WO2007037550A9 - Application therapeutique ou diagnostique du gene tsta3 - Google Patents

Application therapeutique ou diagnostique du gene tsta3

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Publication number
WO2007037550A9
WO2007037550A9 PCT/JP2006/320035 JP2006320035W WO2007037550A9 WO 2007037550 A9 WO2007037550 A9 WO 2007037550A9 JP 2006320035 W JP2006320035 W JP 2006320035W WO 2007037550 A9 WO2007037550 A9 WO 2007037550A9
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WO
WIPO (PCT)
Prior art keywords
cancer
gene
protein
tsta3
tsta
Prior art date
Application number
PCT/JP2006/320035
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English (en)
Japanese (ja)
Other versions
WO2007037550A1 (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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Publication date
Application filed by Link Genomics Inc, Shinichirou Niwa, Yasutaka Makino, Tomoki Ikuta, Kazuya Arai, Takayuki Shindou, Hiromichi Ogura filed Critical Link Genomics Inc
Publication of WO2007037550A1 publication Critical patent/WO2007037550A1/fr
Publication of WO2007037550A9 publication Critical patent/WO2007037550A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a T S T A 3 gene which is a gene specifically amplified in cancer, its therapeutic or diagnostic use, and the like.
  • malignant tumors include lethality caused by generalization through proliferation and invasion and metastasis.
  • Local therapies such as surgical resection or radiotherapy cannot adequately address metastatic cancer, and the development of systemic pharmacotherapy is expected to improve future cancer treatment results. ing.
  • Chemotherapy the current center of cancer drug therapy, often uses cell killing agents that act directly on the DNA and / or RNA of cancer cells and cause them to die.
  • cell killing agents that act directly on the DNA and / or RNA of cancer cells and cause them to die.
  • bone marrow cells For example, bone marrow cells,. Germ cells, hair matrix cells, gastrointestinal epithelial cells and other normal cells with a high level of division, causing 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 epithelial growth factor receptor
  • Iretsa gene: gefitinib
  • a tyrosine kinase inhibitor WO96Z33980
  • HER— which is effective in the treatment of breast cancer.
  • 2 Human epithelial growth factor receptor 2
  • rasuduzumab a humanized monoclonal antibody herceptin
  • Japanese colorectal cancer tends to increase year by year, and the number of deaths is third after lung cancer and stomach cancer.
  • people in their 60s are the most frequent, followed by those in their 50s and 70s.
  • Genetic factors, environmental factors, etc. are thought to be the cause of the increase in colorectal cancer
  • the westernization of eating habits, especially the removal of animal fat is the cause.
  • the development of an effective molecular target drug for colorectal cancer is awaited.
  • about half of the tumor markers (CEA, CA 19-9) used for diagnosis are positive for advanced colorectal cancer, and there is no organ specificity. 'Is desired. Disclosure of the invention '''
  • the present inventors have conducted extensive research and found that the gene that is frequently amplified in cancer (particularly colorectal cancer) is the TSTA3 gene. I found it. Furthermore, the present inventors have found that the growth of cancer cells can be suppressed by inhibiting the expression of TSTA3 protein in colorectal cancer cell lines, and the present invention has been completed. That is, the present invention provides the following cancer therapeutic agent, screening method for candidate sputum having cancer suppressing action, cancer diagnostic agent, cancer diagnostic kit, cancer diagnostic method, and the like. To do.
  • a cancer therapeutic agent comprising a TSTA 3 gene expression inhibitor as an active ingredient.
  • the TSTA3 gene expression inhibitor is
  • the cancer therapeutic agent according to (1) above comprising a substance selected from the group consisting of:
  • a cancer therapeutic agent comprising a TSTA3 protein activity inhibitor as an active ingredient.
  • the cancer therapeutic agent according to (3) above comprising a substance selected from the group consisting of:
  • a method for screening a TSTA3 gene expression inhibitor comprising: (a) contacting a test compound with a cell expressing the TSTA3 gene;
  • 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 an activity inhibitor of T S T A 3 protein comprising:
  • a screening method comprising a step of selecting a compound that binds to the TSTA3 protein.
  • a cancer diagnostic agent comprising the antibody according to (9) above.
  • a cancer diagnostic agent comprising a base sequence that can be hyper-predated under stringent hyper-precipitation conditions in the TSTA 3 gene or a part of the base sequence thereof.
  • a cancer diagnostic kit comprising the antibody according to (9) above.
  • kits for cancer diagnosis containing a polynucleotide comprising a nucleotide sequence capable of being highly hybridized under stringent high-hybridization conditions to the TSTA3 gene or a partial nucleotide sequence thereof.
  • a method for treating cancer comprising the step of administering a TSTA3 gene expression inhibitor to a patient.
  • a method for treating cancer comprising a step of administering a TS TA 3 protein activity inhibitor to a patient.
  • a cancer therapeutic agent containing a TST A 3 gene expression inhibitor as an active ingredient comprising a polynucleotide having the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 A cancer treatment.
  • the present invention provides novel drugs, kits and methods useful for the treatment and / or diagnosis of cancer (for example, colorectal cancer), and screening methods for candidate compounds having cancer-suppressing activity.
  • Figure 1 is a histogram showing the frequency of the TSTA 3 gene relative to the degree of gene amplification in 200 samples from patients with colorectal cancer.
  • Figure 2 shows an optical microscope showing the results of RNA i analysis when siRNA of the TSTA3 gene was transfected into (A) colorectal cancer cell line RK ⁇ and (B) colorectal cancer cell line RKOE 6. It is a photograph (phase contrast image).
  • Figure 3 shows quantitative RNA i effects when (A) colorectal cancer cell line RKO and (B) colorectal cancer cell line 13 ⁇ 41 £ 6 were transfected with siRNA from TSTA3 gene.
  • RT is a graph showing the results of evaluation by PCR.
  • Figure 4 shows the number of viable cells when 5A RNA of 5 to 8 3 genes was transfected into (A) colorectal cancer cell line RKO and (B) colorectal cancer cell line RKOE 6 respectively. It is a graph which shows the result of having evaluated the RNA i effect by a measurement.
  • Fig. 5 is a photograph showing the results of Northern hybridization performed using various normal organ tissues. '
  • Fig. 6 shows optical micrographs (fluorescence images) of part of the cancer cells (6 cells) of each specimen tissue (A to J>) derived from colorectal cancer patients analyzed by the FISH method.
  • Figures 7 and 8 are graphs showing the results of (A) serum derived from colorectal cancer patients and (B) serum derived from healthy subjects analyzed by mass spectrometry, respectively.
  • Figures 8A-C show the correspondence between the peaks shown in Figure 7 and amino acids (or amino acid sequences) as determined by MS / MS analysis.
  • Figure 9 shows the results of a detailed observation of the dynamics of RNAi effects of a cervical cancer cell line HeLa cell line when the TSTA3 gene siRNA was transcribed in time series. It is an optical microscope photograph (differential interference image) shown. BEST MODE FOR CARRYING OUT THE INVENTION
  • the present inventors verified the amplification gene by the array CGH method using a sample derived from a colon cancer patient, and identified a gene amplification region specific to colorectal cancer. Of the regions where amplification occurs frequently in the specimen, HI TSTA3 (T issuespe cifictransplantationa ntigen ⁇ ' ⁇ 3 5 ⁇ ). The gene was found to be highly frequent in specimens from patients with colorectal cancer.
  • TSTA3 T issuespecific translation antigen P 35 B was known as NAD P (H) — a binding protein FX (Mo re 1 1 i, A., eta J. (1977) A rch. B ioch em. B iophys. 1 79, 6 98-705; More ⁇ ⁇ ⁇ , A., eta J. (1 97 7) FE BSL ett. 80, 1 1 4).
