravenously, subcutaneously or intraperitoneally.
  • the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably at intervals of 2 to 5 weeks. Then, antibody-producing cells are collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later.
  • antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, and spleen cells or local lymph node cells are preferred.
  • Cell fusion between antibody-producing cells and myeloma cells is performed in order to obtain a hybridoma.
  • Generally available cell lines of animals such as mice can be used as the myeloma cells to be fused with antibody-producing cells.
  • 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 include mouse myeloma cell lines such as X 6 3 A g. 8. 65 3, NSIZ 1—A g 4-1, NS 0-1 and rat myeloma cell lines such as YB 2-0. It is done.
  • Cell fusion consists of 1 XI 0 6 to 1 XI 0 7 ml antibody-producing cells and 2 X 1 0 5 to 1 in animal cell culture media such as serum-free DM EM, RPMI — 1 6 40 2 X 1 0 6 or Roh m 1 of the myeloma cells were mixed (antibody producing cells and the myeloma cells cell ratio 2: 1 to 3: 1 is preferred), under the promoter present cells fused fusion reaction I do.
  • the cell fusion promoter polyethylene dalycol having an average molecular weight of 1000 to 600,000 Dalton can be used.
  • antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device using electrical stimulation (for example, electoral position).
  • the screening for hybridomas is not particularly limited, as long as the usual method is followed.
  • a part of the culture supernatant contained in a well grown as a hybridoma can be collected and screened by an enzyme immunoassay, a radioimmunoassay, or the like. Cloning of fused cells is performed by limiting dilution.
  • a hybridoma which is a cell producing a monoclonal antibody that reacts with the PPP 1 R 3 D protein, is established.
  • a normal cell culture method As a method for collecting a monoclonal antibody from the hybridoma obtained as described above, a normal cell culture method, ascites formation method, or the like can be employed.
  • hypridoma is cultured in an animal cell culture medium such as RPMI-1640 medium, MEM medium or serum-free medium containing 10% urushi fetal serum under normal culture conditions (for example, 37) 5% C_ ⁇ 2 concentration) 7 cultured ⁇ 1 4 days to obtain the culture supernatant antibody.
  • an animal cell culture medium such as RPMI-1640 medium, MEM medium or serum-free medium containing 10% urushi fetal serum under normal culture conditions (for example, 37) 5% C_ ⁇ 2 concentration) 7 cultured ⁇ 1 4 days to obtain the culture supernatant antibody.
  • intraperitoneally mammalian the same species animal-derived myeloma cells administered eight drive re dormer about 1 XI 0 7 cells is mass proliferating High Priestess do
  • the above antigen is used in mammals such as rats, mice, and rabbits. To be administered.
  • the dose of antigen per animal is 0.1 to LOO mg when no adjuvant is used, and 10 to 100 g when adjuvant is used.
  • adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Immunization is performed mainly by injecting intravenously, subcutaneously or intraperitoneally. Further, the immunization interval is not particularly limited, and immunization is carried out 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably at intervals of 2 to 5 weeks.
  • enzyme immunoassay enzyme immunoassay (ELISA (enz ume— 1 inked mmu nosorbentassy) or EIA (enz ym ei mm unoassay)
  • RIA radioimmunoassay
  • the polyclonal antibody in the antiserum is applied to an affinity column fixed with ⁇ ⁇ ⁇ 1 R 3 D protein, and the antibody that reacts with ⁇ ⁇ ⁇ 1 R 3 D protein (column adsorbed fraction) is collected. To do.
  • the reactivity of the polyclonal antibody in the antiserum against P P P 1 R 3 D protein can be measured by the ⁇ L I S method.
  • the F ab or F ab ' 2 fragment can be prepared by digestion with a conventional protease (eg, pepsin or papain).
  • a conventional protease eg, pepsin or papain.
  • Humanized antibodies are described, for example, by Riec hma nn et al. (Riec hma nn JM o 1 B iol. O ct 5; 2 0 3 (3): 8 2 5-8 1 9 8 8), and J ones et al. Jones et al. Nature 3 2 1: 5 2 2-5 2 5, 1 986 6).
  • Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 1. 6, No. 10, 1 9 8 8”, Japanese Patent Publication No. 3-7 3 2 80, etc. Humanized antibodies are described in, for example, “Nature Genetics, Vol. 1 5, p. 1 4 6-1 5 6, 1 9 9 7 ”,“ Nature Genet
  • the antibody that binds to the PPP1R3D protein of the present invention can be used for the purpose of, for example, suppressing the proliferation of cancer cells or metastasis.
