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WO2007037550A1 - Application therapeutique ou diagnostique du gene tsta3 - Google Patents

Application therapeutique ou diagnostique du gene tsta3 Download PDF

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Publication number
WO2007037550A1
WO2007037550A1 PCT/JP2006/320035 JP2006320035W WO2007037550A1 WO 2007037550 A1 WO2007037550 A1 WO 2007037550A1 JP 2006320035 W JP2006320035 W JP 2006320035W WO 2007037550 A1 WO2007037550 A1 WO 2007037550A1
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WO
WIPO (PCT)
Prior art keywords
gene
tsta
protein
tsta3
antibody
Prior art date
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Ceased
Application number
PCT/JP2006/320035
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English (en)
Japanese (ja)
Other versions
WO2007037550A9 (fr
Inventor
Shinichirou Niwa
Yasutaka Makino
Tomoki Ikuta
Kazuya Arai
Takayuki Shindou
Hiromichi Ogura
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Link Genomics Inc
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Link Genomics Inc
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Publication date
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Publication of WO2007037550A1 publication Critical patent/WO2007037550A1/fr
Publication of WO2007037550A9 publication Critical patent/WO2007037550A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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 gene which is specifically amplified in cans T and its therapeutic or diagnostic use.
  • a characteristic feature of sexual tumors is the growth, invasion, and metastasis of the body through which it can be killed. Sufficient coping with local recurrence such as surgical resection or radiation therapy. Drugs with systemic therapy. Expected to improve future treatment results.
  • Chemotherapy uses cell killing agents that directly act on the cells or RNA and lead to cell death.
  • bone marrow cells germ cells hair matrix cells digestion division Acting on cells and causing strong side effects
  • EGFR epimal growth factor receptor
  • Tyronon kinase inhibitor generic name: Kehuitinif
  • HER-2 human epidermal growth factor receptor 2
  • the present inventors have found that a gene that is frequently amplified or TST in a large intestine canal (which has been extensively studied).
  • the present invention was completed by inhibiting the growth of cancer cells by inhibiting the expression of quality. That is, the present invention provides a screening method for a candidate substance having an inhibitory action on a drug preparation, and a diagnostic method for a diagnostic quinotocan.
  • a screening method comprising a step of selecting the expression regenerative compound as compared with the case where the test compound is not contacted.
  • Radioactive ft isotope Therapeutic protein ⁇ is a low molecular weight drug or a supported hectare as described in (1 0) above.
  • the above or the large intestine is the above (1 3) or (14 diagnostic agent.
  • a quinoto for diagnosis that contains a phototap from a base sequence that can be hyfletized under string-free conditions in the TSTA3 gene or a part of the base sequence.
  • the above or the large intestine is the above (1 6) or (1 7 diagnostic mushrooms.
  • TANA 3 protein activity inhibitor is administered to the subject
  • An anti-rapeutic agent comprising a horincleito having the TSTA 3 gene expression inhibitor as an active ingredient, SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 or SEQ ID NO: A brief description of the 0 side
  • 01 is a histogram showing the frequency of the TSTA 3 gene in 200 specimens from colonic epilepsy.
  • 02 is a photomicrograph (phase contrast image) showing the results of the analysis of STA1 gene s 1 RNA in (A) colorectal cell line RKO and (B) colorectal cancer.
  • Fig. 3 shows the results of (A) large intestine cancellous cell line RKO and (B) large intestine cancer, respectively, s 1 RNA of the TSTA3 gene, transferred 1 RNA, and quantitative RT-PCR results.
  • 06 is an optical micrograph (fluorescence image) of each specimen's tissue (A ⁇ part (6 cells)) derived from colorectal cancer patients analyzed by FISH method.
  • Ayohi B was analyzed by mass spectrometry (A) Colorectal illness Yohi (B) Class A A (: was determined by MS / MS analysis 07 shows the correspondence with noo acid (or amino acid sequence).
  • TSTA3 T issue specific tr an sp on an tigen P 35B was known as NADP (H) — Bonder FX (Morelli, A eta 1 rch B ioc hem Biop s 179 698 re 1 1 l A eta 1 (1977) FEBS L et 4) Research on mice Tissue-specific tr an spla an from the fact that obesity astocyt oma cellline) having a mutation in this gene was transplanted into mice and did not become settled by rejection. ti ge n P 35B).
  • FX is now an enzyme involved in the synthesis of GDP— (D) monomannos (L) — fucose. Fucose is the terminal of many cell surface and secreted N-lycans. (Sm 1 th, PL et 2) J Cell B iol 158 801—815) Serine ectln) (Lowe JB) 1 15-126 Carl ow DA, eta 1 (20 6841-6848).