  • NAD P H
  • FX binding protein FX
  • FX is now known to be an enzyme involved in the synthesis of GDP-(L)-fucose from GDP-(D)-mannose. Fucose is modified in many cells, such as the surface and end of secreted protein N-, O-lipid-binding glycans (Smith, PL, eta 1. (2 0 0 2) J. C e 1 1 B iol 1 58, .80 1— 8 1 5), which are involved in the recognition of selectins (Lowe, J. B. (200 1) In The Molecular Basisof Blood Diseases, W. B. S aunders Comp any, 3 14— 36 1; Sak amoto, S., eta 1. (1 989) 49, 745— 7 52).
  • FX plays an important role in the final stage of fucose synthesis.
  • FX knockout mice have leucocyteadhesiondefi ciency (LAD) type II congenital disease of concomitant lycosylation — lie (Congenitaldisorderofgly cosylation (CDG)-lie)
  • LAD leucocyteadhesiondefi ciency
  • CDG Congenitaldisorderofgly cosylation
  • FX has been reported that in epithelial cancer cells such as colorectal cancer, there is a correlation between high selectin ligand expression and frequent metastasis and poor prognosis.
  • epithelial cancer cells such as colorectal cancer
  • FX has been reported to play an important role in the biosynthesis of selectin ligands involved in the adhesion between cancer cells and epithelial cells (E she 1, R.
  • colon cancer cell lines overexpressing FX have increased ability to adhere to epithelial cells, and when FX is knocked down, selectin ligand expression is reduced and adhesion to epithelial cells is reduced. And FX are thought to be responsible for the regulation of cancer cell extravasation and metastasis (Zipin, A. ea I. (2004) Cancer Res 64, 657 1- 6578).
  • RNA i RNA chain
  • the present invention provides (1) a cancer therapeutic agent containing a TSTA 3 gene expression inhibitor as an active ingredient, and (2) a cancer therapeutic agent containing a TSTA 3 protein activity inhibitor as an active ingredient.
  • TSTA3 gene refers to the rabbit TSTA 3 gene (SEQ ID NO: 1) consisting of 1312 bases registered in the Accession No .: NM_0033 13 in the NC BI nucleotide database.
  • the present invention is not limited to this, for example, the base of the gene, such as a variant that is altered by having one or more base substitutions, deletions, additions, or insertions in the base sequence of the gene.
  • a gene consisting of a polynucleotide comprising a nucleotide sequence that can be hybridized under high hybridization conditions stringent to the sequence or its complementary sequence is also included in the “TSTA3 gene” used herein.
  • Hybridization is a known method or a method similar thereto, such as Molecular Cloning Third Edition, J. Samb rooketa 1., C o 1 d S pr ing Harbo. r La b. Pres s. 2001), etc. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • the “stringent conditions” may be any of low stringency conditions, moderate stringency conditions, and high stringency conditions.
  • “Low-slinging conditions” are, for example, conditions with 5 XS SC, 5 X Denhardt's solution, 0.5% SDS, 50% formamide.
  • the “medium stringent conditions” are, for example, the conditions of 5 XS SC, 5X Denhardt's solution, 0.5% SDS, 50% formamide, and 42.
  • “High stringency conditions” are, for example, conditions of 5 X S S C, 5 X Denhal solution, 0.5% SDS, 50% formamide, 5 O: Under these conditions, it is expected that DNA having higher homology can be efficiently obtained as the temperature is increased. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors affecting the stringency of a hybridization, and those skilled in the art can select these factors as appropriate. By doing so, it is possible to achieve the same stringency.
  • Hybridizable polynucleotides are calculated using homology search software such as FASTA and BLAST using the default parameters. And the nucleotide sequence of SEQ ID NO: 1, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more Polynucleotides having 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity.
  • inhibiting gene expression refers to any of a series of events from gene to protein production (eg, transcription (mRNA production), translation (protein production)). By inhibiting any event, it is meant to inhibit the production of the protein encoded by the gene.
  • cho STA3 protein refers to a rabbit T STA 3 protein consisting of 32 1 amino acid residues (sequence) registered under Accession No .: N P_003304 on the NCB I protein basis. No. 2) and substantially the same activity as this protein (eg, 3,5-epimerase activity, 4 reductase activity (S u 1 1 ivan, FX, eta 1. (1.998) J. B iol. C hem. 273, 8 1 93 -8202; Oh yama, C. eta 1. (1 9 98) J. B io 1. C hem. 27.3, 1 4582-14587; , Eta 1. (2003) Cancer Re s. 63, 628 2-62 89) A mutant protein consisting of an amino acid sequence in which a plurality of amino acid residues are deleted, substituted, inserted, and / or added.
  • the amino acid mutation site and number are not particularly limited as long as the mutant protein retains substantially the same activity as the original protein.
  • the smaller the number of mutations the better.
  • such a mutant protein has approximately 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more of the amino acid sequence of SEQ ID NO: 2.
  • a protein having substantially the same activity as the original protein In general, the larger the homology value, the better.
  • the TSTA3 protein includes a “partial peptide” of the TSTA3 protein.
  • the partial peptide of TSTA3 protein is a partial peptide consisting of a partial amino acid sequence of the amino acid sequence of TSTA3 protein (SEQ ID NO: 2), preferably the same as the activity of TSTA3 protein described above. Any material may be used as long as it has activity.
  • 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 200 amino acids
  • Examples thereof include a polypeptide having an amino acid sequence consisting of residues.
  • these polypeptides contain an amino acid sequence corresponding to the portion involved in the activity of the TSTA3 protein.
  • 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, more preferably about 1 to 10, more preferably More preferably, about 1 to 5 amino acid residues may be changed by deletion, addition, substitution, or insertion. '
  • the TSTA 3 protein used in the present invention can be prepared from cells or tissues expressing the protein. These proteins can be synthesized by a known peptide synthesizer, or can be prepared by a recombinant method using an appropriate host cell selected from prokaryotes or eukaryotes.
  • the TSTA3 protein used in the present invention may be derived from any species, but is preferably derived from human.
  • “Substantially the same activity” indicates that the activities are qualitatively equivalent. Therefore, the enzyme activity (3,5-epimerase activity, 4_reductase activity, etc.) is equivalent (for example, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably Is preferably about 0.5 to 2 times), but quantitative factors such as the degree of activity and the molecular weight of the protein may be different. Enzyme activity was measured by Sullivan, FX, eta I. (1 998) J. Bio 1. C he m. 27 3, 8 1 93-8202; Oh y ama, C. eta 1: (1 998) J-B iol. C h em..
  • s corre 50
  • wo rd lenght 3.
  • cancer therapeutic agent includes anticancer agents, cancer metastasis inhibitors, cancer cell apoptosis inducers, cancer cell proliferation inhibitors, cancer cell infiltration inhibitors, cancer preventive agents, etc. 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 TSTA 3 gene expression inhibitor as an active ingredient.
  • the “TSTA3 gene expression inhibitor” is not limited as long as it inhibits the expression of TSTA3 gene. For example, (i) it inhibits transcription from TSTA3 gene to TS TA3 mRNA. And (ii) a substance that inhibits translation from TSTA3 mRNA to TSTA3 protein. Examples of substances that inhibit transcription from the TSTA3 gene to TSTA3 mRNA include '-
  • examples of poverty that inhibit translation from TSTA3 mRNA to TSTA3 protein include
  • RNA i action on TSTA3 mRNA or a part thereof e.g, siRNA
  • nucleic acid means RNA or DNA.
  • nucleic acid may contain not only pryne and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also have modified sugar moieties, such as one or more hydroxyl groups substituted with halogens, aliphatic groups, or functional groups such as ethers, amines, etc. It may be converted to.
  • RNA i a nucleic acid having an action of inhibiting the expression of TSTA 3 gene by the RNA i effect can be used as an active ingredient.
  • RNA i The term “phenomenon” refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, the expression of the introduced foreign gene and target endogenous gene are both inhibited.