  • a human antibody or a human antibody is preferred in order to reduce immunogenicity.
  • the antibody When used as a diagnostic agent, the antibody may be labeled with a labeling substance for monitoring (eg, radioisotopes, fluorescent substances, etc.) ⁇ If necessary, label with radioactive substances, fluorescent compounds, etc. can do.
  • a labeling substance for monitoring eg, radioisotopes, fluorescent substances, etc.
  • label with radioactive substances, fluorescent compounds, etc. can do.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine, as well as using a bioluminescent compound to label the antibody PPP 1 R 3 D antibody You can also.
  • the presence of a bioluminescent protein is measured by detecting the presence of fluorescence.
  • Important bioluminescent compounds for this purpose are luciferin, luciferase and aequorin.
  • the antibody of the present invention also t can be used to specifically detect the PPP 1 R 3 D protein or the like present in test samples such as body fluids or tissues, the PPP 1 R 3 D protein or the like For preparation of antibody column used for purification, detection of PPP 1 R 3 D protein, etc. in each fraction during purification, analysis of PPP 1 R 3 D protein behavior in the test cells, etc. Can be used for
  • the anti-PPP 1 R 3 D antibody used in the present invention itself may decrease the activity of the antigen in the therapeutic agent or diagnostic agent of the present invention. It can be an agent having a neutralizing activity, but can be used in combination with other agents for producing a therapeutic effect, if necessary. Therefore, the present invention provides, in another aspect, a combination of an anti-PPPP 1 R 3 D antibody and another drug for use in targeted therapy or targeted imaging of cancer (eg, colorectal cancer). And compositions containing such complexes are also provided. According to such an embodiment, the anti-PPP 1 R 3 D antibody used in the present invention is used to label other drugs having a therapeutic effect or a labeling agent for diagnosis, and the PPP 1 R 3 D protein. It can be delivered to highly expressed target sites.
  • 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 radioactive halogen elements such as fluorine-18, iodine- 125 (1251), and iodine-1311. These radioactive halogen elements can also be widely used as radiotherapeutic agents or radiodiagnostic agents by labeling them with antibodies or peptides in the same manner as the above-mentioned radioactive metal elements. For example, 1 25 I or 1 3 1 ® over de of the I by known methods chloramine T method or the like, can be attached to an antibody or antibody fragment.
  • yttrium 1 90 9 0 Y
  • rhenium 1 1 8 6 1 8 6 R e
  • rhenium— 1 8 8 1 88 Re
  • metal chelator When labeling an antibody with a radioactive isotope, a metal chelator is usually used.
  • metal chelating agents include EDTA, DTPA, diaminodithio compound, cyclam, and D0TA.
  • chelating agents may be pre-bonded to the antibody and then labeled with a radioactive metal, or there may be a method of forming a radioactive metal chelate and then binding to the antibody and labeling.
  • site-in force that activates cells responsible for immunity is preferable.
  • toxins such as ricin and diphtheria toxin can be used to directly kill colon cancer cells.
  • a cDNA encoding the fusion protein is constructed by linking the cDNA encoding the therapeutic protein to the cDNA encoding the antibody or antibody fragment, and this DNA is either prokaryotic or eukaryotic.
  • a fusion antibody can be produced by inserting the expression vector into a biological 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 alkylating agents such as nitrogen mustard and cyclophosphamide, antimetabolites such as 5-fluorouracil and mesotrexe, daunomycin, bleomycin, mitomycin C, daunorubicin, Anti-cancer drugs such as antibiotics such as doxorubicin, plant alcohols such as vincristine, vinblastine, vindesine, hormonal drugs such as evening moxifen, dexamethasone (Clinical Oncology (Edited by Japan Clinical Oncology Society 1 9 9 6) Chemotherapy Inc.)), or steroids such as hyethcortisone, prednisone, non-sterolides such as aspirin, indomethacin, immunoregulators such as gold chimare, penicillamine, cyclophosphine Immunosuppression such
  • daunomycin and antibody can be bound by binding between daunomycin and the amino group of the antibody via glutaraldehyde, or by binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carpositimide. How to make Etc.
