WO2004000346A1 - Preventifs et remedes contre le cancer - Google Patents

Preventifs et remedes contre le cancer Download PDF

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Publication number
WO2004000346A1
WO2004000346A1 PCT/JP2003/007926 JP0307926W WO2004000346A1 WO 2004000346 A1 WO2004000346 A1 WO 2004000346A1 JP 0307926 W JP0307926 W JP 0307926W WO 2004000346 A1 WO2004000346 A1 WO 2004000346A1
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WIPO (PCT)
Prior art keywords
protein
cancer
amino acid
acid sequence
present
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PCT/JP2003/007926
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English (en)
Japanese (ja)
Inventor
Yuichi Hikichi
Ryosuke Katsuyama
Yuichi Kakoi
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Takeda Chemical Industries, Ltd.
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Priority to AU2003243951A priority Critical patent/AU2003243951A1/en
Publication of WO2004000346A1 publication Critical patent/WO2004000346A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to an agent for preventing and treating cancer, a diagnostic agent, and the like.
  • a microarray method in which cDNA or oligonucleotides are immobilized has been developed. Techniques for finding disease-specific changes in gene expression have become widespread, and their usefulness has been confirmed. For example, Af fymetrix's GeneChip system is being widely used for diagnosing diseases such as cancer and finding drug discovery target genes.
  • antisense oligonucleotides hybridize to RNAs with complementary sequences and induce RNase H degradation of RMs to inhibit protein translation, or to inhibit direct protein synthesis by hybridization. Bring. Because it is possible to specifically suppress the function of the target gene, it is frequently used as a means of analyzing the function of genes, and some antisense oligonucleotides are being developed for clinical application.
  • RIPK1 Interacting Serine / Threonine Kinase 1 (hereinafter referred to as RIPK1) was discovered, but a molecule showing homology to this RIPK1, RIPK2, has recently been cloned (J. Biol. Chem. 273). , 12296-12300, 1998). Subsequently, RIPK2 binds to apoptosis-related molecules such as Caspase and c IAP, and also binds RIPK2 to MCF7 and 293T. It has been reported that apoptosis is induced by forced expression in cells (J. Biol. Chem. 273, 12296-12300, 1998 and J. Biol. Chem. 273, 16968-16975, 1998).
  • RIPK2 is a molecule that acts downstream of Toll-like Receptor (TLR) -2, 3, 4 and is involved in innate immunity ( Nature 416, 190-194, 2002; Nature 416, 194-199, 2002).
  • TLR Toll-like Receptor
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have found the RIM2 gene as a gene whose expression is significantly increased in cancer tissues, particularly malignant cancer tissues. Furthermore, they found that apoptosis was induced by suppressing the function of the RIPK2 gene, which has been considered to be a factor that induces apoptosis.Based on this finding, the inventors conducted further studies and completed the present invention. Reached.
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a compound that inhibits the activity of a salt thereof, or a salt thereof Prevention and treatment of cancer
  • a prophylactic or therapeutic agent for cancer comprising the antisense polynucleotide according to (3) above,
  • Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary (1), (2), cancer, testis, thyroid, thyroid, brain or hematological
  • the cancer is breast cancer, (1), (2), (5) or
  • a diagnostic agent for cancer comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof;
  • a diagnostic agent for cancer comprising a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof,
  • Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary (8) or cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor
  • a prophylactic / therapeutic agent for cancer comprising a compound having a Receptor-Interacting Serine / Threonine Kinase 2 activity inhibitory activity or a salt thereof,
  • a prophylactic / therapeutic agent for cancer comprising a compound having an inhibitory effect on the expression of Receptor-Interacting Serine / Threonine Kinase 2 or a salt thereof,
  • a prophylactic or therapeutic agent for cancer characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof; Screening method,
  • a method for screening a pharmaceutical compound which comprises using a polynucleotide encoding a protein or a partial peptide thereof, (15b) a compound for preventing or treating cancer, The method according to (15a) above, which is a compound used for the prevention and treatment of Z and a compound having Z or cancer prevention and treatment effects.
  • An agent for preventing or treating cancer which can be obtained by using the screening method according to (13) or (15) or the screening kit according to (14) or (16).
  • an agent for preventing or treating cancer comprising the compound or salt thereof according to (17b) above, (18) a protein or a partial peptide which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a compound which inhibits the activity of the salt or a salt thereof Apoptosis action promoter,
  • a method for screening an apoptosis-activating agent which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof.
  • a compound that inhibits the activity of a protein, or a partial peptide or a salt thereof, having the same or substantially the same amino acid sequence as that represented by SEQ ID NO: 1 in a mammal; Or a salt thereof, or a compound that inhibits the expression of a gene of the protein or an effective amount of a salt thereof, comprising the steps of: • (25) inhibits the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, or expresses a gene of the protein Cancer prevention and treatment methods characterized by inhibiting
  • a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 used in the present invention (hereinafter sometimes referred to as the protein of the present invention or the protein used in the present invention) are cells of human warm-blooded animals (eg, guinea pig, rat, mouse, chicken, egret, bush, hidge, horse, monkey, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, Glands i3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, fat cells, immune cells (eg, Macrophages, T cells, B cells, natural killer cells, obesity cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, cartilage Cells,
  • progenitor cells stem cells or cancer cells, etc.
  • any tissue where these cells are present such as the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, Cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, kidney, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels , Heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joints, skeletal muscle, etc., or a synthetic protein .
  • olfactory bulb amygdala, basal sphere, hippocampus, thalamus, hypothalamus, Cerebral cortex, medulla, cerebellum
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, and more preferably about 50% or more of the amino acid sequence represented by SEQ ID NO: 1.
  • Amino acid sequence homology can be calculated using a homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Search Tool).
  • Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above. However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable. Examples of substantially equivalent activities include, for example, signal transduction activity (eg, RIPK2 intracellular signal transduction activity), phosphorylation activity, and the like. Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) identical.
  • the signal transduction activity and the phosphorylation activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
  • the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the signal transduction activity and phosphorylation activity can be measured by a method known per se, for example, the method described in J. Biol. Chem. 273, 16968-16975, 1998 or a method analogous thereto. . RIPK 2 is autophosphorylated in signaling Activates the NF KB signal.
  • phosphorylation activity can be determined by introducing an expression vector for the protein of the present invention (eg, RI PK2) into cells, solubilizing the cells with an appropriate solubilization buffer, and immunoprecipitation. After obtaining a protein (eg, RI PK2), it is reacted with radiolabeled ATP in an appropriate buffer, and the reaction mixture is subjected to SDS gel electrophoresis, and the protein of the present invention is obtained by autoradiography or the like. (Eg, RI PK 2) can be measured for the degree of autophosphorylation.
  • the signal transduction activity can be determined by introducing an expression vector for the protein of the present invention (eg, RI PK2) into a cell together with a repo overnight gene containing an NF ⁇ B binding sequence in a promoter or an enhancer.
  • an expression vector for the protein of the present invention eg, RI PK2
  • a repo overnight gene containing an NF ⁇ B binding sequence in a promoter or an enhancer.
  • a liquid containing a signal transduction activity or a suppressive substance such as a microorganism cell disruption solution, a microorganism culture supernatant, a eukaryotic cell disruption solution, or a eukaryotic cell culture supernatant is added, and the reporter expression level is known per se.
  • the repo overnight expression level can be measured using, for example, a reporter protein activity using luciferase activity, alkaline phosphatase activity, or the like as an index.
  • Examples of the protein used in the present invention include: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 100, preferably about 1 to 30, An amino acid sequence in which about 1 to 10 amino acids have been deleted, more preferably a number (1 to 5) of amino acids, and 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, 1 to 1) An amino acid sequence to which about 00, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids are added; 3 represented by SEQ ID NO: 1
  • the amino acid sequence contains one or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids.
  • Inserted amino acid sequence, 4 1 in the amino acid sequence represented by SEQ ID NO: 1 Or two or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to 5)) of other amino acids
  • So-called muteins such as proteins containing an amino acid sequence substituted with or an amino acid sequence obtained by combining them are also included.
  • the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
  • the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (capillary end) according to the convention of peptide labeling.
  • the protein used in the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group (-C00H), carboxylate (-C00-), amide (- It may be either C0NH 2 ) or ester (-C00R).