  • Examples of RNA used herein include double-stranded RNA that causes RNA interference of 19 to 30 bases in length, such as ds RNA (doublestrand RNA) siRNA (sma 1 1 interfering RNA) or sRNA (shorthairin RNA). Can be mentioned.
  • RNA can be locally delivered to a desired site by a delivery system such as ribosome, and can be locally expressed using a vector that can generate the above double-stranded RNA.
  • ds RNA, si RNA or sh RNA The method for preparing and using such double mRNA (ds RNA, si RNA or sh RNA) is known from many literatures (Japanese translations of publication 2002-516062; US Publication No. 20 02/086356 A Na ture Ge netics, 24 (2), Fe b., 1 80-183; Genesis, 26 (4), Ap ril, 240-244; Na ture, S pe. 21, 407: 6802, 3 19-20; Genes & De v., Vo l. 16, (8), Ap r.
  • the length of the double-stranded RNA exhibiting the RNA i effect used in the present invention is usually 19 to 30 bases, preferably 20 to 27 bases, more preferably 21 to 25 bases, most preferably 21 to 23. It is a base.
  • siRNA used in Example 3 can be used.
  • antisense succinate or “antisense polynucleotide” has a polynucleotide complementary to at least a part of a target DNA region, and the polynucleotide is the region.
  • the antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • the antisense nucleic acid of the present invention is RNA, DNA, or modified nucleic acid (RNA, DNA). They can be double-stranded DNA, —double-stranded DNA, double-stranded RNA, —double-stranded RNA, or even a DNA: RNA hybrid.
  • modified nucleic acids include sulfur derivatives of nucleic acids, thiophosphate derivatives, and those having resistance to degradation of polynucleotide amides and oligonucleotide amides, but are not limited thereto. Absent.
  • the antisense nucleic acid to be used is ligated downstream of a suitable promoter, and a sequence containing a transcription termination signal is preferably ligated on the 3 ′ side.
  • the nucleic acid thus prepared can be transformed into a desired animal by using a known method.
  • the sequence of the antisense nucleic acid is preferably a sequence that is complementary to the endogenous gene or a part thereof that is possessed by the animal to be transformed, but is not completely complementary as long as the gene expression can be effectively suppressed. May be.
  • an antisense sequence complementary to the untranslated region near the 5 'end of the mRNA of the TS TA 3 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 in inhibiting gene translation is approximately 70% of the target gene transcript.
  • the complementarity is preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. -,
  • the length of the antisense nucleic acid is at least about 10 bases (for example, about 10 to 40 bases), preferably about 15 bases or more. More preferably, it is about 100 bases or more, more preferably 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 IV Gene, edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin, 1993, p. 3 1 9-347)> J. Kawakamieta 1., P harm Tech Japan, Vo l. 8, p. 247, 1 992; Vo l. 8, p. 395, 1 992; S. T. Crookeeta 1., ed., Antisense Reserchand Applications, CRC Pres, 1993, etc.).
  • a nucleic acid having a liposomal activity that specifically cleaves a transcript of the TSTA 3 gene can be used as an active ingredient.
  • ribozyme activity refers to a nucleic acid that specifically cleaves mRNA, which is a transcription product of a target gene.
  • Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but they have an active domain of about 40 nucleotides called hammerhead type or hairpin type. Some have (protein nucleic acid enzymes, 1990, 35, p. 21 91).
  • ribozyme for example, FEBS Lett, 1988, 228, p. 228; FEBS Lett, 1988, 239, p. 285; Protein Nucleic Acid Enzyme, 1990, 35, p. 2191; N uc 1 Ac ids Res, 1989, 17, 7, p.
  • hairpin ribozymes see, for example, Nature, 1 986, 323, p. 349; Nu c 1 Acids Res, 1991, 19, p. 6751: Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, p. 1 12 etc. can be referred to.
  • the expression of the gene is inhibited. Can be harmful ..
  • a compound other than a nucleic acid that inhibits the transcriptional activity of the TSTA 3 gene can be used as an active ingredient.
  • a compound is, for example, a compound that binds to a factor involved in expression / transcription of the TSTA 3 gene.
  • Such compounds may be natural or synthetic compounds.
  • Such a compound can be obtained by a screening method described later.
  • the present invention also provides a cancer therapeutic agent containing a TSTA 3 protein activity inhibitor.
  • TS.TA 3 protein activity inhibitor includes, for example,
  • TSTA3 protein (as opposed to a T'STA3 protein variant with dominant negative properties, or
  • antibody means an antibody that reacts with the full length or fragment of a protein.
  • the form of the antibody of the present invention is not particularly limited, so long as it binds to the TSTA 3 protein of the present invention, in addition to the above polyclonal antibody and monoclonal antibody, a human antibody, a humanized antibody by gene recombination, and further Antibody fragments and antibody modifications are also included.
  • Antibodies that bind to the TSTA 3 protein can be prepared by methods known to those skilled in the art. Details of the anti-TSTA 3 antibody will be described later.
  • TSTA3 protein mutant having a dominant negative property to TSTA 3 protein means that the activity of endogenous wild-type TSTA 3 protein is lost by expressing the gene encoding it. In other words, it refers to a protein having a function of reducing (see Kunihiro Tsuchida, Gene Activity Inhibition Experiment, edited by Yoshikazu Tahira, Yodosha (2001) 26-32).
  • a compound other than the above antibody or mutant that binds to TSTA3 protein can be used as an active ingredient.
  • Such compounds are, for example, compounds that bind to TSTA3 protein and inhibit its activity.
  • Such a compound may be a natural product or a synthetic compound. Such a compound can be obtained by the screening method described below.
  • the substance capable of inhibiting the activity of the TSTA 3 protein of the present invention described above can be used as a cancer therapeutic agent. 2. Screening method for substances that inhibit the activity or expression of TSTA3 protein
  • 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 TSTA3 protein and the test compound.
  • a compound that binds to TSTA3 protein is expected to have an effect of inhibiting the activity of TSTA3 protein.
  • the compound preferably binds to the active site of TSTA 3 protein.
  • a TSTA 3 protein is brought into contact with a test compound.
  • the TSTA 3 protein can be used, for example, in a purified form of TS TA3 protein, in a form expressed intracellularly or extracellularly, or affinity, depending on the indicator for detecting binding to the test compound. It may be in a form bound to one column.
  • the test compound used in this method can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.
  • the binding between the TSTA3 protein and the test compound is then detected.
  • test compound used for this method.
  • natural compounds, organic compounds, inorganic compounds, single compounds such as proteins, peptides, etc. as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, oceans
  • compound libraries include biological extracts and plant extracts. Not determined.
  • the binding between the TS TA3 protein and the test compound can be detected by, for example, a label attached to the test compound bound to the TSTA3 protein. It is also possible to detect changes in TSTA3 protein activity caused by the binding of test compounds of TSTA3 protein expressed intracellularly or extracellularly as an indicator! ).
  • the binding activity between a protein and a test compound can be measured by a known method (for example, Su 1 1 i Van, FX, eta I. (1 998) J. B iol. Ch em. 273 , 8 1 93 -8202; Oh y am a, C. eta I. (1 998) J. Bio I. Chem. 2 7 3, 1 458.2— 1 4587; No da, K., eta J. (2003) Cancer Res. 63, 6282-6289).
  • test compound that binds to TSTA 3 protein and inhibits its activity is then selected.
  • the compound isolated by this method is expected to have a cancer suppressive action and is useful as a cancer therapeutic agent. ,.
  • Another embodiment of the screening method of the present invention is a method using TSTA 3 gene expression as an index.
  • a compound is brought into contact with a cell expressing the TSTA 3 gene.
  • a cell expressing the TSTA 3 gene examples include cells derived from humans, mice, cats, dogs, combs, hidges, birds, pets, livestock, etc., but are not limited to these.