  • FX plays an important role in the final stage of the fucose synthesis system (D) — GDP synthesized from Mannose by GDP—Mannose 4 6—Techret dratase) (GMD) — 4—Kenoichi (D) — FX converts mannose into GDP— 4—keto 6—theol course (3, 5—Echimerase) and NADP DP—4—keto 6—theokine (L) —converts galactose to GDP (4—Le (Sulliv an F 1 (1 998) JB iol Ch em 273 81 93- y ama C eta 1 (1998) JB iol Ch e 4582- 14587)
  • FX nocout mice have leuk dh esi on deficiency (LAD) t yp e silylated congenital disease—lie (Con genitaldiso (I z um l, Y eta 1 (1995) 216 215 lrah ama, T eta I (1993) C an cer 9-1 334 Th urin U, eta 1 (2002 Hy br ⁇ d om ⁇ cs 21 1 1 1— 1 16) FX has an important role in the biosynthesis of selectin licant involved in cell adhesion (E s he l R eta 1 (2000) JB m 275 1 2833-12840 E s he l R et 2) Int J Can cer 98 803-810).
  • the present inventors treat mandarin by suppressing the expression of TSTA3 gene, which has also been confirmed to suppress the proliferation of spermatogonia by suppressing RNA 1 expression of TSTA3 gene. It is also possible to measure the expression level of the TSTA3 gene.
  • TSTA3 gene refers to the human TSTA3 gene consisting of 1312 bases, which is Accession No NM—0033 in NCB I nuclease (SEQ ID NO: 1). Also included in this specification is a gene consisting of ⁇ ⁇ which consists of a nucleotide sequence that can be deflated under the conditions of a stringer or a complementary sequence in the base sequence of the gene or its complementary sequence that may be altered by having a deletion addition or insertion. It is called “TSTA3 gene” to be used.
  • the hyphenation is carried out according to a known method or according to a molecular clinching numb (Molecular Clon id Ed iti on JS amb ro ok eta 1 Ci ng Har bo lab Labs 2001). Come over. You can also use a commercially available life rally according to the method described in the instruction manual.
  • “severe conditions” may be low stringent conditions, medium stringent conditions, or stringent conditions.
  • “Low stringency is 5XSSC 5 X Tenhard solution 0 5% SDS 50 32 ° C.
  • “ Medium stringent condition ” is SC 5X Tenhard solution 0 5% SDS 50% formamide.
  • “Stringent conditions” is, for example, 5 XS Slt solution 0 5% SDS 50% formamide 50
  • the nucleotide sequence of SEQ ID NO: 1 is, for example, 70% or more ⁇ 5 or more 85% or more 90% or more 91% or more 92% or more 93 or more 95% or more 96% or more 97% or more 98% or more 99 I can open it.
  • inhibition of gene expression refers to a tan-haku inherited from a gene by inhibiting any of the events (eg, transcription (mRNA generation), translation (tan-haku-mu)). It shall mean to inhibit the production of quality.
  • TSTA3 protein refers to human TSTA3 protein consisting of 321 amino acid residues that are accessi on No NP—003 in NCB I evening hose and substantially similar to this protein.
  • Activity for example, 3 5 (ep ime rase)
  • Activity 4 Reyu cact (Re duc an FX eta 1 (1998) J hem 273 8193-8202 Oh yama C et 98) JB iol Ch em 273 14582 -1458 K eta 1 (2003) Can cer Res 63 89)), and the deletion of one or more amino acid residues from this sequence.
  • a mutant protein consisting of an amino acid sequence.
  • Amino acid mutation site and number in the above-mentioned mutant protein As long as the protein has substantially the same activity as the original protein, the number of mutations is, for example, 1-50, 1-40, 1-30 Pieces And having substantially the same quality of activity as the original protein, the larger the homology value, the better.
  • TSTA3 protein contains “TSTA3 protein”.
  • the partial amino acid sequence of TS amino acid sequence (SEQ ID NO: 2) is preferably used.
  • Any of the TSTA3 proteins of any of the following may be used, such as at least 20 amino acids in the sequence amino acid sequence, preferably at least 70, more preferably at least 10 or at least 200 amino acids.
  • An amino acid sequence consisting of residues. are preferably listed.
  • These polymorphs contain an amino acid sequence corresponding to the portion involved in the activity of the protein. 1 or more in the row (e.g., about 1 to 20 is more preferable, even more preferably 1 to 5) Or Te that has been modified by amino acid residues or deletion or insertion of degrees).
  • the TSTA 3 protein used in the present invention can be prepared from the original weave. These seeds can also be synthesized by a synthesizer, prokaryotic or eukaryotic, or prepared by a recombinant method using appropriate Shuo cells. Is it okay?
  • Can c res les 63, 6282 Can it be done in accordance with the method such as 6289? For example, it can be measured in accordance with the screen described later.
  • cancer treatment agent is used to mean an anti-cancer agent, a cancer metastasis inhibitor, a phacosinos inducer, a cancer cell growth inhibitor, a cancer cell invasion inhibitor, and the term “cancer”.