  • R in the ester e.g., methyl, Echiru, n- propyl, isopropyl, C, such as n- butyl, _ 6 alkyl groups, such as cyclopentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl cyclo, for example, phenyl, alpha-naphthyl of which C 6 _ l2 7 aryl group, e.g., benzyl, one Nafuchiru C, such as phenylene Lou C Bok 2 ⁇ alkyl group or alpha-naphthylmethyl, such as phenethyl, such as _ 2 alkyl groups C 7 _ 14 Ararukiru group, such as pin bar opening Iruokishimechiru group is used.
  • C such as n- butyl
  • _ 6 alkyl groups such as cyclopentyl
  • a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the protein used in the present invention the amino acid residue (e.g., main Chionin residues) of N-terminal Amino group protecting groups (e.g., formyl group, _ 6 Ashiru groups such as C Myu6 Arukanoiru such Asechiru group ), N-terminal dalluminin residue generated by cleavage in vivo, oxidized with lipamine, substituents on the side chains of amino acids in the molecule (eg-0H , -SH, amino group, imidazole group, I Ndoru group, in Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C such Asechi group, such as C WINCH 6 Ashiru groups such as _ 6 Arukanoiru group)
  • a suitable protecting group e.g., formyl group, C such Asechi group, such as C WINCH 6 Ashiru groups such as _ 6 Arukanoiru group
  • protein used in the present invention include, for example, protein containing an amino acid sequence represented by SEQ ID NO: 1.
  • the partial peptide of the protein used in the present invention examples include: It is a partial peptide of the protein used, and preferably may be any peptide having the same properties as the protein used in the present invention described above. Specifically, for the purpose of preparing the antibody of the present invention described below, a peptide having the 27th to 578th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 and the like can be mentioned. For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more of the constituent amino acid sequences of the protein used in the present invention. Peptides having more than one amino acid sequence are used.
  • one or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids in the amino acid sequence are deleted.
  • 1 or 2 or more (preferably, about 1 to 20, more preferably, about 1 to 10, and more preferably, about 1 to 5) amino acids are added to the amino acid sequence.
  • 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, 1 to 5) amino acids are inserted into the amino acid sequence.
  • one or more (preferably about 1 to 10, more preferably several, and more preferably about 1 to 5) amino acids in the amino acid sequence are replaced with other amino acids. It may be.
  • the partial peptide used in the present invention may be any of the ester (-C00R).
  • the partial peptide used in the present invention includes those having a carboxyl group (or carboxylate) in addition to the C-terminal, N-terminal amino acid residues ( (E.g., methionine residue) whose amino group is protected by a protecting group, glutamine residue generated by cleavage of N-terminal side in vivo and pyrodartamine oxidation, substitution on the side chain of amino acid in the molecule is Also included are those protected with an appropriate protecting group, and complex peptides such as so-called glycopeptides to which sugar chains are bonded.
  • a carboxyl group or carboxylate
  • N-terminal amino acid residues (E.g., methionine residue) whose amino group is protected by a protecting group, glutamine residue generated by cleavage of N-terminal side in vivo and pyrodartamine oxidation, substitution on the side chain of amino acid in the molecule is Also included are those protected with an appropriate protecting group, and complex peptides such as so-
  • the partial peptide used in the present invention can also be used as an antigen for producing an antibody.
  • salts with physiologically acceptable acids eg, inorganic acids, organic acids
  • bases eg, alkali metal salts
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid.
  • the protein, its partial peptide, or a salt thereof used in the present invention can be produced from the above-mentioned human or warm-blooded animal cell or tissue by a known method for purifying a protein, or a DNA encoding the protein. Can also be produced by culturing a transformant containing It can also be produced according to the peptide synthesis method described below.
  • the human or mammalian tissues or cells are homogenized, then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography. Purification and isolation can be performed by combining chromatography such as chromatography.
  • a commercially available resin for protein synthesis can be usually used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM.
  • Resin 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2,, 4, dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2 ′, 4,1-dimethoxyphenylmethyl resin F moc aminoethyl) phenoxy resin.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods.
  • a protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial Obtain peptides or their amides.
  • the protected amino acid is added directly to the resin along with a racemization inhibitor (eg, HOB t, HO OB t), or as a symmetrical anhydride or H ⁇ B t ester or H ⁇ OB t ester After the protected amino acid is activated in advance, it can be added to the resin.
  • a racemization inhibitor eg, HOB t, HO OB t
  • the solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • solvents known to be usable for the protein condensation reaction for example,
  • Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylvinylidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; Sulfoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof are used.
  • the reaction temperature is appropriately selected from a range that is known to be used for a protein bond formation reaction, and is usually appropriately selected from a range of about ⁇ 20 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole so that subsequent reactions are not affected. .
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isopolnyoxycarbonyl, 4-methoxybenzyloxycarbonyl, C11Z, Br-Z, a Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenyl Phosphinothio oil, Fmoc, and the like are used. '
  • the lipoxyl group can be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.).
  • alkyl esterified eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
  • Alkyl esterification eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarponyl It can be protected by hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • groups appropriately used for the esterification for example, low-grade (C ,. 6) Arukanoiru groups such Asechiru group, Aroiru group such Benzoiru group, Benjiruokishika Ruponiru group, and a group derived from carbonic acid such as ethoxycarbonyl group Used.
  • a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz l, C 1 2 - Bz l, 2- two Torobenjiru, B r- Z, such as t one-butyl is used.
  • protecting group for histidine imidazole for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Tos 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Examples of the activated carbonyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (for example, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and ester with H'OB t).
  • active ester for example, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and ester with H'OB t.
  • Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or anhydrous hydrogen fluoride, Acid treatment with sulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc. Reduction by a system is also used.
  • the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 ° C. to 40 ° C.
  • anisol for example, anisol, phenol, thioanisole, methacresol, paracresol
  • a force thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like.
  • the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4- In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • an amide of a protein or partial peptide for example, first, after amidating and protecting the ⁇ -hydroxyl group of the amino acid at the carboxy terminal, a peptide (protein) chain is added to the amino group side with a desired chain length.
  • the protein or partial peptide from which only the K-amino group protecting group at the ⁇ -terminal of the peptide chain has been removed, and the protein or partial peptide from which only the C-terminal carboxyl group-protecting group has been removed And condensing these proteins or peptides in a mixed solvent as described above. Details of the condensation reaction are the same as described above.
  • the crude protein or peptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain the desired amide of the protein or peptide.
  • an ester of a protein or peptide for example, After condensing the ⁇ -hydroxyl group of an amino acid with a desired alcohol to form an amino acid ester, a desired protein or peptide ester can be obtained in the same manner as in the case of a protein or peptide amide.
  • the partial peptide used in the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein used in the present invention with an appropriate peptidase.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, a partial peptide or an amino acid capable of constituting the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting peptide is removed to produce the desired peptide. can do.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • the partial peptide used in the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, 'liquid chromatography', and recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, when the partial peptide is obtained by a salt, the known method or Can be converted into a free form or another salt by a method according to the above.
  • the polynucleotide encoding the protein used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the protein used in the present invention.
  • it is DNA.
  • the DNA include genomic DNA, genomic DNA library, the above-described cell-tissue-derived cDNA, Any of the above-described cell / tissue-derived cDNA library and synthetic DNA may be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • reverse RNA was directly prepared using a preparation of total RNA or mRNA fraction from the above-mentioned cell 'tissue.
  • RT-PCR method transcriptase polymerase chain reaction
  • Examples of the DNA encoding the protein used in the present invention include a DNA containing the base sequence represented by SEQ ID NO: 2 or the base sequence represented by SEQ ID NO: 2 and high stringency. Any DNA that contains a nucleotide sequence that hybridizes under the following conditions and encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 described above. It may be something. Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 under conditions of eight istrines include, for example, about 50% or more, preferably about 6% with the nucleotide sequence represented by SEQ ID NO: 2. 0% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more. DNA or the like is used.
  • Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Saibrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.
  • High stringent end conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70: preferably about 60 to 6
  • the conditions at 5 ° C are shown.
  • the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
  • DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1 a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.
  • the polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention.
  • any of a genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA may be used.
  • Examples of the DNA encoding the partial peptide used in the present invention include, for example, a DNA containing a part of a DNA containing the base sequence represented by SEQ ID NO: 2, or a base represented by SEQ ID NO: 2.
  • a DNA containing a base sequence that hybridizes with the sequence under high stringent conditions, and a DNA containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention is used.