  • a viral vector modified so as to bind to the anti-PPP 1 R 3 D antibody of the present invention can be used (for example, adenoviral vector (Wang, P., eta 1. ( 1 9 9 5) Soma tic Celand Molec. Genet. 2 1, 4 2 9-44 1), retroviral vector (Naviaux RK, eta 1. (1 9 9 6) J. Virol 7 0 , 5 7 0 1-5 7 0 5), and lentiviral vectors (N aldini, L. (1 9 9 8) Curr. O pin.
  • adenoviral vector Wang, P., eta 1. ( 1 9 9 5) Soma tic Celand Molec. Genet. 2 1, 4 2 9-44 1
  • retroviral vector Naviaux RK, eta 1. (1 9 9 6) J. Virol 7 0 , 5 7 0 1-5 7 0 5
  • lentiviral vectors N aldini, L. (1 9 9 8) Cur
  • B iotechno include 9, 4 5 7-4 6 3)
  • Such viral vectors include cell proliferation-related genes, apoptosis-related genes, immune regulatory genes, and other target sites (eg, colon cancer), for example, to induce apoptosis of cancer cells.
  • a gene that produces a therapeutic effect is incorporated.
  • a viral vector that binds to an anti-PPP 1 R 3 D antibody, when administered together with an anti-PPP 1 R 3 D antibody to a patient in need of gene therapy, ie, an antigen recognized by the anti-PPP 1 R 3 D antibody (ie, PPP 1 R 3 D) can be targeted to the existing site.
  • the anti-PPPP1R3D antibody and the other drug can be combined chemically or genetically.
  • “chemical bond” includes ionic bond, hydrogen bond, covalent bond, bond by intermolecular force, bond by hydrophobic interaction, etc.
  • “gene engineering bond” For example, when the fusion protein consisting of an antibody and a therapeutic protein is produced using a technique such as genetic recombination, the binding mode between the antibody and the therapeutic protein is included. To do.
  • Therapeutic agent containing a PPP 1 R 3 D gene expression inhibitor of the present invention a cancer therapeutic agent containing a PPP 1 R 3 D protein activity inhibitor, the anti-PPP 1 R 3 of the present invention
  • Anti-PPP 1 R 3D antibody to be used is either a viral or non-viral vector carrying a radioisotope, therapeutic protein, small molecule drug, and therapeutic gene, or any combination of these and chemical
  • a therapeutic agent that has been genetically engineered can be formulated based on a known 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.
  • Examples of the dosage form of the therapeutic agent of the present invention include tablets, powders, pills, powders, granules, fine granules, soft * hard capsules, film coating agents, and pellets as oral preparations.
  • Agents, sublingual agents, pastes, etc., parenteral agents include injections, suppositories, transdermal agents, ointments, plasters, liquids for external use, etc.
  • the optimum dosage form can be selected.
  • An inhibitor of PPP1R3D protein activity (or expression of PPP1R3D gene) as an active ingredient may be contained in the preparation in an amount of 0.1 to 99.9% by weight.
  • the dose of the active ingredient of the drug of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally for patients (as 60 kg) About 0. l mg per day :: I, 0 00 mg, preferably about 1.0: L 0 0 mg, more preferably about 1.0 to 50 mg It is.
  • the single dose varies depending on the administration subject, target organ, symptom, administration method, etc.
  • the patient (for 60 kg) ) About 0.1 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg is preferably administered intravenously. is there.
  • the final decision can be made as appropriate based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.
  • the preparations thus obtained should be administered to, for example, humans and other mammals (eg rats, rabbits, hidges, bushes, bushes, cats, dogs, monkeys, etc.) Can do.
  • mammals eg rats, rabbits, hidges, bushes, bushes, cats, dogs, monkeys, etc.
  • An 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, kidney cancer, bladder).
  • cancer for example, colorectal cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder.
  • Prevention / treatment of uterine cancer eg cervical cancer, uterine body cancer
  • testicular cancer thyroid cancer
  • knee cancer ovarian cancer
  • brain tumor blood tumor, etc.
  • the agent of the present invention is a P P P 1 R 3 D protein activity inhibitor or P
  • the agent of the present invention may contain both a PPP 1 R 3 D protein activity inhibitor and a P P P 1 R 3 D gene expression inhibitor.
  • the antisense nucleic acid when used, the antisense nucleic acid is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, or the like. Thereafter, it can be administered according to known means.
  • Antisense nucleic acids can be used alone or with a physiologically recognized carrier. They can also be formulated and administered via a catheter such as a gene gun or a hydrogel catheter.