  • tumor is used as a term having the same meaning.
  • nucleic acid means an acid meaning RNA or DNA, which may contain not only furin and hiriminone bases but also those having modified heterocyclic bases. These fluorinated furin and hiliminone.
  • the acylated furin and furanized furin and hiriminone or other complex. Refers to a phenomenon in which a foreign gene and an inherently endogenous ft gene are harmed by double introduction having the same or similar sequence as the target gene sequence.
  • the RNA used here is a double-stranded RNA that produces a 30-base long RNA, such as dsR1 estr and RNA) s ⁇ RNA (sma1 1 ⁇ ngng RNA) or sRNA (shorthairpin).
  • RNA can also be localized to the target system of the rephosome, and can also be expressed locally using the above double-stranded RNA or hectare, such as (ds RNA sl RNA or s hRNA) Preparation Methods Known from various literatures (Special Table 2002- 516062)
  • the term “antisense nucleic acid” or “antisense nucleic acid” refers to a nucleic acid having a complementary chito in at least a part of a DNA region of interest and at least one of the region. I mean.
  • the nucleic acid may be RNA DNA or modified nucleic acid (RN). They may be double-stranded DNA—double-stranded DNA—and DNA RNA hyphenated.
  • the modified nucleus is a nucleic acid sulfur derivative. Thiophosphate derivatives Furthermore, it is not limited to force that has resistance ft to the degradation of foliate and oriconucleocytoamis.
  • the antisense nucleic acid used is linked to a sequence containing a transcription termination signal downstream or on the appropriate side of the appropriate promoter.
  • This nucleic acid can be transformed into a desired animal using known methods. Sequence is the endogenous gene of the animal to be transformed More preferably about 80% or more, more preferably about 90% or more and about 95% or more complementarity. ,
  • the length of the sense nucleic acid is at least about 10 bases or more (for example, 10 is preferably about 15 bases or more, more preferably about 100 salts or more, preferably There are about 500 bases or more.
  • Antisense nucleic acid can be designed as follows (for example, Hirashima and Inoue Neonatal acid IV gene replication and expression The Japan Biochemical Society, Tokyo Chemical Dojin 1 19-347) JK a wa k am ⁇ eta 1 P ha J ap an Vo l 8 p 247 1 992 Vo l 8
  • the therapeutic agent for cans of the present invention uses, as an active ingredient, a nucleic acid having a refosym activity that cleaves into the TSTA 3 gene.
  • rehosym activity refers to a nucleic acid that specifically cleaves the mRNA of a gene used as a kenot. I mean. Is there a rehosym I intron type or Ml RN A contained in RNa se P, or is it larger than a chito? Some have a hammer type or hea hin 0 active nucleotides (Tanha 1990) 35 p 2 1 9 1). Hammerhead Rehosaime FEBS L ett 1988 228 p 228 F t 1988 239, p 285 Protein Nucleic Acid Enzyme 19 Come to harm
  • the present invention can use as an active ingredient an inhibitor of the transcriptional activity of the TSTA 3 gene.
  • a compound is a compound that binds to a factor involved in the expression and transcription of the 3 gene.
  • the product may be a natural product or a synthetic compound. Such compounds can be obtained by the link method.
  • Cancer containing 1 2 T S T A 3 protein activity inhibitory substance provides a therapeutic agent for cannula containing TSTA 3 protein.
  • TSTA3 protein activity inhibitor TSTA3 protein activity inhibitor
  • antibody refers to the entire 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 T protein of the present invention, the above monoclonal antibody monoclonal human antibody humanized antibody by gene recombination and the antibody product thereof are also included.
  • Antibodies that bind to TSTA 3 protein may be prepared by methods known to those skilled in the art.
  • the TSTA3 protein activity ft can be used as the above-mentioned antibody or mutant active ingredient that binds to the TSTA3 protein.
  • Such compounds are examples of compounds that bind to proteins and inhibit their activity, which may be natural or synthetic. Such compounds can be obtained by the method described below.
  • the present invention relates to screening of candidate compounds having an anti-cane action.
  • One preferred embodiment is a method using TSTA 3 protein and a test compound. It is expected to have an effect of inhibiting the activity of the compound protein that normally binds to TSTA 3 protein. This is preferably binding to the active site of TSTA3 protein.
  • the TSTA3 protein is brought into contact with the test compound. The protein responds to the finger to detect the binding with the test compound.
  • Purified form of protein It can be expressed intracellularly or extracellularly or bound to an affinity column. This method can be used by appropriately labeling the compound as necessary, and as a knowledge, radiolabel, fluorescence knowledge, etc. can be mentioned. Not determined.
  • the binding of the TSTA3 protein to the test compound is detected by, for example, the knowledge attached to the test compound bound to the T protein.