  • the DNA that can be hybridized with the base sequence represented by SEQ ID NO: 2 has the same meaning as described above.
  • D which completely encodes the protein and partial peptide used in the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of cloning and expression of the DNA encoding them).
  • DNA amplified by a PCR method using a synthetic DNA primer containing a part of the nucleotide sequence encoding the protein of the present invention, or a DNA incorporated into an appropriate vector is used as the DNA of the present invention.
  • the method of hybridization is, for example,
  • the DNA base sequence can be converted using the ODA-LA PCR method using PCR, a known kit, for example, Mutan TM -super Express Km (Takara Shuzo), Mutan TM- K (Takara Shuzo), or the like. , Gapped duplex method, Kunkel method and the like, or a method analogous thereto.
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by
  • the vector examples include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast (eg, p SH19, pSH15), pacteriophage such as phage ⁇ , animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, pXTl, pRc / CMV, pRcZRSV, pcDNA I / N eo or the like is used.
  • Escherichia coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast eg, p SH19, pSH
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRo when an animal cell is used as a host, SRo; Promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • CMV cytomegalovirus
  • trp promoter one lac promoter, re cA promoter, AP L promoter - ter, 1
  • rho P promoter one such as T7 promoter
  • the host is a strain of the genus Bacillus, SP01 promoter
  • yeast such as overnight, SPO2 promoter, penP promoter, and the like
  • PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, and the like are preferable.
  • the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection promoter, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired. Can be used.
  • Selection methods include, for example, a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter abbreviated as Amp 1 ) If there is), the neomycin resistance gene (hereinafter, Ne o sometimes abbreviated as r, G418 resistance), etc. in the like.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
  • the PhoA 'signal sequence and the OmpA * signal sequence are used.
  • the host is a Bacillus genus
  • the ⁇ -amylase signal sequence and subtilisin signal sequence are used.
  • Signal sequence, SUC2 signal sequence, etc. If the host is an animal cell, use insulin 'signal sequence, Hi-I interferon signal sequence, antibody molecule, signal sequence, etc. it can.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia include, for example, Escherichia coli.
  • Bacillus bacteria examples include, for example, Bacillus' subtilis (Bacillus).
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD—5D, 20B—12, Schizosaccharomyces pombe NC YC 1913, NCYC2036, and Pichia pastoris (Pichia pastor is) K M71 or the like is used.
  • insect cells for example, when the virus is Ac NPV, the cell line derived from the larvae of the night moth (Spodoptera frugiperda cell; S ; cell), the MG1 cell derived from the midgut of Trichoplusia ni, High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • S sleep moth
  • MG1 cell derived from the midgut of Trichoplusia ni
  • High Five TM cells cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • BmNPV a cell line derived from silkworm (Bombyx mori N cell; BmN cell) is used.
  • Sf9 cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J.L., et al., In Vivo, 13, 213-217, (1977)) and the like are used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].
  • animal cells for example, monkey cell COS-7, Vero, Chinese cell, Muster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chinini-zhamster cell CHO (hereinafter, CHO (dh fr)) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
  • the yeast can be transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). Can be. Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nitrogen sources inorganic substances and others.
  • carbon sources include glucose, dextrin, soluble starch, and sucrose.
  • nitrogen sources include ammonium salts, nitrates, corn chip liquor, peptone, potato zein, meat extract, soybean meal, and potato extract.
  • the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • an M9 medium containing glucose and casamino acids is preferable.
  • a drug such as 3 / 3-indolylacrylic acid can be added to make the promoter work efficiently if necessary.
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration or stirring may be added.
  • the cultivation is usually performed at about 30 to 40 for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
  • the culture medium When culturing an insect cell or a transformant in which the host is an insect, the culture medium is supplemented with Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)). Those appropriately added are used.
  • the pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)]
  • a DMEM medium [Virology , Vol. 8, 396 (1959)]
  • RPMI 1640 medium [The] journal of the American Medical Association 199, 519 (1967)]
  • 199 medium for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , Vol. 8, 396 (1959)], RPMI 1640 medium [The] journal of the American Medical Association 199, 519 (1967)], 199 medium
  • the pH is about 6-8.
  • the cultivation is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • the cells or cells When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and Z or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is appropriately used.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-1100 TM.
  • Purification of the protein contained in the culture supernatant or extract obtained in this manner is a separation known per se. Purification methods can be combined appropriately. As these known separation and purification methods, solubility such as salting-out and solvent precipitation is used. PT / JP2003 / 007926
  • the protein thus obtained when the protein thus obtained is obtained as a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein is obtained as a salt, a method known per se or It can be converted to a free form or other salts by a method analogous thereto.
  • the protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein modifying enzyme before or after purification.
  • an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the presence of the protein of the present invention thus produced can be measured by, for example, enzymatic immunoassay western blotting using a specific antibody.
  • the antibody against the protein or partial peptide or a salt thereof used in the present invention is any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. You may.
  • An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes simply referred to as the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, It can be produced according to a known antibody or antiserum production method.
  • the protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site capable of producing an antibody upon administration.
  • Antibody production ability upon administration TJP2003 / 007926
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be given to increase the level.
  • Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times.
  • warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.
  • a warm-blooded animal immunized with an antigen for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them.
  • the hybridoma producing the monoclonal antibody can be prepared by fusing the animal-producing cells obtained with myeloma cells of the same or different species.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • myeloma cells examples include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is used at a concentration of about 10 to 80%.
  • the cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C., preferably 30 to 37, for 1 to 10 minutes.
  • a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier.
  • a solid phase eg, a microplate
  • an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse) or protein A
  • a monoclonal antibody bound to the solid phase A monoclonal antibody bound to a solid phase is prepared by adding a hybridoma supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, adding a protein labeled with a radioactive substance, an enzyme, or the like. And a method for detecting antibody.
  • the selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as the hybridoma can grow.
  • RPMI 164 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd. ))
  • serum-free medium for hybridoma culture SFM-101, Nissui Pharmaceutical Co., Ltd.
  • the culture temperature is usually from 20 to 40 ° C, preferably about 37.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. Cultivation can usually be performed under 5% CO2.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption and desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) Can do it.
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption and desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) Can do it.
  • the polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting the antibody-containing substance of the antibody and separating and purifying the antibody.
  • the type of carrier-protein and the mixing ratio of carrier and hapten depend on the efficiency of antibody against hapten immunized by cross-linking with carrier.
  • any substance may be cross-linked at any ratio.
  • serum serum albumin, psiloculopurine, hemocyanin, etc. may be used in a weight ratio of about 0.1 to 2 with respect to 1 hapten.
  • a method of coupling at a rate of 0, preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant ⁇ Incomplete Freund's adjuvant may be administered to enhance body production during administration. The administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.
  • Polynucleotide encoding the protein or partial peptide used in the present invention eg, DNA (hereinafter, in the description of antisense polynucleotides, these DNAs may be abbreviated as the DNA of the present invention).
  • the nucleotide sequence of the polynucleotide of the present invention eg, DNA
  • Any antisense polynucleotide may be used as long as it contains a basic or substantially complementary base sequence or a part thereof and has an action capable of suppressing the expression of the DNA. Antisense DNA is preferred.
  • the nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). And about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more.
  • the antisense polynucleotide in the case of the antisense polynucleotide thus obtained, it is at least about 70%, preferably about 80%, complementary to the complementary sequence of the nucleotide sequence (for example, the nucleotide sequence near the start codon) of the N-terminal site of the protein of the present invention. % Or more, more preferably about 90% or more, and most preferably about 95% or more of the antisense polynucleotide which is directed to RNA degradation by RNase H.
  • an antisense polynucleotide containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or a part thereof Preferably, for example, an antisense polynucleotide containing a nucleotide sequence complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or a part thereof (more preferably, a nucleotide sequence represented by SEQ ID NO: 2 A base sequence complementary to the base sequence of DNA containing the base sequence to be prepared, or an antisense polynucleotide containing a part thereof.
  • An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphorus such as phospho-thiolate, methylphosphonate, and phosphorodithionate. It may be substituted by an acid residue.