  • a cell expressing the TSTA3 gene a cell expressing the endogenous TSTA3 gene or a cell in which the exogenous TSTA3 gene is introduced and expressing the gene can be used.
  • Cells expressing an exogenous TST A 3 gene can usually be prepared by introducing an expression vector into which a TSTA 3 gene has been inserted into a host cell.
  • the expression vector can be prepared by general genetic engineering techniques.
  • test compound used in this method there are no particular limitations on the test compound used in this method, but for example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, and chemical compounds Compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, etc. are used.
  • the “contact” of a test compound to cells expressing the TSTA 3 gene is usually performed by adding the test compound to the culture medium of the cells expressing the TSTA 3 gene. It is not limited to.
  • the test compound is a protein or the like, “contact” can be carried out by introducing a DNA vector expressing the protein into the cell.
  • the expression level of the TSTA3 gene is then measured.
  • “gene expression” includes both transcription and translation.
  • the gene expression level can be measured by methods known to those skilled in the art. For example, mRNA is extracted from cells expressing the TST A 3 gene according to a conventional method, and the transcription level of the gene is obtained by carrying out the Northern high pridase method or RT-PCR method using this mRNA as a cocoon. Can be measured.
  • the promoter region of the TS TA 3 gene can be isolated according to a conventional method, and the downstream of the marker gene (for example, luciferase, GFP, galactosidase, etc.
  • the transcription level of the gene can be detected using indicators such as luminescence, fluorescence, and color development)
  • the transcription level of the gene can also be measured by observing the activity of the marker gene. It is also possible to measure the translation level of the gene by collecting protein fractions from cells expressing the TSTA3 gene and detecting the expression of each TSTA3 protein by electrophoresis such as SDS-PAGE. . Furthermore, it is also possible to measure the translation level of a gene by detecting the expression of the protein by performing Western blotting using an antibody against T S T A3 protein.
  • the antibody used for detecting the TSTA 3 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.
  • the present invention also provides an anti-TSTA3 antibody, a cancer therapeutic agent containing this antibody, and the like.
  • the cancer therapeutic agent is used for cancer targeted therapy or targeted drug delivery.
  • anti-TSTA 3 antibody includes an antibody that specifically binds to TSTA3 protein (including fragments (partial peptides) or salts thereof).
  • the TSTA3 antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.
  • the antibody class is not particularly limited, and includes antibodies having any isotype such as IgG, Ig, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.
  • the term “antibody” used herein is used to include any antibody fragment or derivative.
  • F ab, F ab ′ 2 , CDR humanized antibody, multifunctional antibody, single chain antibody (Sc Fv) and so on.
  • 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, Har 1 ow E. & Lane D., Anti bod y, Cold Spring Laboratory Pres (1 988)). See).
  • the protein used as the sensitizing antigen is usually TSTA3 protein or a salt thereof.
  • the TSTA3 protein also includes a partial peptide, which is not limited to, for example, a fragment of the amino acid sequence of SEQ ID NO: 2, for example, 20 or more, 40 or more 60 or more, 80 or more, 100 or more partial peptides having a continuous amino acid sequence portion.
  • these fragments for example, amino (N) terminal fragments and carboxy (C) terminal fragments are used.
  • one or more (preferably about 1 to 10, more preferably several (1 to 6)) amino acid residues in the above amino acid sequence are deleted or substituted. Even inserted and / or added Good.
  • salts of TSTA 3 protein or partial peptides used herein include salts with inorganic acids (eg, hydrochloric acid, sulfuric acid) or salts with organic acids (eg, oxalic acid, formic acid, propionic acid), etc. Is used.
  • the TSTA3 protein of the present invention used as a sensitizing antigen for antibody acquisition is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or human, and particularly preferably a protein derived from a human. .
  • TSTA3 protein partial peptide thereof or salt thereof (in the present specification, these are collectively referred to as “TSTA3 protein”) as an antigen. It is administered to magpies.
  • the dose of the antigen per animal is 0.1 to 10 Omg when no adjuvant is used, and 1 to 100 g when the 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.
  • the immunization interval is not particularly limited, and immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several days to several weeks, preferably 2 to 5 weeks.
  • antibody-producing cells are collected 1 to 60 days after the last immunization day, preferably 1 to 14 days later.
  • Examples of antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, with spleen cells or local lymph node cells being preferred.
  • the myeloma cell and the antibody-producing cell are fused.
  • Cell fusion can be carried out in animal cell culture media such as serum-free DM EM, RPMI-1640 medium, and 1 X 10 6 to 1 X 10 7 Zm 1 antibody-producing cells and 2 X 1 0 5 ⁇ 2 X 10 6 m 1 myeloma cells are mixed (cell ratio of antibody-producing cells to myeloma cells 2:;! ⁇ 3: 1 is preferred) in the presence of a cell fusion promoter.
  • a cell fusion promoter polyethylene glycol having an average molecular weight of 100 to 600 daltons 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 cell suspension is diluted appropriately with, for example, urine fetal serum-containing RPM 1-16-1640 medium, etc., and then 3 ⁇ 10 5 @ / we 11 on a microtiter plate. Slowly, add selective medium to each well, and then replace the selective medium appropriately. As a result, cells that grow from about 14 days after the start of culture in a selective medium can be obtained as a hybridoma. 'Next, the culture supernatant of the growing hybridoma is screened for the presence of antibodies that react with TSTA 3 protein. The screening of the high-pridoma 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 hyperidoma can be collected and screened by an enzyme immunoassay, a radioimmunoassay or the like. Cloning of fused cells is performed by a limiting dilution method or the like. Finally, a hybridoma, a cell that produces monoclonal antibodies that react with the TSTA 3 protein, is established.
  • Hypridoma is cultured with RPMI— 1 6 40 0 containing 10% urine fetal serum.
  • Earth in an animal cell culture medium such as MEM medium or serum-free medium, normal culture conditions (eg if 37: 5% C_ ⁇ 2 concentration) 7 with: L '4 days of culture, on the culture supernatant Obtain antibodies.
  • the antigen described above is administered to mammals such as rabbits, mice, and rabbits.
  • the dose of antigen per animal is 1 to 1 mg when Ajuban is not used, and 10 to 1000 .g when Ajhan 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 the immunization is performed 1 to 10 times, preferably 2 to 5 times at intervals of several weeks from the day, preferably at intervals of 2 to 5 weeks.
  • EL I SA enzyme immunoassay
  • EIA enzyme immmunoassay
  • RIA radioimmunoassay
  • an antibody that reacts with the TSTA 3 protein (column adsorbed fraction) is collected by applying the polyclonal antibody in the antiserum to an affinity column fixed with the TSTA 3 protein.
  • the reactivity of polyclonal antibodies in the antiserum against TSTA3 protein can be measured by the EL ISA method or the like.
  • the F ab or F ab ′ 2 fragment can be prepared by digestion with a protease (eg, pepsin or papain) by a conventional method.
  • a protease eg, pepsin or papain
  • Humanized antibody R i 'echmann et al. Riechmann. JM o 1 B iol. Oc t 5; 203 (3): 825—8, 1 988
  • J ones et al. J on es et al. Nature 321: 522— 525, 1986
  • Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 1. 6, No. 10, 1 988”, Japanese Patent Publication No. 3-73280, and humanized antibodies, for example, ⁇ ature Ge netics, V o '15, p. 146— 1 56, 1 997 ”,“ Nature Ge netics, Vo l. 7, p. 1 3-21, 1994 ”, Special Table Hei 4 504365 No., International Application Publication No. WO 94-2558 5, etc., “Nikkei Science, June, 40 to 50, 1995”, “Nature, Vol. 368, p. 85.6-859, 1 994 ", JP-T 6-500233, and the like, respectively.
  • the antibody that binds to the TSTA3 protein of the present invention can be used for the purpose of, for example, proliferation of cancer cells or suppression of metastasis.