  • the change in TSTA 3 protein activity can be used as an indicator.
  • the binding activity between the protein and the test compound can be determined by known methods (eg Sulliv an FX, e 998) JB iol Ch em 273 81 93 Ohyama C eta 1 (1 998) JB em 273 14582-14587 No daa 1 (2003) Cancer Res 63 63).
  • Another embodiment of the screening method of the present invention is a method for TSTA3 inheritance.
  • the cells are further brought into contact with cells expressing the TSTA 3 gene.
  • the origin of the “cells” used is human mouse urchin hysson chicken and henoto cells that are not derived from livestock, etc. Cells or exogenous tt T ST Compound life rally Gene life rally expression product Cell extraction Fermentation microorganism product Marine organism extract Plant extract etc.
  • test compound or protein A method of introducing a DNA hector expressing a protein into the cell.
  • the expression level of the TSTA 3 gene “gene expression” includes both transcription and translation.
  • Hell can be measured by methods known to those skilled in the art. It is possible to measure the transcriptional reherence of the A3 gene by extracting the mRNA from cells expressing the A3 gene according to a conventional method, and using the Norse hybri-isen method or RT-PC. Disclose the floor motor region of TA 3 gene according to the conventional method.
  • the transcription level of the gene can also be determined by looking at the TSTA3 gene expression from the cells expressing the TSTA3 gene and detecting the expression of the TSTA3 protein by SDS-PAGE. It is also possible to measure the residue by detecting the expression of the protein by Western Frono using an antibody against TSTA 3 protein Detection of T STA 3 protein 3 Anti-TSTA3 antibody and therapeutic agent containing this antibody, complex
  • the present invention also contains an anti-TSTA3 antibody. In one preferred embodiment of the present invention, the above therapeutic agents are used or for enthusiastic drug delivery.
  • the “anti-TSTA3 antibody” is an anti-TSTA3 antibody that specifically binds to TS TA3 Tanja (including partial hechito) or a salt thereof).
  • the anti-TSTA3 antibody used in the present invention is a monoclonal antibody or monoclonal antibody. May be.
  • the antibody class also includes any antibody, such as IgG IgM Ig Ig gD or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of the above.
  • the term used here is meant to include any antibody fragment or derivative.
  • the antibody of the present invention can be produced by a known method. This method is well known in the art (see, for example, Har 1 ow E & Antibody, Cold Spring Harborory Pres (1 988)).
  • the protein used as the sensitizing antigen in the present invention is a protein or a salt thereof.
  • the above TSTA3 protein contains chito, isn't it limited? Good.
  • the TSTA protein used here or its part is used, for example, as a salt with a mechanical acid (eg, hydrochloric acid or sulfuric acid) or a salt with acetic acid, oxalic acid or fluoric acid).
  • the TSTA 3 protein of the present invention to be used for the antibody is preferably derived from a mammal, for example, a mouse derived from a human or a mouse.
  • Dust STA 3 protein as described above
  • TSTA3 Ranoto mouse Usaki antigen per animal
  • FCA Freund Hunt
  • F IA Freund Hunt
  • aluminum hydroxide etc.
  • the interval between administrations is particularly limited. Several days to several weeks, preferably 2 to 1 to 10 times, preferably 2 to 5 times. After 0 days from immunization, antibody-producing cells are preferably collected after 1 to 14.
  • Anti-spleen cells Linha node cells Peripheral blood cells can be listed Where Linha node cells are preferable
  • Antibody-producing cells and myeloma cells The cell line YB 2Z0 and the Rano myeloma cell line. Next, the above-mentioned myeloma cells and antibody-producing cells are fused. Serum-free DMEM RPM I—1640 medium animal 1 X 1 0 6 ⁇ 1 X 10 7 cells Zm 1 antibody-producing cells and 2 X 10 Zml myeloma cells are mixed (antibody-producing cells and myelo 2 1-31 are preferred) Melt in the presence of cell fusion promoter It is possible to use an average molecular weight of 1000 to 6000 tarton recall as a cell fusion promoter. It is also possible to fuse antibody-producing cells with commercially available cell fusion devices that use electrical stimulation (eg Noyon).
  • electrical stimulation eg Noyon
  • Hyfuri-Nink is well following normal methods. Especially limited is part of the culture supernatant contained in wells grown as hyfritoma Immunoassay Radiation I Screening cells by radioimmunoassay etc. Cloning is performed by the limiting dilution method. And most Earth MEM medium or through e Te animal cell culture media in to serum medium such as 37 ° C 5% C_ ⁇ 2 concentration) Te cultured for 7 to 14 days to get that.
  • the antibody titer was measured by enzyme immunoassay (ELISA (enz ume lmmuno sorbentassy) or EIA (enzynoassay)) radio tt immunoassay (RIA rad ⁇ o ⁇ ss ay), etc. Search on the day of showing the antibody titer.