  • the sugar (deoxyliposome) of each nucleotide may be substituted with a chemically modified sugar structure such as 2,1-methylation, and the base portion (pyrimidine, purine) may also be chemically modified. Any one may be used as long as it hybridizes to DNA containing the nucleotide sequence represented by SEQ ID NO: 2.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • an antisense polynucleotide capable of inhibiting the replication or expression of the protein gene of the present invention has been cloned or T JP2003 / 007926
  • nucleotide 29 It can be designed and synthesized based on the nucleotide sequence information of the DNA encoding the determined protein.
  • a polynucleotide nucleic acid
  • Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention, and polynucleotides capable of specifically hybridizing with the protein-related RNA of the present invention can be used in vivo and in vitro.
  • corresponding means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene.
  • corresponding means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene.
  • correspondence between a nucleotide, base sequence or nucleic acid and a peptide (protein) is a nucleotide
  • the palindrome region at the 'end and the hairpin loop at the 3' end can be selected as preferred regions of interest, but any region within the protein gene can be selected as the region of interest.
  • the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region is as follows: The relationship between the target nucleic acid and the polynucleotide capable of hybridizing with the target is
  • Antisense polynucleotides are polynucleotides containing 2-dexoxy D-ribose, polynucleotides containing D-reports, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or non-polynucleotides.
  • Other polymers having a nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers containing special bonds provided that such polymers are found in DNA and RNA
  • Base pairing including nucleotides having a configuration that allows base attachment
  • Bridges and may also be unmodified polynucleotides (or unmodified oligonucleotides), or even those with known modifications, such as those with a label known in the art, capped, Methylated, one or more natural nucleotides replaced by analogs, modified intramolecular nucleotides, e.g., uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, Those with a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), eg, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signals) Peptides, poly-L-lysine, etc.) and sugars (eg, Compounds with side groups such as monosaccharide, etc.), capped, Methylated, one or more natural nucleotides replaced by analogs, modified intramolecular nucleotides,
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or ethers, It may be converted to a functional group such as amine.
  • the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides.
  • the antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Sense nucleic acid toxicity Make it smaller.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes and increase nucleic acid uptake (eg, , Phospholipids, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond.
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids, which prevent degradation by nucleases such as exonucleases and RNAses.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention.
  • the nucleic acid can be applied to cells by various methods known per se.
  • the protein or partial peptide of the present invention or a salt thereof hereinafter sometimes abbreviated as the protein of the present invention
  • the DNA encoding the protein or partial peptide of the present invention hereinafter referred to as the D NA
  • an antibody against the protein or partial peptide of the present invention or a salt thereof hereinafter referred to as “the present invention”.
  • an antibody sometimes abbreviated as an antibody
  • the antisense polynucleotide of the DNA of the present invention hereinafter sometimes abbreviated as the antisense polynucleotide of the present invention
  • a medicament containing an antisense polynucleotide of the gene encoding the protein of the present invention, a compound that inhibits the activity of the protein of the present invention or a salt thereof, or an antibody against the protein of the present invention may be, for example, a large intestine.
  • Cancer breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid gland It can be used as a prophylactic / therapeutic agent for cancer such as cancer, brain tumor or blood tumor.
  • compounds or salts thereof that modulate (preferably inhibit) the activity of the protein of the present invention include, for example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, and biliary tract.
  • a prophylactic and therapeutic agent for cancer such as cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor, or blood tumor can do.
  • it is a prophylactic / therapeutic agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen, etc.)-Independent, HER2-negative, etc.
  • the compound or a salt thereof that inhibits the activity of the protein of the present invention can also be used, for example, as an apoptosis action regulator (preferably an apoptosis action promoter).
  • the protein of the present invention is useful as a reagent for screening a compound or its salt that regulates the activity of the protein of the present invention.
  • the present invention provides a method for screening a compound or a salt thereof that regulates the activity (eg, ligand binding activity, signal transduction activity, etc.) of the protein of the present invention, characterized by using the protein of the present invention. More specifically, for example, (i) the ligand binding activity or signal transduction activity of a cell capable of producing the protein of the present invention; A method for screening a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention, which is characterized by comparing the ligand binding activity or the signal transmission activity of the mixture, is used.
  • the ligand binding activity or signal transduction activity can be determined by a method known per se, for example, J. Immunol. 166 (1), pp. 15-19, 2001 Measure and compare according to the method described in or above.
  • the protein expression vector of the present invention is prepared by adding an NF ⁇ B binding sequence to a promoter or enhancer. And (ii) a reporter gene containing the protein expression vector of the present invention in the promoter or enhancer of the NF ⁇ B binding sequence.
  • the amount of reporter expression is compared. The repo overnight expression level can be measured by using a reporter protein activity using luciferase activity, alkaline phosphatase activity or the like as an index.
  • the protein expression vector of the present invention may be used to bind the NF ⁇ B binding sequence to a promoter or enhancer. Transfection into cells with the reporter gene contained therein. If necessary, (a) a solution containing ligands such as microbial cell disruption solution, microbial culture supernatant, eukaryotic cell disruption solution, and eukaryotic cell culture supernatant (B) culturing with the addition of a substance having the same binding activity as the ligand itself or (c) a natural ligand; and (ii) using the protein expression vector of the present invention to promote the binding sequence of NFKB.
  • ligands such as microbial cell disruption solution, microbial culture supernatant, eukaryotic cell disruption solution, and eukaryotic cell culture supernatant
  • Transfected cells or microbial cell culture supernatant, eukaryotic cell lysate, eukaryotic cells Youe liquid containing the ligand, such as supernatants, by adding a substance having a (b) a ligand itself or (c) a natural ligand comparable binding activity, trial Measure and compare reporter expression levels when cultured in the presence of the test compound.
  • the expression level of the repo overnight can be measured by using the repoprotein activity using luciferase activity, alkaline phosphatase activity and the like as an index.
  • a host transformed with a vector containing a DNA encoding the protein of the present invention described above is used.
  • a host for example, animal cells such as COS 7 cells, CHO cells, and HEK293 cells are preferably used.
  • a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used.
  • the method for culturing cells capable of expressing the protein of the present invention is the same as the above-described method for culturing the transformant of the present invention.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • the ligand binding activity or signal transduction activity in the case of (ii) is increased by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of (i).
  • the test compound to be used is a compound that promotes the activity of the protein of the present invention, wherein the ligand binding activity or signal transduction activity in the case (ii) above is about 20% or more, preferably A test compound that reduces the activity of the protein of the present invention by 30% or more, more preferably about 50% or more, can be selected as the compound that inhibits the activity.
  • the compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low-toxic drug for enhancing the action of the protein of the present invention.
  • the compound having an activity of inhibiting the activity of the protein of the present invention is used as a safe and low-toxicity drug for suppressing the physiological activity of the protein of the present invention, for example, a prophylactic / therapeutic agent for cancer, an apoptosis promoting agent and the like. Useful.
  • Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts And compounds selected from plasma and the like.
  • the salt of the compound includes the peptide of the present invention described above. The same ones as used in the above are used.
  • compounds or salts thereof that regulate (preferably inhibit) the expression of the gene encoding the protein of the present invention include, for example, breast cancer and colon Cancer, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid It can be used as a prophylactic and therapeutic agent for cancer such as cancer, kidney cancer, brain tumor and blood tumor.
  • it is a prophylactic / therapeutic agent for cancers (eg, breast cancer, etc.) that are hormone-independent (eg, estrogen, etc.)-Independent, HER2-negative, etc.
  • the compound that inhibits the expression of the gene encoding the protein of the present invention or a salt thereof can be used, for example, as an apoptosis-controlling agent (preferably, an apoptosis-promoting agent).
  • the DNA of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the expression of a gene encoding the protein of the present invention.
  • the screening method includes (iii) culturing cells capable of producing the protein of the present invention, and (iv) culturing cells capable of producing the protein used in the present invention in the presence of the test compound. There is a screening method which is characterized by comparing with the case.
  • the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases (ii) and (iv) is measured and compared.
  • test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.
  • the amount of the protein is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, and measuring the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. can do.
  • the mRNA amount can be measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a probe, or
  • the gene expression in the case of the above (iv) is about 20% or more, preferably 30% or more, as compared with the case of the above (iii).
  • Test conjugates that inhibit at least about 50%, more preferably at least about 50%, can be selected as compounds that inhibit the expression of the gene encoding the protein of the present invention.