  • a combination of a virus vector such as a recombinant adenovirus particle and an anti-PPP 1 R 3 D antibody when used for cancer treatment, these may be used alone, Is used with a pharmaceutically acceptable carrier.
  • a carrier a carrier as described above and a water isotonic solution 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. Examples of dosage forms include emulsions, syrups, capsules, tablets, granules, injections, ointments and the like.
  • Anti-P of the present invention include emulsions, syrups, capsules, tablets, granules, injections, ointments and the like.
  • PP 1 R 3D antibody When administering viral vector particles or pharmaceutical compositions containing them for treatment, it is usual to administer 10 3 to 10 15 viral particles at a time per adult. Is preferred, but may vary depending on the disease state and the target cell 'tissue properties.
  • the administration frequency may be once to several times a day, the administration period may be from 1 day to several months or more, and one to several doses are set as one set, and many sets are administered intermittently over a long period of time May be.
  • the virus vector particles or virus vector nucleic acid molecules used in the present invention can be used for detection of specific cells and / 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 viral vector and transfecting it into an appropriate host cell can be combined with an anti-PPP 1 R 3 D antibody in tumor cells.
  • an anti-PPP 1 R 3 D antibody in tumor cells.
  • the present invention also provides a diagnostic agent for cancer.
  • the cancer diagnostic agent of the present invention comprises: (a) an antibody against the PPP 1 R 3 D protein, or (b) a stringent hybridization with the PPP 1 R 3 D gene or a partial nucleotide sequence thereof. It contains a polynucleotide comprising a base sequence that can hybridize under conditions.
  • the diagnostic method using the anti-PPP 1 R 3 D antibody of the present invention includes, for example, (a) a step of contacting a biological sample derived from a subject with an antibody against the PPP 1 R 3 D protein, and (B) detecting and / or quantifying the binding between the antibody in the sample and the PPP 1 R 3 D protein or its partial peptide or salt thereof.
  • the labeled anti-PPP 1 R 3 D antibody is used to detect the binding between the PPP 1 R 3 D protein or a fragment thereof and the anti-PPP 1 R 3 D antibody. And / or quantified.
  • subject-derived biological sample refers to a subject-derived tissue, cell, or body fluid (for example, blood (including whole blood, plasma, serum, etc.), urine, lymph, saliva, sweat, semen, etc. ) including.
  • a “subject” is usually a human subject who is or is expected to undergo a cancer screening, such as a human subject who has or is suspected of having cancer. included.
  • cancers examples include 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 (example: cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, knee cancer, ovarian cancer, brain tumor, blood tumor, etc., especially colorectal cancer preferable.
  • Immunoassays to detect the expression of PPP 1 R 3 D in a biological sample from a subject as described above are suspected of having cancer (eg, colorectal cancer).
  • a biological sample taken from a subject at risk of cancer is contacted with an anti-PPPP 1 R 3 D antibody under conditions that produce specific antigen-antibody binding, and then immunospecific binding by the antibody Includes measuring the amount.
  • Such antibody binding is used to detect the presence and / or increased expression of the PPP 1 R 3 D protein.
  • detection of increased PPP 1 R 3 D protein expression is an indicator of disease state.
  • the level of PPP 1 R 3 D protein in the 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-PPPP1R3D 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 can be appropriately determined by those skilled in the art using conventional test methods.
  • the antibody is conjugated to an enzyme, such as that used in the enzyme immunoassay (EIA) [V oi 1 er, “Enzyme-labeled immunosorbent assay” (ELISA), 1 9 7 8, Diagnostic Horizons, 2: 1-7, M icrobiological A ssociates Q uarterly Pub 1 ication, Walkersvi 1 1 e. MD; J. CI in. Pat.
  • EIA enzyme immunoassay
  • Enzymes that can be used to attach a detectable label to an antibody include, but are not limited to, peroxidase and alkaline phosphatase. This detection can also be achieved by a colorimetric method using a chromogenic substrate for the enzyme.
  • RIA radioimmunoassay
  • sandwich immunoassay immunometric method
  • FIA fluorescence immunoassay
  • TRFIA time-resolved fluorescence immunoassay
  • EIA enzyme Immunoassay
  • LIA Luminescent immunoassay
  • ELIA Electrochemiluminescence immunoassay
  • Latex agglutination Immunoprecipitation Atssey, Precipitation reaction, Gel diffusion precipitation reaction, Immunodiffusion assay
  • Examples include an agglutination assay, a complement binding assay, an immunoradiometric assay, a fluorescence immunoassay, and an immunoassay selected from the group consisting of protein A immunoassay (WO 0 0Z 1 4).