  • enzyme immunoassay enzyme immunoassay
  • EIA enzyme immunoassay
  • RIA rad ⁇ o ⁇ ss ay radio tt immunoassay
  • the anti-blood clot polyclonal antibody For example, R lec hmann et al. (Riec hmann J Oct 5 203 (3) 825-8 1988) and Jone es et al. Nature 321 522-525 1986 can be prepared by one of the methods.
  • Antibodies that bind to TA 3 protein which can be produced by referring to“ Naturture Vol 368 p 856—859 199-500233 ”, are used for the purpose of cancer cell growth, etc.?
  • a human antibody it is preferable to use a human antibody to reduce the immunogen ft.
  • the antibody When used as a diagnostic agent, the antibody may be labeled as a radioisotope or a fluorescent substance for monitor links. Radioactive materials The most common compounds that can be identified are fluorescent compounds such as fluorescein isothionoa, rotamitrin and fluorescamine. Similarly, the bioluminescent compound TSTA3 antibody can be labeled. Bioluminescence ft tan haku Measured by detecting the presence of light. Heavy for this knowledge purpose 3 2 Complex containing anti-TSTA 3 antibody
  • the anti-TSTA 3 antibody used in the present invention itself be a neutralizing agent (agent) that weakens the activity of the antigen in the blocking agent of the present invention or combined with an agent that exhibits a therapeutic effect as necessary You can use it. Therefore, the present invention also provides a complex containing an anti-TSTA 3 antibody and another drug for use in targeting therapy or desensitization in humans (eg, large intestine). . According to such an embodiment, the anti-TSTA 3 antibody to be used can be used as an intelligent site for highly expressing TSTA 3 protein, as well as other agents that have therapeutic effects.
  • Examples of the “other leaf agent” used in the present invention include an elemental therapeutic protein, a low molecular weight drug, and a genetically-targeted non-viral hector.
  • examples of the “radioisotope” include radioactive halokens such as fluorinone 125 ( 125 I) and iodine-131. These radiohalogen elements are also known in the same way as the above-mentioned radiometal elements, and are widely used as radiotherapeutic agents or radio14 diagnostic agents.
  • 125 I or 131 I can be sterilized by chloramine T method, etc. It can be conjugated to an antibody fragment.
  • therapeutic proteins in the present invention are preferably immunity to the site of immunity, for example, human interleukin—macrofern colony stimulating factor human macrofernolo human interleukin 1 2 and the like.
  • toxins such as ricin and nophtheria toxin for colon colonization.
  • the fusion antibody with protein the antibody or antibody fragment is co-coated with the DNA coated with the protein. It is possible to construct an antibody by constructing A and introducing this DNA into a prokaryotic or eukaryotic expression hex.
  • Low molecular drug is used in this specification to mean “radiation ft isotope” or “drugs other than metallurgy or diagnostic compounds”.
  • a gene that exerts a therapeutic effect when it does not induce nose in a spurious part of a cell growth-related gene such as a gene related to cell proliferation (eg, large intestine) (therapeutic gene), a viral hector that binds to an anti-TSTA3 antibody When administered to a person in need of anti-TSTA3 child therapy, anti-TSTA 3 antibody (i.e., TSTA3) or activating to the existing site Get the gene.
  • “chemical bond” includes ionic bond, hydrogen bond, bond by interfacial force, and bond by hydrophobic interaction.
  • Engineerering bond includes, for example, an antibody and a therapeutic protein. This includes the mode of binding between the antibody and the treatment when produced using any technology. 4 Formulation and administration method
  • Therapeutic treatment containing the TSTA 3 gene expression inhibitor of the present invention The therapeutic agent containing the activity inhibitory substance of porphyry activity Anti-T-containing therapeutic agent of the present invention or the anti-TSTA 3 anti-antibody used in the present invention Flavoring W Storage Stabilizer Buffering Agent Suspending Agent Tonicity Agent Binder Fluidity Accelerating Agent Can it be used as a flavoring agent?
  • Examples of the dosage form of the blacksmithing agent of the present invention are, for example, as an oral agent, pill, powder, granule, fine granule, soft hard cuff cell agent, film coret, sublingual agent, first agent, etc. parenteral, injection, suppository, suppository Cement plaster External liquids etc. are mentioned. Those skilled in the art can select the optimal dosage form according to the administration route. TST activity (or expression of TSTA 3 gene) inhibitor as an active ingredient can be included in the preparation at 09% by weight.
  • Oral administration is generally about 0 lmg to 1 OO Omg per day. 10 Omg, more preferably about 10-50 mg.
  • Target organ Symptom Administration method for example, the form of injection is usually normal (for example, 6 O kg) About 0 01 to 3 Omg, preferably about 0 1 to 2 I will come. In the case of animals other than humans, the above dose of 60 kg can be administered.