  • the screening kit of the present invention contains cells capable of producing the protein or partial peptide used in the present invention or a salt thereof, or the protein or partial peptide used in the present invention.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a test compound as described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract.
  • salt of the compound those similar to the aforementioned salts of the protein of the present invention are used.
  • a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention and a compound or a salt thereof that regulates (preferably inhibits) the expression of a gene encoding the protein of the present invention are, for example, colon cancer, Breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, It is useful as a prophylactic / therapeutic agent for cancer, such as ligament cancer, brain tumor or hematological tumor, and as an apoptosis promoter.
  • they are agents for preventing or treating cancers (eg, breast cancer, etc.) that are hormone-independent (eg, estrogen, etc.)-Independent, HER2-negative, etc., and for promoting apoptosis.
  • cancers eg, breast cancer, etc.
  • hormone-independent eg, estrogen, etc.
  • HER2-negative e.g.
  • apoptosis e.g., HER2-negative, etc.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). ), Syrups, emulsions, suspensions and the like.
  • Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and tablets for tablets.
  • compositions for parenteral administration for example, injections, suppositories, etc. are used. Injections are intravenous, subcutaneous, intradermal, intramuscular, intravenous, intraarticular. And dosage forms such as agents. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol ( For example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (po lyoxye thy 1 ene (50mo 1) adduc t of
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • a suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a conventional suppository base.
  • the above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form adapted to the dose of the active ingredient.
  • dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories, and usually 5 to 50 mg / dosage per dosage unit dosage form.
  • the injection contains 5 to 100 mg of the above-mentioned antibody, and other dosage forms contain 10 to 25 mg of the above-mentioned antibody.
  • compositions may cause an undesired interaction due to the combination with the above antibody.
  • other active ingredients may be contained.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (eg mice, rats, puppies, sheep, bush, pussi, puma, birds, cats, dogs, Monkeys, chimpanzees, etc.) orally or parenterally.
  • warm-blooded animals eg mice, rats, puppies, sheep, bush, pussi, puma, birds, cats, dogs, Monkeys, chimpanzees, etc.
  • the dose of the compound or a salt thereof varies depending on its action, target disease, subject to be administered, route of administration, and the like.
  • a compound or a salt thereof that regulates the activity of the protein of the present invention for the purpose of treating breast cancer When the compound is orally administered, generally in an adult (assuming a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1 to 10 Omg per day.
  • the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like, but, for example, regulates the activity of the protein of the present invention for the purpose of treating breast cancer.
  • a compound or a salt thereof is administered to an adult (with a body weight of 6 O kg) usually in the form of an injection, the compound or a salt thereof is about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg per day. More preferably, about 0.1 to 1 Omg is administered by injection to the cancerous lesion. In the case of other animals, the dose can be administered in terms of the weight per 6 O kg.
  • an antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. In particular, it can be used for quantification by sandwich immunoassay.
  • the present invention provides a method for quantifying the protein of the present invention in a test wave, characterized by measuring In the quantification method (ii) above, it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention. .
  • the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
  • the antibody molecule itself may be used, or the F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of protein) in the test solution. Any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and this is calculated from a standard curve prepared using a standard solution containing a known amount of antigen. .
  • nephelometry, competition method, immunometric method and sandwich method are preferably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [125 iota], [131 1], [3 ⁇ 4], and [14 c] used.
  • the above enzyme a stable enzyme having a large specific activity is preferable.
  • 3-galactosidase,) 3-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • Carriers such as agarose, dextran, and cellulose Examples include soluble polysaccharides, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction another labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is an antibody having different binding sites to the protein of the present invention. Is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the San Deutsch method, for example, a competition method, an immunometric method, or a nephrometry.
  • a competition method the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) and the recognized antigen (B) bound to the antibody are separated.
  • F labeled antigen
  • B recognized antigen
  • BZF separation Measure the amount of labeling of either B or F, and quantify the amount of Bobara in the test wave.
  • a soluble antibody was used as the antibody
  • B / F separation was performed using polyethylene glycol
  • a liquid phase method using a second antibody against the antibody was used as the first antibody.
  • a solid phase method using a soluble first antibody and a solid phase antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. Reaction with an excess amount of the labeled antibody, and then adding immobilized antigen to the unreacted labeled antibody. After binding the body to the solid phase, the solid and liquid phases are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.
  • the amount of insoluble sediment resulting from the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser-nephrometry utilizing laser scattering is preferably used.
  • the protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, documents, etc.
  • the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
  • an increase or decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It can be diagnosed as having cancer, such as kidney cancer, brain tumor or hematological tumor, or is more likely to be affected in the future.
  • the antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or a tissue.
  • a subject such as a body fluid or a tissue.
  • the detection of the protein of the present invention in each fraction during purification, and the analysis of the behavior of the protein of the present invention in test cells, etc. Can be used.
  • the DNA of the present invention can be used, for example, as a probe to produce human or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein of the present invention or its partial peptide in monkeys, chimpanzees, etc.).
  • human or warm-blooded animals e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein of the present invention or its partial peptide in monkeys, chimpanzees, etc.
  • it is useful as a diagnostic agent for genes such as decreased expression, increased DNA or mRNA or excessive expression.
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, the known Northern Hybridization ⁇ PCR-SSCP method (Genomics, Vol. 5, pp. 874-879, Q989), Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)).
  • PCR-SSCP method for example, colon cancer, breast cancer, lung cancer, and prostate Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, visceral cancer, It can be diagnosed as having a high possibility of cancer such as a brain tumor or a blood tumor.
  • the antisense polynucleotide of the present invention which can complementarily bind to the DNA of the present invention and suppress the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo.
  • ligand binding activity or signal transduction activity such as colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, and spleen
  • cancer such as cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor .
  • the antisense polynucleotide of the present invention can be used, for example, as an apoptotic action regulator (preferably an apoptotic action promoter).
  • the antisense polynucleotide When used as the above-mentioned prophylactic / therapeutic agent or modulator, it can be formulated and administered according to a method known per se.
  • the above-mentioned antisense polynucleotide is inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus association virus vector, etc. It can be administered orally or parenterally to mammals (eg, rats, egrets, sheep, sheep, bush, birds, cats, dogs, monkeys, etc.).
  • the antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be made into an aerosol and administered topically into the trachea as an inhalant.
  • the above-mentioned antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and is intravenously or subcutaneously. Etc. may be administered.
  • the dose of the antisense polynucleotide varies depending on the disease to be treated, the subject of administration, the route of administration, and the like.For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating breast cancer, the dose is generally used for adults. (Weight 60 kg), the antisense polynucleotide is administered in an amount of about 0.1 to 10 O mg per day. 03 007926
  • the antisense polynucleotide can be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.
  • RNA containing a part of the RNA encoding the protein of the present invention a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a lipozyme containing a part of the RNA encoding the protein of the present invention, etc.
  • the expression of the gene of the present invention can be suppressed and the function of the protein used in the present invention or the DNA used in the present invention in vivo can be suppressed, for example, for example, colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, thyroid cancer Prevention and treatment of cancer such as cancer, brain tumor or blood tumor, preferably prevention of cancer (eg, breast cancer, etc.) independent of hormones (eg, estrogen), HER2 negative, etc.Furthermore, it can be used as an apoptosis action regulator or the like.
  • the double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
  • the lipozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of RNA encoding the protein of the present invention.
  • a part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known lipozyme.
  • RNA or lipozyme When used as a therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. (5) a drug containing the antibody of the present invention
  • the antibody of the present invention which has the activity of neutralizing the activity of the protein of the present invention, includes, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer It can be used as a prophylactic or therapeutic agent for cancers such as renal, bladder, uterine, ovarian, testicular, thyroid, knee, brain or blood tumors it can. Preferably, it is a prophylactic / therapeutic agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen, etc.)-Independent, HER2-expressing negative, etc. Further, the antibody of the present invention can also be used, for example, as an apoptosis action regulator (preferably, an apoptosis action promoter).
  • an apoptosis action regulator preferably, an apoptosis action promoter
  • the prophylactic or therapeutic agent for the above-mentioned diseases which comprises the antibody of the present invention, has low toxicity, and is used as it is as a liquid or as a pharmaceutical composition in an appropriate dosage form, in humans or mammals (eg, rat, rabbit, etc.). It can be given orally or parenterally (eg, intravenously, subcutaneously, etc.) to cattle, higgins, bushes, puppies, cats, dogs, monkeys, etc.).