  • 2 2 7 Gazette page 39, line 25, 4 to page 4, 2nd line, line 8, EP 1 1 1 1 04 7 A, paragraph 2, [0 1 1 5], page 19, line 3 to line
  • various diseases related to PPP 1 R 3 D protein dysfunction can be diagnosed. can do. For example, if an increase in the concentration of PPP 1 R 3 D protein is detected, for example, the disease may be caused by overexpression of PPP 1 R 3 D protein (eg, cancer (eg, large intestine cancer)) Can be diagnosed as being highly prone or likely to be affected in the future.
  • PPP 1 R 3 D protein eg, cancer (eg, large intestine cancer)
  • the anti-PPP 1 R 3D antibody of the present invention can also be used for in vivo diagnosis.
  • the preparation and use of antibody preparations that can be used herein are well known in the art.
  • antibody-chelating agents are described in Nuc 1. Med. Biol. 1 9 9 0 1 7: 24 7- 2 54.
  • a probe or primer designed based on the base sequence of the PPP 1 R 3 D gene can be used.
  • a diagnostic method can, for example, (a) be hybridized under a high-hydidization condition that is stringent in the biological sample derived from the subject and the base sequence of the PPP 1 R 3 D gene or a fragment thereof.
  • quantifying can, for example, (a) be hybridized under a high-hydidization condition that is stringent in the biological sample derived from the subject and the base sequence of the PPP 1 R 3 D gene or a fragment thereof.
  • the DNA (or gene fragment thereof) of the PPP 1 R 3 D gene in a biological sample derived from a subject is detected and determined or quantified using the probe.
  • 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 even longer It can be a polynucleotide fragment.
  • low, medium or high stringency conditions as described above may be used.
  • “a base sequence that can be hyperpredified under a high predation condition that is stringent to the base sequence of the PPP 1 R 3 D gene or a fragment thereof” includes the PPP 1 R 3 D gene.
  • a base sequence (antisense polynucleotide) complementary to the base sequence of the fragment is also included.
  • Methods of probe and nucleic acid hydridization are known to those skilled in the art, for example, WO 8 90 6 6 98, EP—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, EP—A 0 1 7 3 2 5 1, EP—A 0 1 2 8 0 1 8.
  • a polynucleotide probe or primer can be used to detect or quantify the target sequence using known techniques.
  • Such known methods include, for example, Southern hybridization, Northern hybridization, RT-PCR method, PCR-SSCP method (Genomics, Vol. 5, 8 7 4-8 7 9 (1 9 8 9)), Proceedingsofthe National Academy or Sciences of United States of America, Vol. 8 6, 2 7 6 6-2 7 7 0 (1 9 8 9) ), FISH method, DNA chip or array CGH (Combolic Genetic Hybridization) method can be used. Quantitative detection can be performed by quantitative RT-PCR.
  • the array C GH method applies the chromosome C GH method (K allioniemi, A. eta 1. (1 9 9 2) Science 2 5 8, 8 1 8-8 2 1); Using a DNA chip in which genomic DNA fragments (BAC, PAC, YAC, etc.) covering the chromosomal region are densely spotted, the cancer-derived DNA and normal DNA labeled with different dyes are placed on the slide. This is a method to detect DNA copy number abnormalities in cancer with high resolution by simultaneously hybridizing to the genomic DNA fragment and detecting its binding state (Pinke 1, D. eta 1. (1 9 9 8) N at. G enet. 2 0, 2 0 7-2 1
  • the mRNA level of the PPP 1 R 3 D in the cell is determined based on the standard gene (housekeeping gene (eg, S haper, N. L. et al., J. Mammary G land B iol. N eop 1 asia 3 (1 9 9 8) 3 1 5-3 2 4; Wu, YY and Rees, J. L., A cta D erm: V enereol. 8 0 (2 0 0 0) 2— 3) mRNA levels and preferably RT-PCR Can also be compared.