  • the therapeutic agent of the present invention is a can (eg, large intestine, stomach, lung, gland, esophagus, liver, biliary tract, spleen, kidney, bladder) Is preferably used in the large intestine
  • the leaf preparation of the present invention contains a substance that inhibits the activity of T STA 3 protein or a substance that inhibits the expression of a child as an active ingredient, it can be used as an anti-cancer agent, a photonos inducer of cancer cells, and the like.
  • the target type of cancer or cancer is not limited to a specific type.
  • expression of the leaf 3 protein activity inhibitory substance Ayohi T S T A 3 gene of the present invention may be inhibited.
  • an antisense nucleic acid when used in the therapeutic agent of the present invention, the acid may be administered according to a known method such as a single virus or a suitable virus such as a retrovirus, a virus, a virus, a virus, a virus, a virus, and a virus.
  • Antisense nucleic acids can be formulated with physiologically acceptable carriers and administered via a gene gun or catheter such as a hash.
  • an anti-TSTA 3 antibody such as a recombinant atenovirus particle may be used for cancer therapy.
  • a pharmaceutically acceptable carrier As such a carrier, it is already aquatic Ordinarily, 10 3 to 10 15 viruses at a time per adult is preferable? Disease state ⁇ w cells Depending on the nature of the tissue, the dose may be once to several times a day. One to several injections over several months may be given intermittently over a long period of time as one senot.
  • the viral hectare hectare nucleic acid molecule used in the present invention can be used for diagnosis of the state of specific cells and tissues.
  • a virus-hector particle obtained by incorporating a marker gene that can be detected into a virus into a suitable host cell can be used to detect and diagnose anti-TSTA3 antibodies and cells.
  • a suitable host cell can be used to detect and diagnose tumor cells by combining anti-detectable knowledge.
  • the present invention also provides a diagnostic agent.
  • a clear can diagnostic agent has (a) an antibody against TSTA 3 protein S T A 3 gene or a part of its base sequence having a holinus consisting of a base sequence that can be hyfitized under stringent conditions.
  • the antibody against TSTA 3 protein can be identified.
  • TSTA 3 protein is determined in the test solution. Bind or detect STA3 antibody binding or / or quantified.
  • subject-derived biological sample includes a subject-derived fluid (eg, blood (including whole blood, plasma, serum, etc.) urine, phosphorus, semen, etc.). “Subjects” also include human subjects who are usually or are suspected of undergoing canine screening, and who are suspected of being affected. Examples of such cans: stomach, lung, milk, prostate, esophageal, liver, kidney, bladder, uterus (eg, cervical, uterine or thyroid, knee, ovary, tumor, blood tumor, etc. It is preferable.
  • a subject-derived fluid eg, blood (including whole blood, plasma, serum, etc.) urine, phosphorus, semen, etc.
  • Subjects also include human subjects who are usually or are suspected of undergoing canine screening, and who are suspected of being affected. Examples of such cans: stomach, lung, milk, prostate, esophageal, liver, kidney, bladder, uterus (eg, cervical, uterine or thyroid, knee, ovary, tumor, blood tumor, etc. It is prefer
  • One embodiment of the immunoassay is contacted with a nitrocellulosic support or carrier for the purpose of immobilizing all proteins present in the life such as serum samples.
  • the support is then treated with a buffered anti-TSTA3 antibody that is detectable.
  • antibody-chelating agents are described in Nuclo 1 1 9 9 0 1 7 247-2 54.
  • an antibody having a paramagnetic 14 ion as a label used as a Menonique is described in Ma gnetic Resonance in Me dici 2 2 33 9-342
  • a flow or flyer having the base sequence of the T S T A 3 gene can be used. Specifically, for example, (a) a biological sample derived from a subject and a base sequence of a TSTA3 fragment, a stringentene-like high-rise loop, and a reflowable base sequence (flow) process and (b) ) Detecting and / or detecting the presence of the polynucleotide and T or a fragment thereof in the test W.
  • TSTA 3 (or a gene fragment thereof) in a biological test derived from a subject is detected using the above-mentioned flow.
  • the length of the base sequence used as the flow is, for example, 1 2 or more bases 1 8 bases or more 2 1 bases or more 24 bases or more 2 7 bases or more No. 98 EP— A0200362 US Pat. No. 2,915 08
  • the quantification can be carried out using a known technique using a cytochrome or flymer for the T S T A 3 gene.