  • the antibody of the present invention may be administered as it is, or may be administered as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the antibody of the present invention or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
  • compositions for parenteral administration include injections, suppositories, etc., and injections include intravenous, subcutaneous, intradermal, intramuscular, and intravenous injections. Shapes may be included.
  • Such an injection can be prepared according to a known method. Injections can be prepared, for example, by dissolving, suspending or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid commonly used for injections.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable solubilizing agents, for example, alcohol (e.g., ethanol), polyalcohol (e.g., , Propylene glycol, polyethylene glycol), nonionic surfactants [eg, Polysorbate 80, HC0-50
  • a suppository for rectal administration may be prepared by mixing the antibody or a salt thereof with a usual suppository base.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation.
  • carriers and excipients for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient.
  • Such dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories.
  • the content of the antibody is preferably 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injection, and 10 to 25 Omg for other dosage forms.
  • the dosage of the prophylactic / therapeutic or diagnostic agent (pharmaceutical) containing the antibody of the present invention varies depending on the administration target, target disease, symptoms, administration route, and the like.
  • the antibody of the present invention is used in a single dose, usually about 0.01 to 2 OmgZkg body weight, preferably about 0 :! to about 1 OmgZkg body weight, and more preferably about 0.1 to 5 mgZkg body weight.
  • Is administered by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.
  • the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • a composition is provided as a dosage form suitable for oral or parenteral administration (eg, intravascular injection, subcutaneous injection, etc.).
  • compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • the “compound having an activity of regulating (preferably inhibiting) the activity of RI PK2” may be any compound having an activity of regulating (preferably inhibiting) the activity of RIPK2, such as colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, hepatic cancer It is used as a prophylactic / therapeutic agent for cancer such as cancer, brain tumor, or blood tumor.
  • Any compound can be used as long as it has a K2 expression regulating (preferably inhibiting) action, such as colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, Used as a prophylactic and therapeutic agent for cancer such as spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor, or blood tumor Can be
  • the prophylactic / therapeutic agent is produced in the same manner as described above.
  • the present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (may be abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the formula:
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, spermatozoa, For germ cells and the like including progenitor cells, calcium phosphate is preferably used at the stage of embryonic development in non-human mammal development (more preferably, at the stage of single cells or fertilized egg cells and generally before the 8-cell stage). It can be produced by transferring the desired DNA by the method, electric pulse method, lipofection method, coagulation method, microinjection method, particle gun method, DEAE-dextran method, etc.
  • the exogenous DNA of the present invention intended for somatic cells, organs of living organisms, tissue cells, and the like can be transferred and used for cell culture, tissue culture, and the like.
  • non-human mammals for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, eight-muster, ma., Males, rats and the like are used.
  • mice for example, C57B LZ6 strains, DBA2 strains as pure strains, etc.
  • a cross line a B6C3F line, a BDFi line, a B6D2F line, a BALBZc line, an ICR line, etc., or a rat (for example, Wistar, SD, etc.) are preferable.
  • mammal in the recombinant vector that can be expressed in mammals, human and the like can be mentioned in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by a non-human mammal, but to the DNA of the present invention once isolated and extracted from a mammal.
  • mutant DNA of the present invention those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, substitution with another base, etc. DNA that has been used is used, and also includes abnormal DNA.
  • a mutation for example, mutation
  • the abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
  • the exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the target animal.
  • a DNA linked to a downstream of a promoter capable of expressing the DNA in animal cells is used. It is generally advantageous to use it as an A construct.
  • various mammals having the DNA of the present invention having a high homology to the human DNA eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.
  • a target mammal for example, a mouse fertilized egg
  • various promoters capable of expressing the derived DNA it is possible to create a DNA transgenic mammal that highly expresses the DNA of the present invention.
  • Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as ⁇ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or a baculovirus.
  • animal viruses such as E. coli are used.
  • a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • promoters that regulate DNA expression include: (1) a promoter of DNA derived from a virus (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.). Yuichi, 2 Promoters derived from various mammals (humans, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), for example, albumin, insulin II, ⁇ roplakin II, Erasuyuze, erythropoietin, Endothelin, muscle creatine kinase, glial fibrillary acidic protein, daryuthion S-transferase, platelet-derived growth factor / 3, keratin Kl, 1 ⁇ 10 and 14, collagen I and II , Cyclic AMP-dependent protein kinase / 3 I-subunit, dystrophin, Tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor
  • Myosin light chains 1 and 2 myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle ⁇ -actin, prebub enkephalin ⁇ , A promoter such as vasopressin is used.
  • cytomegalovirus promoter which can be highly expressed in the whole body, human peptide chain elongation factor 1 (EF-1) promoter, human and chicken j3 actin promoter, and the like are preferable.
  • the vector preferably has a sequence (generally called terminator) that terminates transcription of the target messenger RNA in the DNA-transferred mammal.
  • terminator a sequence that terminates transcription of the target messenger RNA in the DNA-transferred mammal.
  • it is derived from viruses and various mammals.
  • the sequence of each DNA can be used, and preferably, SV40 terminator of Simian virus or the like is used.
  • the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are used to further express the target exogenous DNA at 5 'upstream of the promoter region, the promoter region and the translation region. It is also possible to ligate between them or at the 3 'downstream of the translation region.
  • the normal translation region of the protein of the present invention is derived from a liver, a kidney, a thyroid cell, a fibroblast derived from a human or various mammals (for example,- ⁇ egret, dog, cat, guinea pig, hamster, rat, mouse, etc.). All or part of genomic DNA from DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cell, and fibroblast-derived RNA can be used as raw materials. I can do it.
  • a foreign abnormal DNA can produce a translation region obtained by mutating a normal protein translation region obtained from the above cells or tissues by a point mutagenesis method.
  • the translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • Production after DNA transfer The presence of the exogenous DNA of the present invention in the germ cell of the product means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal and somatic cells .
  • the offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.
  • the non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it.
  • Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal.
  • Excessive presence of the exogenous DNA of the present invention in the germinal cells of the animal after transfer of DNA indicates that all of the offspring of the animal produced have the extraneous DNA of the present invention in all of their germinal and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has an excess of the exogenous DNA of the present invention in all of its germ cells and somatic cells.
  • the non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and finally promotes the function of endogenous normal DNA, thereby finally obtaining the protein of the present invention. May develop hyperfunction, and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention and to examine a method for treating these diseases. It is possible.
  • a prophylactic / therapeutic agent for a disease associated with the protein of the present invention Cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid gland It can also be used for screening tests for prophylactic and therapeutic agents for cancer such as cancer, kidney cancer, brain tumor, and blood tumor.
  • the non-human mammal having the foreign abnormal DNA of the present invention After confirming that sex DNA is stably maintained, the animal can be subcultured as a DNA-bearing animal in a normal breeding environment. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • the DNA construct with the promoter can be prepared by ordinary DNA engineering techniques. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal.
  • the presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germinal and somatic cells.
  • a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all offspring have the DNA.
  • the abnormal DNA of the present invention is highly expressed, and the function of the protein of the present invention is ultimately achieved by inhibiting the function of endogenous normal DNA.
  • Inactive refractory disease may occur and can be used as a model animal for the disease.
  • using the abnormal DNA-transferred animal of the present invention it is possible to elucidate the pathological mechanism of the function-inactive refractory of the protein of the present invention and to examine a method for treating this disease.
  • the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of the normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention (dominant negative activity). Action).
  • the agent for preventing or treating the protein of the present invention or a functionally inactive refractory agent for example, Colorectal, breast, lung, prostate, esophageal, stomach, liver, biliary, spleen, kidney, bladder, uterine, ovarian, testicular, It can also be used for screening tests for prophylactic and therapeutic agents for cancers such as thyroid cancer, knee cancer, brain tumor, and blood tumor.
  • ⁇ ⁇ Isolation and purification of the mutant protein of the present invention and production of its antibody can be considered. Furthermore, using the DNA-transferred animal of the present invention, it is possible to examine clinical symptoms of a disease related to the protein of the present invention, including a functionally inactive refractory disease of the protein of the present invention. More detailed pathological findings in each organ of the disease model related to the protein of the present invention can be obtained, which will contribute to the development of new treatment methods and the research and treatment of secondary diseases caused by the disease. it can.