  • a rabbit antibody or rabbit antibody is preferable in order to reduce immunogenicity.
  • the antibody When used as a diagnostic agent, the antibody may be labeled with a labeling substance for monitoring or the like (for example, a radioisotope, a fluorescent substance, etc.). If necessary, it can be labeled with radioactive substances, fluorescent compounds, and the like. Among the most common firefly labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Similarly, an antibody TSTA3 antibody can be labeled using a bioluminescent compound. The presence of 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 can be used to specifically detect TSTA 3 protein or the like present in a subject such as a body fluid or tissue.
  • preparation of antibody columns used to purify TSTA3 protein, detection of TS TA 3 protein in each fraction during purification, analysis of TS TA 3 protein behavior in test cells, etc. Can be used for.
  • the anti-TSTA3 antibody used in the present invention is a drug (a gent) having a neutralizing activity that attenuates the activity of an antigen in the therapeutic agent or diagnostic agent of the present invention. Although it is possible, it can be used in combination with other drugs for producing a therapeutic effect as needed. Therefore, the present invention provides, in another embodiment, a complex of an anti-TS TA 3 antibody and another drug for use in targeted therapy or targeted imaging of cancer (eg, colorectal cancer), A composition containing such a complex is also provided. According to such an embodiment, the anti-TSTA 3 antibody used in the present invention is used to transfer another drug that exhibits therapeutic efficacy or a labeling agent for diagnosis to a target site that highly expresses the TSTA 3 protein. Can be delivered.
  • Examples of the “other drug” used in the present invention include a virus vector or a non-viral vector for gene transfer to a target such as a radioisotope, a therapeutic protein, or a small molecule drug. Illustrated.
  • radioisotopes examples include radiohalogen elements such as fluorine-18, iodine-1,25 ( 125I ), and iodine-131. These radiohalogens be labeled antibody Ya base petit de like the radioactive metallic element described above, if e widely be utilized.
  • radioactive Osamu therapeutic agent or a radioactive diagnostic agent Yodo of with 125 I or 131 I is chloramine T method It can be bound to an antibody or antibody fragment by a known method such as technetium-99m, indium-1 1 1 and gallium 67 ( 67 Ga) for diagnostic purposes.
  • Metal chelate Known metal chelating agents include EDTA, DTP A, diaminodithio compounds, cyclam, and DOTA, etc. These chelating agents are pre-bound to the antibody and then labeled with a radioactive metal. In some cases, after forming a radioactive metal chelate, the antibody is bound and labeled.
  • examples of the “therapeutic protein” are preferably cytokines that activate cells responsible for immunity, for example, human leukin 2, human granulocyte, one macrophage, one colony stimulating factor, human macrophage colony Stimulating factors, heat interleukin 1 2 and the like.
  • cytokines such as ricin and diphtheria toxin can be used to directly kill colon cancer cells.
  • a fusion antibody can be produced by inserting the expression vector into a prokaryotic or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • “Small molecule drug” is used herein to mean a diagnostic or therapeutic compound other than “radioisotope”, “therapeutic protein” and the like.
  • small molecule drugs include alkylating agents such as nitrogen mustard and cyclophosphamide, antimetabolites such as 5-fluorouracil and methotrexe, daunomycin, bleomycin, mitomycin C, daunorubicin
  • Anti-cancer agents such as antibiotics such as doxorubicin, plant alkalo such as vincristine, vinblastine, vindesine, hormonal agents such as evening moxifen, dexamethasone Cancer and Chemotherapy Co., Ltd.)), or steroids such as Hyde mouth cortisone and prednisone;
  • methods for binding daunomycin and antibody include binding between daunomycin and the amino group of the antibody via glutaraldehyde, or binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carpositimide. Is given.
  • viral vector a viral vector modified so as to be able to bind to the anti-TSTA 3 antibody of the present invention can be used (for example, adenoviral vector). Yuichi (Wang, P., eta 1. (1 995) Soma tic C e 1 1 and M o 1 ec. Gene t-. 2 1, 429— 441), Rerovirus vector ⁇ — ( Na viaux RK, eta 1. (1 996) J. V irol 70, 570 1—— 5705), wrench Wills vector (N a 1 dini, L. (1998) C urr. Op i n. B iotechno 1. 9 , 457-463)).
  • virus vectors can be used to induce apoptosis of cancer cells at target sites (eg, colon cancer) such as cell proliferation-related genes, apoptosis-related genes, and immunoregulatory genes.
  • target sites eg, colon cancer
  • a gene that produces a therapeutic effect is incorporated.
  • Viral vectors that bind to anti-TSTA3 antibodies can be targeted to sites where antigens recognized by anti-TSTA3 antibodies (i.e., TSTA3) are present when administered to patients in need of gene therapy along with anti-TSTA3 antibodies. it can.
  • the anti-TSTA 3 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 engineered bond” For example, it includes the binding mode between an antibody and a therapeutic protein when a fusion protein comprising an antibody and a therapeutic protein is produced using a technique such as genetic recombination. 4.
  • a cancer therapeutic agent containing the TSTA3 gene expression inhibitor of the present invention a cancer therapeutic agent containing a TSTA 3 protein activity inhibitor, a therapeutic agent containing the anti-TSTA3 antibody of the present invention, or
  • the anti-TSTA 3 antibody used in the present invention comprises a radioactive vector, a therapeutic protein, a small molecule drug, and a viral vector or non-viral vector carrying a therapeutic gene, or any combination thereof.
  • a therapeutic agent chemically or 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, colorants, Flavors, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity promoters, flavoring agents, etc., but are not limited to these, Other commonly used carriers can be used as appropriate.
  • Specific examples include calcium anhydride, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl chloride, and decylaminoacetate.
  • Polyvinylpyrrolidone gelatin, medium chain fatty acid ⁇ glyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, and the like.
  • Examples of the dosage form of the therapeutic agent of the present invention include tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film coating agents, pellets, Sublinguals, pacifiers, etc.
  • Examples of parenterals include injections, suppositories, transdermal agents, ointments, plasters, liquids for external use, etc. You can choose the dosage form.
  • An inhibitor of T S T A 3 protein activity (or TSTA3 gene expression) 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.
  • oral administration generally, for example, to a patient (as 6 O kg)
  • it is about 0.1 mg to l, 00 Omg, preferably about 1.0 to 10 Omg, more preferably about 1.0 to 5 Omg per day.
  • the single dose varies depending on the subject of administration, target organ, symptom, administration method, etc. It is convenient to administer about 0.1 to 3 Omg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg by intravenous injection per day.
  • the final decision can be made as appropriate based on the judgment of the doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.
  • the preparation thus obtained is administered, for example, to rabbits and other mammals (eg rabbits, rabbits, hidges, bushes, bushes, cats, dogs, monkeys, etc.) Can.
  • rabbits and other mammals eg rabbits, rabbits, hidges, bushes, bushes, cats, dogs, monkeys, etc.
  • 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 of uterine cancer eg cervical cancer, endometrial cancer
  • testicular cancer thyroid cancer
  • knee cancer ovarian cancer
  • brain tumor blood tumor, etc.
  • the agent of the present invention contains a TSTA 3 protein activity inhibitor or TSTA 3 gene expression inhibitor as an active ingredient, it can be used as an anticancer agent, a cancer metastasis inhibitor, a cancer cell apoptosis inducer, and the like.
  • the target cell, tissue, organ, or cancer type is not limited to a specific type.
  • the agent of the present invention may contain both a T S T A 3 protein activity inhibitor and a T S T A 3 gene expression inhibitor.
  • an antisense nucleic acid is used in the therapeutic agent of the present invention
  • the antisense nucleic acid is used alone or after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc.
  • an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc.
  • Antisense nucleic acids can be formulated alone or with a physiologically acceptable carrier and administered via a catheter such as a gene gun or hydrogel catheter.