  • a known method for example, Furi Seisenyon Nosan Haifuri Yusei Noyon RT— R— SS CP method (Genomics 5th 874-87)) Proceeding ng soft he Na ti on e my of Science he Un itedof Ame rica 86 ⁇ 2766 ⁇ 2770 (19
  • the array CGH method is a chromosomal CGH method (Ka 1 1 1 on 1 em 1 a 1 (1992) Science 258 818-821 method). There is a method to detect high-resolution DNA coherency in high resolution by detecting the binding state of the DNA derived from normal DNA and normal DNA using Shonoto's DNA chinoff and performing the high-speed detection when the chemome DNA on the slite. eta 1 (1 998) Na t Ge net 20
  • TSTA 3 gene is overexpressed by the above-described method (eg, DNA mRNA), it is possible that there is a disease caused by TS (eg, cane (eg, large intestine)) It is possible to diagnose that it is possible to be affected in the future.
  • TS eg, cane (eg, large intestine)
  • the presence of a fragment of a target protein in a test sample is identified using a mass spectrometer (MS).
  • MS mass spectrometer
  • the determination of the non-acid sequence can be used to determine whether or not the T protein exists in the biological test derived from the subject + the mass.
  • the mass of the trial +4 can be identified from the results of the mass analysis to identify the individual amino acids of the protein and heptagon.
  • Ionization can be used in various ways, such as Matrix Astheno Tracer Teethofushi (MALD I) Elect Mouth Souffle Ionization (ESI) Qi) Field Desorption (FD) Ion Ionization
  • MALD I Matrix Astheno Tracer Teethofushi
  • ESI Elect Mouth Souffle Ionization
  • FD Field Desorption Ionization
  • MALD I Matrix Astheno Tracer Teethofushi
  • ESI Elect Mouth Souffle Ionization
  • FD Field Desorption
  • MAL time-of-flight (t ime offli gh t TF) mass spectrometry mass spectrometry
  • QMS quadrupole type
  • ion tranov type magnetic field type
  • the TSTA 3 gene or the TSTA 3 gene in a biological test derived from a subject containing a hyfuri group sequence under stringent-enhanced hyphenation conditions is detected and Z or quantified as a marker.
  • Kinoto These mushrooms are used to detect the markers described above for immunological techniques or hyphenation.
  • can marker refers to a molecule that is not present in a subject's body fluid (eg, saliva, saliva, sweat, semen, etc.) Good Indicates or indicates the presence of presence in the body fluid or cells or tissues of the subject
  • the quinoto contains components that detect and detect TSTTA 3 protein in body fluid samples from subjects and its parts, for example, TSTA 3 protein or ELISA or quantification.
  • Such components can be used, for example, tissue sections.
  • Such antibodies may be radiofluorimetric colorimetric or fermented.
  • the quinoto of the present invention contains a labeled secondary antibody In addition to a functional base sequence, etc., it may contain a container. The laher associated with the container may be used to detect drugs or large intestine cankers. Other items such as instructions for use may also be provided.
  • Example 1 Array C GH method of colonic cannabis-specific amplified gene This example identifies colonic canal-specific gene amplification region Sample 200 samples of sampled sample Ayohi Array CGH-based test Results BSTA C l 1 -661A1 used TSTA 3 located in BAC C l 1 -661A1 2 Using gene information (NC BI http // www ncbih go vZ) (T issuectranspl antation an tigen P 35 Accession on No NM_00331 3) It was found that the gene was large and frequently high (01 Table 2). Indicates frequency. In addition, Table 2 below shows the mean value of 20 degrees (G / R value) and frequency of TSTA 3 gene colonic persecutor and GZR value or 1 2 average value.
  • TSTA3 gene is a large sample of 200 samples. (Table 2)
  • the amplification degree of a gene cell line derived from a high-frequency gene intestinal canal was verified in those who suffered from large intestine.
  • the cultured cell lines were RKO and RK, which are cell lines derived from the large intestine. Extracted from cultured cells using B 1 o d & C e l l Cu l t ure (Q I AGEN) according to Frotocol with Kinoto
  • Table 3 shows the degree of amplification of the TSTA 3 gene in cell lines derived from large intestine. As shown, it was found that the TSTA 3 gene located in BARP 1 1 -661 A 12 also increased in the colon canal cell line.
  • AREA CGH Quantitative PCR was performed using SYBR Green RT-PCR Re-agplied Biosyst ems) and Froto 7500 Real-Time PCR System (Ap posyst erns). Framer used the following PE RON for synthesis).
  • Table 4 shows the relative values of TS TA 3 gene amplification in large intestine-derived cell lines versus B DNA (normal). As shown, amplification is occurring in the T S T A 3 gene region even in cell lines (Table 4).
  • the cell line was cleared from ATCC, and cultured according to the Frotocol marked with ⁇
  • the RNA selected a specific 2 lmer in the gene and synthesized its sequence A (commissioned to Q I AGEN)
  • s 1 RNA was introduced into RK0 cells using L ipofect am (Invitrogen), and 50 nM siRNA was introduced into the cells according to the standard.