  • the standard gene housekeeping gene (eg, S haper, N. L. et al., J. Mammary G land B iol. N eop 1 asia 3 (1 9 9 8) 3 1 5-3 2 4; Wu, YY and Rees, J. L., A cta D erm: V enereol. 8 0 (2 0 0 0) 2—
  • the target sequence (DNA, mRNA, etc.) is detected and quantified by the method described above and overexpression of the PPP 1 R 3 D gene is confirmed, for example, overexpression of PPP 1 R 3 D It can be diagnosed that there is a high possibility that the disease is caused (for example, cancer (eg, colorectal cancer)) or that the disease is likely to be affected in the future.
  • cancer eg, colorectal cancer
  • the presence of the target protein or fragment thereof in the test sample can be identified using a mass spectrometer (MS). That is, by using a mass spectrometer, the amino acid sequence of a target protein or a fragment thereof can be determined, and it is determined whether or not a PPP 1 R 3 D protein is present in a biological sample derived from a subject. can do.
  • Mass spectrometry is a method for determining the mass of a sample by ionizing a sample such as a protein or peptide using MS, separating it according to the obtained mass Z charge (mZz), and measuring its intensity. . From the results of mass spectrometry, individual amino acids constituting the amino acid sequence of proteins and peptides can be identified.
  • MALD I matrix assisted laser desorption
  • ESI electrospray ionization
  • EI gas phase
  • FD field desorption
  • MALDI matrix assisted laser desorption
  • ESI electrospray ionization
  • FD field desorption
  • MALDI matrix assisted laser desorption
  • ESI electrospray ionization
  • EI gas phase
  • FD field desorption
  • MALDI matrix assisted laser desorption
  • ESI electrospray ionization
  • CI gas phase
  • FD field desorption
  • LC-ESI MSZMS Q-T FMS
  • MALD I-TO FMS etc.
  • amino acid sequencing methods using sequencers eg, gas phase sequencers
  • the present invention also provides a kit for detecting and Z or quantifying a PPP 1 R 3 D protein or a fragment thereof in a body fluid sample of a subject containing an anti-PPP 1 R 3 D antibody as a cancer marker. . Furthermore, the PPP 1 R 3 D gene or the PPP 1 R 3 D gene in a biological sample derived from a subject, which contains a base sequence that can be hybridized under high hybridization conditions, to the base sequence of the PPP 1 R 3 D gene or a part thereof. It also provides a kit for detecting and quantifying the fragment as a single cancer. These kits are used to detect cancer markers by the above-described immunological techniques or hyperpredation methods.
  • cancers examples include colorectal 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, knee cancer, ovarian cancer, brain tumor, blood tumor, etc., but colorectal cancer is particularly preferred.
  • cancer marker refers to a subject's body fluid (eg, blood, urine, lymph, saliva, sweat, semen, etc.) or cells or tissues that are not derived from normal tissues, or This refers to a molecule that is selectively upregulated in cancer cells or tissues, and the presence of the molecule in the body fluid or cells or tissues of a subject indicates or suggests the presence of cancer.
  • the kit of the first embodiment contains a component for detecting and / or quantifying PPP 1 R 3 D antigen (including PPP 1 R 3 D protein and its partial peptide) in a body fluid sample from a subject.
  • PPP 1 R 3 D antigen including PPP 1 R 3 D protein and its partial peptide
  • Such components detect the level of PPP 1 R 3 D in, for example, tissue sections or body fluid samples such as blood and urine. And or used to quantify obtain.
  • Such antibodies may be labeled with radioactivity, fluorescence, colorimetry, or enzyme labeling.
  • the kit of the present invention may contain a labeled secondary antibody.
  • the kit according to the second embodiment contains a polynucleotide having a base sequence that can be hybridized under hybridization conditions stringent to the base sequence of the PPP 1 R 3D gene or a part thereof.
  • the kit of the present invention may contain the above-described polynucleotide immobilized on a DNA chip.
  • the kit of the present invention includes an anti-PPP 1 R 3D antibody, a PPP 1 R 3D gene or a part of its base sequence, a stringent high sequence, a base sequence that can be hybridized under hybridization conditions, and the like.
  • containers and labels may be included.
  • the label on or associated with the container may indicate that the drug is used to detect colorectal cancer markers.
  • other items such as instructions for use may be further included.
  • Example 1 Identification of colon cancer specific amplification gene by array C GH method
  • sample preparation of 200 colorectal cancer specimens and verification based on the array CGH method were performed.
  • FIG. 1 is a histogram showing the frequency of amplification of PPP 1 R 3 D gene in 200 colorectal cancer patients.