  • a virus vector such as a recombinant adenovirus particle and an anti-TSTA 3 antibody
  • these may be used alone, but generally pharmaceutically.
  • an acceptable carrier used with an acceptable carrier.
  • the above-described carrier and aqueous isotonic solutions such as water, physiological saline, glucose, human albumin and the like are preferable.
  • additives, preservatives, preservatives, balances and the like that are commonly used in pharmaceutics can also be added.
  • the pharmaceutical composition thus prepared can be administered by an appropriate administration form and administration route depending on the disease to be treated.
  • administration forms include emulsions, syrups, force capsules, tablets, granules, injections, ointments and the like.
  • Administration of the anti-TSTA3 anti-virus vector particle of the present invention or a pharmaceutical composition containing the same for treatment If, once the normal adult human per 0 3 -: 1.0 While 15 preferred to administer the virus particles may be varied depending on the nature of Jo Ya target cells' tissue disease.
  • the number of doses may be from once to several times a day, and the administration period may be from one day to several months or more, with one to several doses as one set, and multiple sets are intermittently administered over a long period of time. May be given.
  • virus vector single particle or virus vector single nucleic acid molecule used in the present invention can be used for detection of specific cells and Z or tissues, or diagnosis of disease states.
  • a viral vector particle obtained by integrating a detectable marker gene into a nucleic acid molecule of a viral vector and transfecting it into an appropriate host cell is combined with an anti-TSTA 3 antibody in tumor cells.
  • an anti-TSTA 3 antibody in tumor cells.
  • it can be used to detect and diagnose tumor cells by combining a detectable label with an anti-TSTA3 antibody.
  • the present invention also provides a diagnostic agent for cancer.
  • the diagnostic agent for cancer of the present invention comprises: (a) an antibody against TSTA3 protein, or (b) TSTA3 gene or a partial nucleotide sequence thereof under high hybridization conditions under stringent hybridization conditions. It contains a polynucleotide consisting of a base sequence capable of soybean.
  • the diagnostic method using the anti-TSTA 3 antibody of the present invention includes, for example, (a) a step of bringing a subject-derived biological sample into contact with an antibody against TSTA3 protein, and (b) in the sample. And a step of detecting and / or quantifying the binding between the antibody and TSTA3 protein or a partial peptide thereof or a salt thereof.
  • a labeled anti-TSTA 3 antibody is used, and the TSTA 3 protein or fragment thereof and anti-T Binding to STA 3 bodies is detected and / or quantified.
  • subject-derived biological sample eg, blood (including whole blood, plasma, serum, etc.), urine, lymph fluid, saliva, sweat, semen, etc.) Including.
  • 'subject' is usually a human subject who will or will want to undergo a cancer screening, including human subjects suspected of having or suspected of having cancer. It is. Examples of such cancers are large cancer, stomach cancer, lung cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterus. Cancers (eg, cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, knee cancer, ovarian cancer, brain tumor, blood tumor, etc., especially colorectal cancer Is preferred.
  • Immunoassays to detect the expression of TSTA 3 in biological samples derived from subjects as described above are collected from subjects suspected of having cancer (eg, colorectal cancer) or at risk for cancer. And subjecting the biological sample to contact with an anti-TSTA 3 antibody under conditions that cause specific antigen-antibody binding, and then measuring the amount of immunospecific binding by the antibody. Using such antibody binding, the presence of T S T A 3 protein and Z yet increased expression is detected. In this case, detection of increased T S T A 3 protein expression is an indicator of disease state. If necessary, the level of T S T A 3 protein in the biological sample may be compared with the level of healthy individuals who do 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-TSTA 3 antibody.
  • the solid support is then washed twice with buffer to remove unbound antibody.
  • the amount of bound antibody on the solid support is measured according to well-known methods.
  • the detection conditions suitable for each measurement can be appropriately determined by those skilled in the art using conventional test methods.
  • the antibody is conjugated to an enzyme, such as that used in the enzyme immunoassay (EIA) [V oi 1 er, A.
  • EIA enzyme immunoassay
  • ELISA enzyme immunoassay
  • the enzyme that binds to the antibody is reacted with a suitable substrate, preferably a chromogenic substrate, in such a way as to produce a chemical molecule that can be detected, eg, spectrophotometrically, by fluorescence measurement with visible means.
  • a suitable substrate preferably a chromogenic substrate
  • Enzymes that can be used to attach a detectable label to an antibody include, but are not limited to, peroxidases and alkaline phosphatases. This detection can also be accomplished by a colorimetric method using a chromogenic substrate for the enzyme.
  • RIA radioimmunoassay
  • sandwich immunoassay immunometric method
  • FIA fluorescence immunoassay
  • TRF time-resolved fluorescence immunoassay
  • IA enzyme immunoassay
  • LIA luminescence immunoassay
  • ECL IA electrochemiluminescence immunoassay
  • latex agglutination immunoprecipitation assay
  • immunoprecipitation assay sedimentation reaction method
  • gel diffusion sedimentation reaction method Immunodiffusion assays, agglutinin assays, complement binding assays, immunoradiometric assays, fluorescent immunoassays, and immunoassays selected from the group consisting of protein A immunoassays (WO00) No. 14227, page 39, line 25 to page 4, page 2, line 8, line 8, EP 1 1 1 1047 A2, paragraph [01 1 5], page 19, line 35 to page 20, line 47, etc.) .
  • various diseases associated with TSTA 3 protein dysfunction can be diagnosed by using the TSTA3 protein quantification method in vivo using the antibody of the present invention. For example, if an increase in the concentration of TSTA 3 protein is detected, for example, it is likely that the disease is caused by overexpression of TSTA 3 protein (eg, cancer (eg, colon cancer)) or will be affected in the future. Can be diagnosed as likely to Note that the anti-TSTA3 antibody of the present invention can also be used for diagnosis with ⁇ ni Vo.
  • the preparation and use of antibody preparations that can be used herein are well known in the art. For example, an antibody anti-respiratory agent is described in Nu cl. Med d Bio, 1 990 17: 247-254. An antibody having a paramagnetic ion as a label used in magnetic resonance imaging is described in, for example, agnetic Resonance in Medicine 199 1 22: 339-342.
  • a probe or primer designed based on the base sequence of the TSTA 3 gene can be used:
  • diagnostic methods include, for example: (a) Hybridization under stringent conditions in a biological sample derived from a subject and the base sequence of a TST A3 gene or a fragment thereof A step of contacting a polynucleotide (probe) having a possible base sequence, and (b) detecting and / or quantifying a hybridization between the polynucleotide in the sample and the TSTA 3 gene or a fragment thereof. Step '.
  • DNA of the TSTA 3 gene (or a gene fragment thereof) in a biological sample derived from a subject is detected and / or quantified using the probe.
  • the length of the base sequence used as a probe is, for example, 12 bases or more, 15 bases or more, 18 bases or more, 2 1 bases or more, 24 bases or more, 27 bases or more, 30 bases or more, or a longer length. It can be a polynucleotide fragment.
  • the hybrid conditions may use the low, medium or high stringency conditions described above.
  • the “base sequence that can be hybridized under a hybridizing condition that is intelligent to the base sequence of the TSTA 3 gene or a fragment thereof” includes the base sequence of the TSTA 3 gene or a fragment thereof.
  • Complementary base sequences antisense polynucleotides
  • Probe and nucleic acid hybridization methods are known to those skilled in the art, for example, WO 89/066. No. 98, EP—AO 200362, US Pat. No. 2,9 1 5,082, EP—A 0063879, EP-A 0 1 732 5 1, and EP—AO 1 280 18.
  • the target sequence can be detected or quantified using a known polynucleotide probe or primer for the T S T A3 gene using a known technique.
