WO2003061697A1 - Agents preventifs/remedes contre le cancer - Google Patents

Agents preventifs/remedes contre le cancer Download PDF

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Publication number
WO2003061697A1
WO2003061697A1 PCT/JP2002/013642 JP0213642W WO03061697A1 WO 2003061697 A1 WO2003061697 A1 WO 2003061697A1 JP 0213642 W JP0213642 W JP 0213642W WO 03061697 A1 WO03061697 A1 WO 03061697A1
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WIPO (PCT)
Prior art keywords
cancer
protein
amino acid
acid sequence
present
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PCT/JP2002/013642
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English (en)
Japanese (ja)
Inventor
Yuichi Hikichi
Ryosuke Katsuyama
Yuichi Kakoi
Satoshi Nishizawa
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Takeda Chemical Industries, Ltd.
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Publication of WO2003061697A1 publication Critical patent/WO2003061697A1/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, and the technology for finding disease-specific changes in gene expression has spread, and its usefulness has been confirmed.
  • Aiiymetrix's GeneChip system is being used extensively for diagnosing diseases such as cancer and discovering drug discovery target genes.
  • antisense oligonucleotides become complementary to RNA with complementary sequences. It hybridizes and induces RNAse degradation by RNAse H to inhibit protein translation, or it also results in direct inhibition of protein synthesis by hybridization. Since it is possible to specifically suppress the function of the target gene, it is frequently used as a means for analyzing the function of a gene, and some antisense oligonucleotides are being developed for clinical application.
  • TLRs Toll-like receptors
  • TLR 2 and TLR 6 recognize lipoproteins and glycolipids
  • TLR4 recognizes lipopolysaccharide
  • TLR5 It has been reported that the recognition of Flagellin and that of Ding 119 are related to the recognition of 0 DNA (Nature Immunology 2 (8), 675-680, 2001).
  • TLR 1 (Proc Natl Acad Sci USA 95 (2), 588-593, 1998) is involved in the recognition of microbial products in cooperation with TLR2 (J Immunol. 165 (12), 7125- 7132, 2000) and that it inhibits the action of the TLR 2 and TLR 6 heteroreceptors (J Inunu nol. 166 (1), 15-19, 2001). More recently, analysis of TLR 1 knockout mice have been made, TLR1, at the same time it is reconfirmed acting Oite both the TLR 2 and ligand recognition, P am 3 CSK 4 as a new TLR 1 ligand Trisyl peptides (J Immunol. 169 (1), 10-14, 2002) and OspA (Nat Med.
  • TLR 2 and TLR4 have been reported. Analysis of TLR 2 and TLR4 is also progressing in the study of signal transduction factors.For example, from the analysis of Receptor- interacting serine / threonine kinase-2 (RI PK2) knockout mice, Although shown to work in transmission (Nature. 416 (6877), 190-194, 2002), very little is known about the TLR1 signal.
  • RI PK2 Receptor- interacting serine / threonine kinase-2
  • paclitaxel which is frequently required as a pile cancer drug, exhibits a lipopolysaccharide-like effect on mouse macrophages, and Kawasaki et al. Found that this effect was due to TLR4 and MD2 signals (J Biol Chem. 275 (4), 2251-2254, 2000). However, they report that both TLR4 and mouse MD2 are required, and that this MD2 is species-specific and that human MD2 does not show the LPS-like effect of paclitaxel. Most of these studies on the TLR1 gene involve cells of the immune system, and there is no report on the relationship between the TLR1 gene and cancer (solid cancer).
  • Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, Ovarian cancer, testicular cancer, thyroid cancer, thyroid cancer, brain tumor or hematological tumor, the prophylactic or therapeutic agent described in the above (1) to (5),
  • a cancer diagnostic 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 cancer diagnostic agent comprising a DNA 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,
  • the diagnostic agent according to (7) or (8) which is ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor, or blood tumor,
  • a prophylactic / therapeutic agent for cancer comprising a compound having a To 11-like receptor activity inhibitory activity or a salt thereof,
  • a prophylactic / therapeutic agent for cancer comprising a compound having a To 11-like receptor expression inhibitory activity or a salt thereof,
  • a cancer characterized by using a protein or a partial peptide thereof or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Screening methods for prophylactic and therapeutic agents,
  • An agent for preventing or treating cancer which can be obtained by using the screening method according to (12) or (14) or the screening kit according to (13) or (15).
  • Apoptosis inducer (19) an apoptosis-inducing agent 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,
  • An apoptosis-inducing agent characterized by using a DNA 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. Screening method,
  • (21) a compound or a salt thereof that 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; Pile cancer drug sensitivity enhancer
  • (22) a compound or a salt thereof that inhibits the expression of a protein or a partial peptide or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1;
  • a protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 to a mammal, Administering a compound or a salt thereof that inhibits the activity of the partial peptide or a salt thereof, or an effective amount of a compound or a salt thereof that inhibits the expression of the gene of the protein or the partial peptide or a salt thereof.
  • the present invention also provides use of a compound or a salt thereof that inhibits the activity of a salt thereof, or a compound or a salt thereof that inhibits gene expression of the protein or its partial peptide or a salt thereof.
  • FIG. 1 shows the results of examining the effect of paclitaxel on apoptosis induction.
  • the protein containing the sequence may be a human warm-blooded animal (eg, guinea pig, rat, mouse, chicken, egret, bush, hidge) Cells (eg, hepatocytes, spleen cells, nerve cells, glial cells, knee / 3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelium) Cell, smooth muscle cell, fibroblast, fiber cell, muscle cell, fat cell, immune cell (eg, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, Acidocytes, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, breast cells
  • a human warm-blooded animal eg, guinea pig, rat, mouse, chicken, egret, bush, hidge
  • Cells
  • Brain various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, knee, 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, testicle, ovary, placenta, uterus It may be a protein derived from bone, joint, skeletal muscle, or the like, or may be a synthetic protein.