  • Table 2 shows the amplification degree (GZR value) and frequency of 200 PPP specimens of the PPP 1 R 3 D gene in colorectal cancer patients.
  • the average value shows the average value for samples with a GZ R value of 1.2 or more.
  • the PPP 1 R 3 D gene was amplified in 64.5% of 20 0 colorectal cancer patients, with an average amplification of 1.8. .
  • the maximum value was 2.7, and remarkable amplification occurred very frequently.
  • Example 2 Verification of gene amplification in a colon cell line derived from the large intestine
  • the degree of amplification in a colon cancer-derived cultured cell line was verified for a high-frequency gene region in colon cancer patients.
  • Cultured cell lines were DLD-1 and RKO E6, which are colon cancer-derived cell lines. Genomic DNA was extracted from the cultured cells according to the protocol attached to the kit using Blood & CellCu1ture DNAKite (QIAGEN).
  • Table 3 shows the degree of amplification (GZR value) of the PPP 1 R 3 D gene in a cell line derived from colorectal cancer. As shown, in colorectal cancer-derived cell lines Also found that amplification occurred in the PPP 1 R 3 D gene, located in BAC C lone RP 1 1— 2 8 G 1 8.
  • the PPP 1 R 3 D gene which was frequently amplified in 200 colorectal cancer patients, was a colon cancer-derived cell line, and gene amplification was observed at the genomic level.
  • RNA i analysis was performed and the phenotype was observed.
  • the cell line was purchased from ATCC and cultured according to the attached protocol.
  • s i RN A a specific 21 mer within the gene was selected and s 1 RNA was synthesized with the sequence as the target (commissioned to QIAGEN).
  • siRNA was introduced into cells using RNA i FECT (QIAGEN) and siRNA of ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ according to the attached protocol.
  • RNA i FECT QIAGEN
  • siRNA of ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ according to the attached protocol As a control, N egative Control 1 siRNA (QI AG EN) was used. The cells were observed under an inverted microscope for 4 days after introduction into the cells.
  • cDNA was synthesized according to the attached protocol using Super Sc r i p t I I I F i r s t — S t r a n d S y n t h e s i S S y s t e rn f o r R T-P C R (I n v i t ro g e n).
  • Quantitative RT-PCRR was performed using this cDNA as a saddle. Quantitative PCR was performed using SYBRG reen RT—PCRR eagents (A p 1 ied B iosys te rn s) according to the attached protocol. iosyst em s). Primers synthesized the following sequences (* commissioned to PERON). Primer sequence:
  • RNAi analysis of the PPP1R3D gene was performed using DLD-1 and RKO E6, which are cell lines derived from colorectal cancer.
  • Figures 2A and 2B show the observation images on day 4 after Transfeetion of siRNA of R ⁇ ⁇ 1 R 3 D gene in DLD_ 1 and RK ⁇ E 6 cells, respectively (upper: X 4 0; lower row: X 2 0 0).
  • a, b, and c are s i R N A 3 species of the P P P 1 R 3 D gene, and N C represents a negative control.
  • phenotypic observations showed that the number of cells in both cells a, b, and c was significantly reduced compared to N egative Control (NO) (Fig. 2).
  • Figure 3A and Figure 3B show that the cells were collected 24 hours after Transfeetion of siRNA of PPP 1 R 3 D gene in D LD-1 and RKOE 6 cells, respectively, and quantitative RT-PCR was performed. The results are shown. As an endogenous control, the relative ratio to NC was shown using the relative ratio of GAPDH. As shown, as a result of confirming the effect at the RNA level with quantitative RT_PCR, the expression level of all three types of siRNA was markedly decreased in all cells (Fig. 3).
  • Fig. 4 A and Fig. 4 B show that the number of viable cells was measured with the measurement reagent using the cells on day 4 after Transfeetion of siRNA of PPP 1 R 3 D gene to DLD-1 and RKOE 6 cells, respectively. Results are shown. The graph shows the relative amount to NC. As shown, N egative Control (N Compared to C), the number of cells was significantly reduced (Fig. 4). In all of the cells, all three types of siRNA (a, b, c) of the PPP 1 R 3 D gene were significant in the t-test (P> 0.01).
  • Example 4 Functional analysis by RNAI analysis using a colon-derived normal cell line
  • the RNAI analysis was performed using a cell line derived from a normal tissue of the large intestine. Verify that is cancer specific.