  • each organ is removed from the DNA-transferred animal of the present invention, and after minced, it is possible to obtain free DNA-transferred cells, culture them, or systematize the cultured cells using a protease such as trypsin. It is possible.
  • the protein of the present invention can be identified, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism can be examined, and its abnormality can be examined. It is an effective research material for elucidation.
  • the DNA transgenic animal of the present invention in order to use the DNA transgenic animal of the present invention to develop a therapeutic agent for a disease associated with the protein of the present invention, including a refractory inactive type of the protein of the present invention, Using a quantitative method or the like, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease. Also, using the DNA transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a method for treating a DNA associated with the protein of the present invention.
  • the present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention.
  • the DNA is inactivated by introducing a reporter gene (eg, a / 3-galactosidase enzyme gene derived from Escherichia coli), and the repo allele gene is a promoter of the promoter of the present invention.
  • a reporter gene eg, a / 3-galactosidase enzyme gene derived from Escherichia coli
  • the repo allele gene is a promoter of the promoter of the present invention.
  • the non-human mammal according to (6) which can be expressed under control,
  • test compound is administered to the animal described in (7), and the expression of a reporter gene is detected.
  • a screening method is provided.
  • a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression ability of the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA substantially does not have the ability to express the protein of the present invention (hereinafter referred to as the knockout DNA of the present invention).
  • Non-human mammalian embryonic stem cells hereinafter abbreviated as ES cells).
  • non-human mammal the same one as described above is used.
  • the method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence or inserting or substituting another DNA by a genetic engineering technique. . These mutations can, for example, shift the reading frame of codons or disrupt the function of Promote or Exon. What is necessary is just to prepare the knockout DNA of the present invention.
  • Non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or 1acZ (j3-galactosidase gene), cat Inserting a reporter gene or the like represented by (chloramphenicylacetyltransferase gene) disrupts exon function, or terminates transcription of the gene in the intron portion between exons.
  • a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or 1acZ (j3-galactosidase gene), cat Inserting a reporter gene or the like represented by (chloramphenicylacetyltransferase gene) disrupts exon function, or terminates transcription of the gene in the intron portion between exons
  • the resulting A DNA chain having a DNA sequence constructed so as to disrupt the gene (hereinafter, abbreviated as “gathering vector 1”) is introduced into the chromosome of the animal by, for example, a homologous recombination method.
  • gathering vector 1 A DNA sequence on or near the DNA of the present invention used for Southern hybridization analysis or a targeting vector and a DNA sequence on a targeting vector other than the DNA of the present invention used for preparing a targeting vectorcan be obtained by analyzing by the PCR method using as primers and selecting the knockout ES cells of the present invention.
  • the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like for example, those already established as described above may be used.
  • BDFi mice C57BL / 6 and DBAZ2 and C57BL / 6 and DBAZ2
  • BDFi mice have the advantage of high number of eggs collected and robust eggs, and have the background of 'C57BL / 6 mice, The ES cells obtained using this were used to generate C57 It can be used to advantage in that loss can replace its genetic background with C57BL / 6 mice.
  • blastocysts 3.5 days after fertilization are generally used.However, it is more efficient to collect 8-cell embryos and culture them up to blastocysts. Many early embryos can be obtained.
  • Either male or female ES cells may be used, but male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.
  • a method for determining the sex of ES cells a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be given as an example.
  • this method conventionally, for example G-banding method, it requires about 1 0 6 cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 5 0)
  • the primary selection of ES cells at the initial stage of culture can be performed by gender discrimination, and the early selection of male cells can greatly reduce the labor required at the initial stage of culture.
  • the secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method.
  • Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but they must be carefully subcultured because they tend to lose their ability to generate individuals.
  • a suitable feeder cell such as STO fibroblasts
  • a carbon dioxide incubator preferably 5% carbon dioxide, 95% air or 5% oxygen, in the presence of LIF (1 to 10,000 U / ml)
  • Culture at about 37 ° C with 5% carbon dioxide gas and 90% air.
  • trypsin / EDTA solution usually 0.001 to 0.5% trypsin / 0.1 l
  • a 5 mM EDTA preferably, about 0.1% tris / L M EDTA
  • Such subculture is usually performed every 1 to 3 days. At this time, cells are observed, and morphologically abnormal cells are found. If so, the cultured cells should be discarded.
  • ES cells can be transformed into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. It is possible to differentiate [M.]. Evans and MH
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.
  • non-human mammal those similar to the aforementioned can be used.
  • the non-human mammal deficient in DNA expression of the present invention can be obtained, for example, by introducing the evening-getting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell and introducing the evening-getting vector into a non-human mammal.
  • the DNA of the present invention is inactivated by homologous recombination to replace the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination, thereby obtaining the DNA of the present invention. Can be knocked out.
  • Cells in which the DNA of the present invention has been knocked out can be analyzed by Southern hybridization analysis or DNA analysis using a DNA sequence on or near the DNA of the present invention as a probe. It can be determined by analysis by PCR using, as a primer, the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse used for the targeting vector.
  • the cell line in which the DNA of the present invention has been inactivated is cloned by homologous gene recombination, and the cell line is cloned at an appropriate time, for example, at the 8-cell stage.
  • the chimeric embryo is injected into a human mammalian embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal.
  • the resulting animal will be artificially altered from cells having a normal DNA locus of the present invention. It is a chimeric animal composed of both cells having different DNA loci of the present invention.
  • all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual.
  • the individuals obtained in this manner are usually individuals deficient in the hetero-expression of the protein of the present invention, and mated with individuals deficient in the hetero-expression of the protein of the present invention.
  • a homozygous deficient individual can be obtained.
  • a transgenic non-human mammal having a chromosome into which a gettering vector has been introduced can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method. Compared to a transgenic non-human mammal, it can be obtained by selecting a gene having a mutation in the DNA locus of the present invention by homologous recombination.
  • the animal individuals obtained by mating should also be confirmed to be knocked out of the DNA, and reared in a normal breeding environment. Can be.
  • the germline can be obtained and maintained according to a standard method. That is, by mating male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained.
  • the obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and plural homozygotes are obtained.
  • homozygous and heterozygous animals having the inactivated DNA are bred and passaged.
  • the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.
  • the non-human mammal deficient in expression of the DNA of the present invention lacks various biological activities that can be induced by the protein of the present invention, a disease caused by inactivation of the biological activity of the protein of the present invention. Since it can be a model, it is useful for investigating the causes of these diseases and examining treatment methods.
  • the non-human mammal deficient in expression of the DNA of the present invention can be used for screening for a compound having a therapeutic / preventive effect against diseases caused by the deficiency or damage of the DNA of the present invention.
  • the present invention comprises administering a test compound to a non-human mammal deficient in expression of the DNA of the present invention, and observing and measuring changes in the animal.
  • a test compound to a non-human mammal deficient in expression of the DNA of the present invention, and observing and measuring changes in the animal.
  • the non-human mammal deficient in DNA expression of the present invention used in the screening method includes the same ones as described above.
  • Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound.
  • a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in the organs, tissues, disease symptoms, etc. of the animal are used as indicators.
  • the test compound can be tested for its therapeutic and prophylactic effects.
  • test compound for example, oral administration, intravenous injection and the like are used, and it can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like.
  • the dose of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
  • testis For example, colon, breast, lung, prostate, esophagus, stomach, liver, biliary tract, spleen, kidney, bladder, uterus, ovary, testis
  • a test compound is administered to a non-human mammal deficient in DNA expression of the present invention. Then, the difference in the degree of onset of cancer and the degree of cure of cancer in the non-administered test compound group are observed over time in the above tissues.
  • test compound when administered to a test animal, When the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound has a therapeutic / preventive effect on the above-mentioned diseases. It can be selected as a compound.
  • the compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect on a disease caused by a deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic or therapeutic agent. Further, a compound derived from the compound obtained by the above screening can also be used.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkalis). Salts with metals and the like are used, and especially preferred are physiologically acceptable acid addition salts.
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid
  • citric acid malic acid
  • succinic acid benzoic acid
  • methanesulfonic acid benzenesulfonic acid, etc.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the drug containing the protein of the present invention described above.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs). , Monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.
  • the compound when the compound is orally administered, generally, breast cancer of an adult (assuming a body weight of 60 kg) is used.
  • the single dose of the compound varies depending on the administration subject, the target disease and the like.