  • s 1 RNA was introduced into RKOE 6 cells FECT (QI AGEN) Then, 100 ⁇ of sl RNA was introduced into the cells according to the rules. Ne gatlve C on RNA (QI AGEN) was used for anti-Akira. Inverted for 4 days after introduction into cells
  • Quantitative RT quantitative RT—PCR method was used to determine the effect of s 1 RNA from cells 24 hours after introduction of s 1 RN
  • the number of viable cells after introduction of s 1 RNA was measured using Wa ll ac 14 ll abe l / Lumi nes sc ce Coun ter A ki n l mel) using A 1 mar Blue (B e) according to the Flotocol attached. . Result
  • RNA 1 analysis of genes was performed using RKO and RKOE 6, which are cell lines derived from the large intestine.
  • 04 A and 04 B show the results of measuring the number of viable cells with the measuring reagent for s 1 RNA of RKO cells and RKO TA3 gene 4 days after T ran sfection. . Results of measurement of viable cell count as shown Remarkable cell counts (04A and B) compared to the expression a ti V e C o n t r o l (NC). Two types of siRNA of TSTA3 gene (a and t test were significant (p ⁇ 0 0 1).
  • RNA 1 is used in a cell line derived from the normal tissue of the large intestine to confirm that it is specific for the suppression effect of epilepsy genes ⁇ RNA i analysis>
  • the cell line used is CCD 1 8 Co purchased from ATCC.
  • CCD 1 8 Co purchased from ATCC.
  • Following the cultivated Frotocol. Use the siRNA used in Example 3a. Introducing s 1 RNA into cells Carry out in the same way as described in Example 3.
  • TSTA3 gene is an organ-specific expression such as a normal tissue or the like.
  • RNA probe was hone according to the rules.
  • RNA rehering in each normal organ tissue was determined by organ (Heart M (Brain) placenta (Placenta) lung (Liver) skeletal muscle (Skeletal Muscle) kidney (Kidney) knee spleen (Spleen) ) Thymus Prostate Prostate Testicular Intestine Colon Peripheral Blood Leukocyte Stomach Thyroid T (Spinal Cord) Lymph node Trachea ) Gland) Bone Marrow was analyzed by Northan High Fritai, Inc. As mentioned above, it was registered as Accession No NM-003313 sequence or mRNA in the inheritance of TS TA 3 (http // www ncbi nlm nih gov). Results
  • Ultimate Plus LC PACKINGS
  • Stream 0 2% acetonitrile (Wako 062-02901) containing 90% acetonitrile with MERCK 128 acid or 0 57% / nun linear gradient was analyzed.
  • 07 A and 07 B are: (A) Serum and This is the amino acid sequence of the TSTA3 protein (corresponding to the amino acid sequence of positions 10 to 18. Therefore, the fractofragment (SEQ ID NO. In normal hematopoiesis, no ion hekes were found in the corresponding partial sequence (see 08C), so this target molecule TA 3 protein may have increased in cancer-specific blood abundance.
  • SEQ ID NO. In normal hematopoiesis, no ion hekes were found in the corresponding partial sequence (see 08C), so this target molecule TA 3 protein may have increased in cancer-specific blood abundance.
  • RNAi analysis was performed.
  • the introduction of s1 RNA into the cells was carried out by using o 1 imine (Inv trologen) and following the flow of 100 nM s.
  • Nega tlv o 1 si RNA (Q I AGEN) was used.
  • the present invention provides a therapeutic agent for cans, a diagnostic agent, a diagnostic method and a therapeutic method. Therefore, the present invention is useful in the fields of can diagnosis and the like.

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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 Ceased WO2007037550A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001041804A1 (fr) * 1999-12-08 2001-06-14 Ramot University Authority For Applied Research & Industrial Development Ltd. Procede de modulation et de mesure de l'activite enzymatique de fx dans les cellules cancereuses, les reactions inflammatoires et maladies et en auto-immunite
WO2003060470A2 (fr) * 2001-12-21 2003-07-24 Arcturus Engineering, Inc. Profilage de l'expression du cancer du sein
WO2005118869A2 (fr) * 2004-05-28 2005-12-15 Dana-Farber Cancer Institute, Inc. Compositions, kits et procedes pour identifier, evaluer, prevenir et traiter un cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001041804A1 (fr) * 1999-12-08 2001-06-14 Ramot University Authority For Applied Research & Industrial Development Ltd. Procede de modulation et de mesure de l'activite enzymatique de fx dans les cellules cancereuses, les reactions inflammatoires et maladies et en auto-immunite
WO2003060470A2 (fr) * 2001-12-21 2003-07-24 Arcturus Engineering, Inc. Profilage de l'expression du cancer du sein
WO2005118869A2 (fr) * 2004-05-28 2005-12-15 Dana-Farber Cancer Institute, Inc. Compositions, kits et procedes pour identifier, evaluer, prevenir et traiter un cancer

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