  • RNA For the cell line, use CCD 18 CO purchased from AT C. Culture conditions are according to the attached protocol. For s i RN A, the a, b or c sequence of Example 3 is used. For introduction of si RNA into cells, L i pof e cta min e 2200 (Invitrogen) is used, and 25 nM of siRNA is introduced into cells according to the attached protocol. As a control, N ega tiv e C o n t r o ls i RNA (Q I AGE N) is used. Observe under an inverted microscope for 5 days after introduction.
  • Example 5 Evaluation of gene amplification
  • Figure 5 shows optical micrographs (fluorescence images) of some of the cancer cells (6 cells) observed in each sample tissue (A to J). As shown, more than 3 spots were found in cancer cells. It was confirmed that the PPP 1 R 3 D gene region was amplified in 10 specimens whose gene amplification degree (G / R) was 1.2 or more by the array CGH method. It was also shown that gene amplification occurred from the early stage to the late stage of the disease state. This indicates that the P P P 1 R 3 D gene region can be applied not only as a molecular target for cancer drugs but also in cancer diagnosis by FISH.
  • Example 6 Detection of PPP 1 R 3 D protein in blood by mass spectrometry
  • Serum specimens of colon cancer patients 10; L and healthy specimens 10 L are diluted with 500 L of diluted buffer solution (10 mM Tris HC1 ph 7.4 + 150 mM NaCl), then ProteomeLab IgY-12 SC Proteome partitioning kit (BECKMAN COULTER: A24618) was used to remove a large amount of serum proteins such as albumin and globulin. 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.
  • diluted buffer solution 10 mM Tris HC1 ph 7.4 + 150 mM NaCl
  • odoacetamide SIGMA: 144-48-9
  • SIGMA 144-48-9
  • the alkylation reaction was carried out at room temperature for 30 minutes in the dark.
  • add 4 times the volume of cold acetone (with Wako: 014-08681, —20) to the reaction solution, let stand at 80 for 1 hour, and centrifuge at 15,000 xg for 30 minutes to precipitate the protein. It was collected.
  • the recovered protein was dissolved in 80 L of 2M urea + 100 mM ammonium bicarbonate solution. After dissolution, a portion was subjected to measurement of protein concentration by the BCA method. Trypsin (Promega: V511C) was added to the sample protein at 1/50 (w / w), and digestion was performed at 37 ° C for 16 hours. 2) Analysis using Nano-HPLC direct mass spectrometer
  • FIG. 6A and FIG. 6B show the results of analysis of (A) serum derived from colorectal cancer patients and (B) serum derived from healthy subjects by the above method, respectively.
  • Figures 7A-C show the peaks shown in Figure 6 as determined by MS / MS analysis. Shows the correspondence with amino acids (or amino acid sequences).
  • the growth inhibition effect was observed by knocking down the PPP 1 R 3 D gene, but what effect was observed in the cervical cancer cell line was determined by the RNAi analysis method described above. , evaluated.
  • RNAi analysis was performed using the above three siRNAs. Introducing s i R N A into cells using O 1 i g o f e c t am i n e (I n v i t r o g e n)
  • FIG. 8 shows the results of measuring the number of viable cells (MTT assay) with the measurement reagent using the cells on the 4th day after Transfeetion of the PPP1 R 3 D gene siRNA to HeLa cells.
  • the graph shows the relative amount with respect to NC (Negative control siRNA (Qiagen)).
  • NC Negative control siRNA (Qiagen)
  • differential interference images were taken under a microscope, and their dynamics were observed in detail. Specifically, after transfection of siRNA (a, b, c) in HeLa cells, differential interference images of the same field were observed 1, 2, 3, and 4 days later. The results are shown in Fig. 9. NC used negative control siRNA (Qiagen). Compared with NC, the growth rate was suppressed and cell death was induced (partially indicated by arrows). In the figure, a, b, and c correspond to siRNA a, siRNA b, and siRNA c in FIG. 8, respectively. As shown in Fig. 9, induction of cell death was observed in a, b, and c where growth inhibition was observed.
  • NC used negative control siRNA (Qiagen). Compared with NC, the growth rate was suppressed and cell death was induced (partially indicated by arrows).
  • a, b, and c correspond to siRNA a, siRNA b, and siRNA c in FIG. 8, respectively.
  • 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 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 WO2007037533A1 (fr)

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