  • known techniques include, for example, Southern High Hybridization, Northern Hybridization, RT-PCR, PC R-SSCP (Genomics, Vol. 5, 8 7 4-8 7 9 (1 9 8 9)), Proceedingsofthe National 1 Ac ade my of Sciencesofthe U nited States
  • the array CGH method is an application of the chromosomal CGH method (Ka 1 1 ioni em i, A. et 1. (1 992) Science 258, '8 1 8— 8 2 1).
  • a DNA chip spotted with high-density genomic DNA fragments BAC, PAC: YAC, etc.
  • the DNA copy number abnormality in cancer is detected with high resolution by performing hybridization simultaneously and detecting the binding state (Pinke 1, D. eta 1. (1 9 98 ) N at. G enet.
  • the mRNA level of TS TA 3 in cells is standardized.
  • Genes housekeeping genes (eg, Shaper, NL et al., J. Mammary Gland Biol. Neo oplasia 3 (1 99 8) 3 1 5-3 2 4; Wu, YY and Rees, J. and Ac ta Derm. V enereol. 80 (2000) 2-3—and mRNA levels, preferably RT—can also be compared by PCR.
  • the target sequence (DNA, mRNA, etc.) is detected and quantified by the method described above, and overexpression of the TST A 3 gene is confirmed, for example, a disease caused by excessive expression of TSTA3 (for example, cancer ( For example, colorectal cancer))) or likely to be affected in the future.
  • a disease caused by excessive expression of TSTA3 for example, cancer ( For example, colorectal cancer)
  • the presence of a target protein or a fragment thereof in a test sample can be identified using a mass spectrometer (MS).
  • MS mass spectrometer
  • a sample such as a protein or peptide is ionized using MS, separated according to the obtained mass / charge (mZz), and the intensity of the sample is measured to determine the mass of the sample. It is. From the results of mass spectrometry, individual amino acids constituting the amino acid sequence of a protein or peptide can be identified. '
  • ALD I matrix assisted desorption ionization method
  • ESI electrospray ⁇ ⁇ -on method
  • EI, CI gas phase method
  • FD field desorption
  • ion separation an ion separation method compatible with ionization is used.
  • a time-of-flight mass spectrometer (TOF) mass spectrometer or ESI is used.
  • the quadrupole type (QMS), ion trap type, magnetic field type, etc. are used.
  • Mass spectrometers are sometimes used in tandem. Examples include LC-1 ES I MS / MS, Q-TOF MS. MALD I-TOF MS, and the like.
  • sequencer eg, gas phase sequencer
  • the present invention also provides a TSTA in a body fluid sample of a subject comprising an anti-TSTA3 antibody.
  • 3 Provide a kit for detecting and / or quantifying a protein or fragment thereof as a cancer marker.
  • the TSTA 3 gene or a fragment thereof in a biological sample derived from a subject which contains a base sequence that can hybridize under stringent hybridization conditions to the TSTA 3 gene or a part of the base sequence thereof, is cancerated.
  • a kit for detection and / or quantification as a marker is also provided. These kits are used to detect a cancer marker by the above-described immunological technique or hybridization method.
  • 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 (eg cervical cancer, endometrial cancer), testicular cancer, thyroid cancer, spleen cancer, ovarian cancer, brain tumor, blood tumor, etc. Is preferred.
  • cancer marker is not derived from normal tissue in a subject's body fluid (eg, blood, urine, lymph fluid, saliva, sweat, semen, etc.) or cells or tissues Or 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 indicates the presence of cancer.
  • the kit of the first embodiment contains a component for detecting and / or quantifying TSTA3 antigen (including TS′TA3 protein and its partial peptide) in a body fluid sample from a subject.
  • a component for detecting and / or quantifying TSTA3 antigen including TS′TA3 protein and its partial peptide
  • a component for detecting and / or quantifying TSTA3 antigen in a body fluid sample from a subject.
  • TSTA3 protein is detected and / or quantified by EL Isa
  • a component can detect the level of TS TA 3 in a tissue section or body fluid sample such as blood or urine and / or Z or Can be used to quantify.
  • 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 of the second embodiment contains a polynucleotide comprising a base sequence that can hybridize under stringent hybridization conditions to the TSTA 3 gene or a part of the base sequence thereof.
  • the kit of the present invention may contain the above polynucleotide immobilized on a DNA chip.
  • the kit of the present invention is capable of hybridizing to an anti-TSTA3 antibody, TSTA3 gene or a partial base sequence thereof under stringent high-prediction conditions.
  • containers and labels may be included.
  • the label on or with the container may indicate that the drug is used for the best detection of colorectal cancer.
  • other items such as instructions for use may be further included.
  • Example 1 Identification of colon cancer-specific amplified gene by array CGH method
  • TSTA 3 T issue specific trans- lantantant i'g en P 35 B located in BAC C lone RP l 1-661 A 1 2 used (NCB I Accession No .: NM_0033 1 3)
  • the gene was found to be highly frequent in colorectal cancer patients ( Figure 1, Table 2)
  • Figure 1 shows the frequency of TSTA3 gene relative to the amplification level in 200 colorectal cancer patients
  • Table 2 below shows the degree of amplification (GZR value) and frequency of 200 TSTA 3 gene colon cancer patients, with an average value of 1.2 G Average values for the above samples are shown.
  • Example 2 Verification of the degree of gene amplification in a colon-derived cultured cell line
  • the degree of amplification in a large intestine cancer-derived cultured cell line was verified for a high-frequency gene region in colon cancer patients.
  • the cultured cell lines used were RK0 and RKOE 6, which are cell lines derived from colorectal cancer.
  • Genomic DNA was extracted from the cultured cells using B1ood & Ce11CultureDNAKit (QIAGEN) according to the protocol attached to the kit.
  • Table 3 shows the degree of amplification (GZR value) of the TSTA 3 gene in a colon cancer-derived cell line. As shown, in the colon cancer-derived cell line, it was found that amplification occurred in the T S T A 3 gene located in BAC Clone RP 11-66 1 A 12.
  • Quantitative PCR was performed to confirm amplification of the TSTA3 gene region. Constant Quantitative PCR was performed using SYBR Green RT—PCR Reagents (Applied Biosyst em s) according to the attached protocol, 7500 Real-Time PCR PCR ( (Applied Biosyst erns). Primer 1 synthesized the following sequence (commissioned to PE RON) and used it. .
  • Table 4 shows the degree of amplification of the TSTA 3 gene in a colon cancer-derived cell line. Values are relative to the control DNA (normal). As shown, in the colon cancer-derived cell line, it was found that amplification occurred in the TSTA3 gene region.
  • TSTA 3 gene was also amplified in colorectal cancer-derived cell lines (RKO, RKOE 6). Therefore, cultured cells that could be used for functional analysis of TSTA3 gene in cancer were selected.
  • Example 3 Functional analysis by RNA i analysis using colorectal cancer cell lines
  • RNA i analysis was performed using RK ⁇ and RKOE 6), and the phenotype was observed.
  • the cell line was purchased from AT CC and cultured according to the attached protocol.
  • siRNA we selected specific 21mer in the gene and synthesized siRNA targeting the sequence (commissioned to QIAGEN).
  • siRNA introduction into RKO cells Lipofectine 2000 ′ (Invitrogen) was used, and 50 nM siRNA was introduced into the cells according to the attached protocol.
  • siRNA introduction into RKOE 6 cells RNA i FECT (QIAGEN) was used, and 100 nM siRNA was introduced into the cells according to the attached protocol.
  • negati Vetronic Control RNA QI AGEN 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 Sup er s c r i p t I I I F i r s t -S t r a n d Syn t S h e s S i s s t e fo er RT-PCR (I n v i t r o ge n).