  • the compound is usually in the form of an injection and usually used for adult (with a body weight of 6 O kg) breast cancer.
  • the compound When administered to a patient, the compound is administered in a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 mg. It is convenient to administer about 1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.
  • the present invention provides a test compound administered to a non-human mammal deficient in expression of a DNA of the present invention, and detects the expression of a reporter gene.
  • the non-human mammal deficient in expression of DNA of the present invention may be the non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactive by introducing a reporter gene.
  • a gene which can be expressed under the control of a motor for the DNA of the present invention is used.
  • test compound examples include the same compounds as described above.
  • the same gene as described above is used, and a / 3-galactosidase gene (1 acZ), a soluble alkaline phosphatase gene or a luciferase gene is preferable.
  • the repo overnight gene is under the control of the promoter for the DNA of the present invention.
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with datalaldehyde or the like, and phosphate buffered physiological After washing with saline solution (PBS), react with a staining solution containing X-ga 1 at room temperature or around 37 ° C for about 30 minutes to 1 hour, and then tissue samples are washed with ImM ED TAZ PBS solution. By washing, the jS-galactosidase reaction is stopped, and coloration may be observed.
  • mRNA encoding 1 ac Z may be detected according to a conventional method.
  • the compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the DNA promoter activity of the present invention.
  • the compound obtained by the screening method may form a salt
  • the salt of the compound may be a physiologically acceptable acid (eg, an inorganic acid) or a base (eg, an alkali metal). And the like, and particularly preferably a physiologically acceptable acid addition salt.
  • examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, male
  • the compound of the present invention which promotes or inhibits the activity of the promoter against DNA, or a salt thereof can regulate the expression of the protein of the present invention and regulate the function of the protein, such as colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, knee It is useful as a prophylactic and / or therapeutic agent for cancer such as cancer, brain tumor, or blood tumor.
  • a compound derived from the compound obtained by the above screening can also be used.
  • a drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs). , Monkeys, etc.).
  • the dose of the compound or a salt thereof varies depending on the target disease, the subject of the administration, the administration route, and the like.For example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally the adult For a breast cancer patient weighing 60 kg, the compound is administered at about 0.1-100 mg, preferably about 1.0-5 Omg, more preferably about 1.0-2 Omg per day.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • a compound that inhibits the promoter activity of DNA of the present invention is usually administered in the form of an injection to an adult.
  • the compound When administered to a breast cancer patient weighing 6 O kg, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.:! To 20 mg, more preferably about 0.1 to 1 mg per day.
  • Omg is conveniently administered by intravenous injection. In the case of other animals, the dose can be administered in terms of the weight per 6 O kg.
  • the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention. It can greatly contribute to investigating or preventing the causes of various diseases caused by insufficient expression of DNA.
  • genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene). Creating an introduced animal) will allow the specific synthesis of the protein and the study of its effects in living organisms. Furthermore, by binding an appropriate repo overnight gene to a portion of the above promoter and establishing a cell line in which this is expressed, the ability of the protein of the present invention itself to be produced in the body can be specifically promoted or suppressed. It can be used as a search system for low molecular compounds that have an action.
  • bases, amino acids, and the like are indicated by abbreviations based on the abbreviations of the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
  • optical isomer for an amino acid the L-form is indicated unless otherwise specified.
  • DNA Deoxylipo nucleic acid cDNA complementary deoxyliponucleic acid A adenine
  • Tr p Tribute fan
  • Trt Trityl
  • 1 shows the nucleotide sequence of DNA containing the full length gene encoding RIPK2.
  • Example 3 shows the nucleotide sequence of the oligonucleotide used in Example 2.
  • Example 3 shows the nucleotide sequence of a primer used in Example 2.
  • Example 3 shows the nucleotide sequence of a primer used in Example 2.
  • Example 1 shows the nucleotide sequence of a probe used in Example 2.
  • Example 2 shows the nucleotide sequence of a probe used in Example 2.
  • RNA total RNA extracted from two breast cancer tissues and 23 other normal tissues was used as a material. Gene expression analysis was performed using an oligonucleotide microarray (Human Genome U95A, U95B, U95C, U95D, U95E; Affymetrix).
  • Example 2 In order to analyze the effect of RIPK2 gene on the apoptosis observed in breast cancer tissues, RI PK2 antisense oligonucleotide transfection experiments were performed. First, after designing an antisense (SEQ ID NO: 4) for the base sequence represented by SEQ ID NO: 3, a phosphorothioated oligonucleotide was synthesized, purified by HPLC, and used for an introduction experiment (Amersham Pharmacia Biotech). As a control oligonucleotide, the reverse sequence (SEQ ID NO: 5) of the base sequence represented by SEQ ID NO: 4 was similarly phosphorothioated, purified by HPLC and used (Amersham Pharmacia Biotech).
  • the expression level of the RIM2 gene was determined using two types of primers (SEQ ID NO: 6 and SEQ ID NO: 7) and a probe (SEQ ID NO: 8), and the ABI7700 (Perkin-Elmer) manual was used. PCR was performed according to the procedure. The expression level of RIPK2 was subtracted from the data obtained using the non-reverse transcription control from the data obtained using the cDNA, and then corrected with the total RNA amount used for comparison.
  • the expression of the protein of the present invention is increased in cancer tissues, and when the activity of the protein of the present invention is inhibited, cancer cells undergo apoptosis. Therefore, the protein of the present invention and a polynucleotide encoding the protein are diagnostic markers for cancer.
  • Compounds or salts thereof that regulate (preferably inhibit) the activity of the protein, compounds or salts thereof that regulate (preferably inhibit) the expression of the protein gene include, for example, colorectal cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, sarcoma It can be used safely as a prophylactic or therapeutic agent for cancer such as cancer, brain tumors or hematological tumors.
  • the prophylactic / therapeutic agent for cancer eg, breast cancer, etc.
  • hormone-independent eg, estrogen, etc.
  • HER2 expression negative e.g., HER2 expression negative
  • the compound that inhibits the activity of the protein or a salt thereof, or the compound that inhibits the expression of the protein gene or a salt thereof can be safely used, for example, as an apoptosis-regulating agent (preferably, an apoptosis-action promoting agent). You can do that too.
  • the antisense polynucleotide or antibody of the present invention can inhibit the expression of the protein used in the present invention.
  • Therapeutic agent preferably as a preventive agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen) -independent, HER2-negative, etc., or an apoptotic agent (preferably It can be used safely as an apoptosis action promoter).

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Abstract

L'invention concerne un composé, ou son sel, inhibant l'activité d'une protéine dont la séquence en acides aminés est la même ou sensiblement la même que la séquence représentée par la SEQ ID NO: 1, un composé inhibant l'expression du gène codant pour cette protéine, un nucléotide antisens dont la séquence de base, ou une partie de cette séquence, est complémentaire ou sensiblement complémentaire de la séquence de base d'un ADN codant pour cette protéine ou pour un de ses fragments peptidiques, et un anticorps dirigé contre cette protéine, ou contre un de ses fragments peptidiques, ces composés étant utilisables en tant que préventifs et remèdes contre le cancer et les promoteurs d'apoptose.
PCT/JP2003/007926 2002-06-24 2003-06-23 Preventifs et remedes contre le cancer WO2004000346A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040102A1 (fr) * 1998-02-06 1999-08-12 Millennium Pharmaceuticals, Inc. Nouvelles molecules de la famille de proteines liees au card et leur utilisation
WO2000055350A1 (fr) * 1999-03-12 2000-09-21 Human Genome Sciences, Inc. Sequences et polypeptides geniques associes au cancer chez l'homme
WO2001000826A2 (fr) * 1999-06-28 2001-01-04 Millennium Pharmaceuticals, Inc. Nouvelles molecules de la famille de proteines liee a la card et utilisations de ces dernieres

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040102A1 (fr) * 1998-02-06 1999-08-12 Millennium Pharmaceuticals, Inc. Nouvelles molecules de la famille de proteines liees au card et leur utilisation
WO2000055350A1 (fr) * 1999-03-12 2000-09-21 Human Genome Sciences, Inc. Sequences et polypeptides geniques associes au cancer chez l'homme
WO2001000826A2 (fr) * 1999-06-28 2001-01-04 Millennium Pharmaceuticals, Inc. Nouvelles molecules de la famille de proteines liee a la card et utilisations de ces dernieres

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