  • Quantitative RT-PCR was performed using this cDNA as a cage. Quantitative PCR is performed using SYBR G reen RT-PCR Re agents (Applied B iosystems) and according to the attached protocol, it was carried out using 7500 Real-Time PCR S vstem (App 1 ied B iosyst em s). The following sequences were synthesized (consigned to OPERON) and used as primers. ,
  • G 1 ycera 1 dehyde-3-phosphatedeh yd rogenase (GAPDH) C on tro 1 Re agents (Ap plied B io'syst ems) is used as the standard gene for calculating the relative ratio Then, the expression level of GAPDH was determined, and the relative ratio was calculated.
  • si RN A Introduce the number of viable cells using Almar Blue (Biosource) according to the attached protocol and Wa 1 1 ac 1420 Multilabel / Lum inescence C o'n nter ARVO (Pe r ki n E 1 me r)
  • RNAi analysis of TSTA3 gene was performed using RKO and RKOE 6, which are cell lines derived from colorectal cancer.
  • Fig. 2 A and Fig. 2 B show the observed images of the 4th day after Transfeetion of TSTA 3 gene siRNA to R.KO cells and RKOE 6 cells, respectively (upper: X40; lower: X200)
  • a and c are 2 types of siRNA of TSTA 3 gene, respectively, NC indicates a negative control.
  • both a and c s i RNAs showed a marked decrease in cell numbers compared to NO ( Figure 2A and B).
  • Figure 3 A and Figure 3 B show that RKO cells and RKOE 6 cells have TS Shown are the results of quantitative RT_PCR using cells collected 24 hours after transfecting the TA3 gene siRNA. As an endogenous control, relative amount to NC was shown using relative ratio of GAPDH. As shown, the effect of RNA level was confirmed by quantitative RT-PCR. As a result, the expression level of both types of siRNA's was significantly reduced (Figure 3 8).
  • Fig. 4 A and Fig. 4 B show that the number of viable cells was measured with the measurement reagent using the cells on the 4th day after transfection of the TS TA 3 gene si RNA to RKO cells and RKOE 6 cells, respectively. The results are shown.
  • the graph shows the relative amount to NC. As shown, the number of viable cells was measured, and as with the phenotype, the number of cells was significantly reduced compared to (N C) in Negati V e Contro (FIGS. 4A and B). All 2 types of siRNA (a, c) of TSTA3 gene were significant (p ⁇ 0. 0 1) in t test.
  • RNAi analysis is performed using a cell line derived from a normal tissue of the large intestine to verify that the target gene suppression effect is cancer-specific.
  • RNA Q I AG EN. Observe under an inverted microscope for 5 days after introduction.
  • the organ specific expression of the T S T A 3 gene in normal tissues was carried out by the Northern hybridization method known in the art.
  • RNA level expression in normal organ tissues 23 organs (Heart, Brain, Placenta, Lung, Liver, Skeleton) Skeletal Muscle, Kidney, PaDcreas, Spleen, Thymus, Prostate, Testis, Ovary, Small Intestine, Colon ), Peripheral Blood Leukocyte (Stomach), Thyroid, Spinal Cord, Lymph Node, Trachea, Adrenal Gland, Bone Marrow
  • the gene sequence of TSTA 3 can be found in NCBI '(http://www.ncbi.nlm.nih.gov) as Accession No.
  • the sequence of 1,312 bp is registered as mRNA in NM—003313.
  • Figure 5 shows the results. As shown in the figure, a band was found around 1.4 kb in the testis, confirming remarkable expression. In addition, expression was confirmed in all other organs, suggesting that this gene is a universally important gene in normal tissues.
  • the presence of gene amplification in the TSTA3 gene region in cancer cells of the specimen tissue was evaluated by the FISH method known in the art. Using the sample tissue (derived from colorectal cancer patients) that had a gene amplification degree (G / R) of 2 or more according to Array CGH 3 ⁇ 4, Ding 5. Ding 8 3 genes are located 8 (: (:
  • Figure 6 shows optical micrographs (fluorescence images) of some of the cancer cells (for 6 cells) observed in each sample tissue (A to J). As shown, more than 3 spots of signal were found in cancer cells. It was confirmed that the TSTA3 gene region was amplified in 10 samples whose gene amplification degree 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 stage. This indicates that the TSTA3 gene region can be applied not only as a molecular target for cancer drugs, but also in cancer diagnosis by the FISH method.
  • Example 7 Detection of TSTA3 protein in blood by mass spectrometry
  • Serum specimens from colorectal cancer patients .10 and healthy subject serum specimens IOL are diluted with dilution buffer solution (lOmM Tris HC1 ph 7.4 + 150mM NaCI) 500 ⁇ L, then ProteomeLab IgY-12 SC Proteome Partitioning Kit (BEC A COULTER: A24618) was used to remove serum abundant proteins such as albumin and globulin. Dithiothreitol (Wako: 049-08972) was added to the resulting image so that the final concentration was lOm, and the reduction reaction was performed for 6 (T for 30 minutes. After completion of the reaction, the final concentration was lOmM.
  • dilution buffer solution lOmM Tris HC1 ph 7.4 + 150mM NaCI
  • Amid (SIGMA: 144-48-9) was added, and the alkylation reaction was carried out in the light-shielded state at room temperature for 30 minutes After completion of the reaction, 4 times the amount of cold acetone (Wako: 014-08681, -20 ° C), left at 1-80 for 1 hour, and then centrifuged at 15,000 Xg for 30 minutes to recover the protein as a precipitate.
  • the recovered protein was 80 L of 2M urea + 100 mM bicarbonate.
  • Nanocolumn (C18 Pep ap 3 ⁇ m, ⁇ , 75 ⁇ id x) with 0.7 g peptide fragment directly connected to ⁇ -Precolumn cartridge (C18 PepMap300, 5 m, 300A, 300 / im id x 5 mm LC PACKINGS: 163589) 150 mm LC PACKINGS: 160321).
  • Ultimate Plus (LC PACKINGS) was used.
  • the flow rate is 200nL / fflin, and a linear gradient with a concentration gradient of 0.573 ⁇ 4 / niin between 0.
  • FIG. 7A and FIG. 7B show the results of analyzing (A) serum derived from colorectal cancer patients and (B) serum derived from healthy subjects by the above method, respectively.
  • an ion peak corresponding to the fragment ion of the target molecule was clearly observed in the serum from colorectal cancer patients. That is, as shown in Fig. 8B, at least ⁇ end-side partial arrangement of 50,000 I This corresponds to the amino acid sequence of positions 10 to 18 in the amino acid sequence of TSTA 3 protein (SEQ ID NO: 2). Therefore, as shown in FIG. 8A, a target peptide fragment (SEQ ID NO: 11) at amino acid sequences 10 to 21 was identified. Such an ion peak corresponding to the partial sequence was not observed in the analysis using normal serum (see FIG. 8C). Therefore, it was strongly suggested that this target molecule (ie, TS TA 3 protein) may have increased blood abundance specifically in cancer.
  • Example 8 Evaluation of RNAi effect in cervical cancer-derived strain HeLa cell line
  • TSTA 3 gene knockdown showed a growth-inhibiting effect, but we evaluated the effects of cervical cancer cell lines using the RNAi analysis method described above. .
  • RNAi analysis was performed.
  • siRNA introduction of siRNA into the cells, the following was used: ⁇ 1 igofe ctamin e (Invitrogen) was used, and 100 nM of siRNA was introduced into the cells according to the attached protocol.
  • N e g.at i V e C o n t r o 1 si RNA (Q I AGEN) was used. Result.
  • the present invention provides a therapeutic agent for cancer, a diagnostic agent, a diagnostic method, a therapeutic method, a kit used therefor, and the like. Therefore, the present invention is useful in fields such as cancer diagnosis or target 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 TSTA3. 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 TSTA3; un agent diagnostique du cancer et un procédé de diagnostic du cancer utilisant ledit anticorps et analogue.
PCT/JP2006/320035 2005-09-30 2006-09-29 Application therapeutique ou diagnostique du gene tsta3 WO2007037550A1 (fr)

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