  • olfactory bulb amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum
  • spinal cord eg,
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes the amino acid sequence represented by '' of about 50% or more, preferably about 60% or more, more preferably about 50% or more of the amino acid sequence represented by SEQ ID NO: 1.
  • 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, intracellular signal transduction activity of a To11-like receptor (preferably TLR1)), ligand binding activity (eg, To11) Receptor (preferably TLR 1) and its ligand or small molecule-binding activity, and the like.
  • the signal transmission activity and the ligand binding activity may be equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times).
  • quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the ligand examples include a bacterial lipopeptide (preferably a triacylated lipopeptide or the like) or N-palmitoyl-S-di palmitoylglyceryl (Pam 3 ) Cys- Ser- (Lys) 4 obtained by chemical synthesis. (CSK 4 ), N-palmitolyl-S-dilaurylgryceryl (N-Pam-S-Lau 2 ) trisyl peptides such as CSK 4, and outer-surface lipoprotein (OspA) derived from Borrelia burg dorieri.
  • a bacterial lipopeptide preferably a triacylated lipopeptide or the like
  • N-palmitoyl-S-di palmitoylglyceryl Pam 3
  • Cys- Ser- (Lys) 4 obtained by chemical synthesis.
  • CSK 4 N-palmitolyl-S-dilaurylgryceryl
  • OspA outer-surface lip
  • the signal transduction activity and the ligand binding activity can be measured by a known method, for example, the method described in J. Ifflmunol. 166 (1), pp. 15-19, 2001 or a method analogous thereto. .
  • TLR family members activate the NF- ⁇ B signal by ligand binding. Therefore, for example, the activity of the protein used in the present invention (eg, To11-like receptor (eg, TLR1)) depends on the expression vector of the protein, the NF- / B binding sequence and the promoter or the promoter. It is introduced into cells together with the reporter gene etc.
  • Enhansa contains ligands such as microbial cell lysate, microbial culture supernatant, eukaryotic cell lysate, and eukaryotic cell culture supernatant And (b) a substance having the same binding activity as the ligand itself or (c) the natural ligand, and the expression level of the reporter is measured by a known method.
  • the reporter expression level can be measured, for example, by using the repo overnight protein activity using luciferase activity, alkaline phosphatase activity or the like as an index.
  • the apoptotic activity against a pile cancer agent eg, paclitaxel
  • the protein eg, TLR1
  • the signal transduction activity is measured.
  • Apoptosis activity against an anticancer agent eg, paclitaxel, etc.
  • an anticancer agent eg, paclitaxel, etc.
  • a TLR1 signal-related molecule eg, TLR2, MD2, etc.
  • a cell having the ability may be used, or the above activity may be measured by forcibly expressing a TLR1 signal-related molecule (eg, TLR2, MD2, etc.).
  • 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 10 amino acids have been deleted, more preferably about 1 to 5 amino acids, and 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 100 amino acids).
  • amino acid sequence represented by SEQ ID NO: 1 an amino acid sequence into which two 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 have been inserted, 1 SEQ ID NO: 1 or 1 in the amino acid sequence represented by Is an amino acid in which two or more (eg, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been substituted with other amino acids) It also includes so-called muteins, such as proteins containing a sequence or an amino acid sequence combining them.
  • the position of the insertion, deletion or substitution is not particularly limited.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (capilloxy terminus) 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 (one COOH), a carboxylate (one COO-), an amide ( — It may be either CONH 2 ) or ester (one COOR).
  • R in the ester e.g., methyl, Echiru, n- propyl, isopropyl
  • C WINCH 6 alkyl group such as n- butyl, for example, C 3, such as cyclohexyl cyclopentyl, cyclohexylene - 8 cycloalkyl group, for example, phenyl, alpha-naphthyl C 6 _ 12 7 aryl group, such as, for example, benzyl, phenyl, such as phenethyl - - 2 alkyl or alpha-naphthylmethyl etc.
  • Ararukiru groups such as alkyl groups, such as pivaloyl I Ruo carboxymethyl group is found using.
  • a protein in which the lipoxyl group is amidated or esterified is also included in the protein used in the present invention. included.
  • 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, such as C Bok 6 Arukanoiru such Asechiru group (: I 6- Glycyl group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, Substituent on the side chain of amino acid in the molecule (for example, 1 OH, one SH, amino group, imidazole group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., protected by a formyl group, a C WINCH 6 Ashiru group such as C 6 Arukanoiru group such Asechiru group) Or complex proteins such as so-called glycoproteins to which sugar chains are bound.
  • Amino group protecting groups e.g., formyl group, such as C Bok 6 Arukanoiru such Asechir
  • the protein used in the present invention include, for example, a protein (TLR1) containing the amino acid sequence represented by SEQ ID NO: 1.
  • the partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and preferably has the same properties as the protein used in the present invention described above. Any one may be used.
  • 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.
  • 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.
  • one or more amino acid sequences preferably, About 1 to 20 amino acids, more preferably about 1 to 10 amino acids, and even more preferably about 1 to 5 amino acids are added, or 1 or 2 or more amino acids (preferably , About 1 to 20 amino acids, more preferably about 1 to 10 amino acids, still more preferably a number (1 to 5) amino acids, or 1 or 2 or more (preferably (About 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids may be substituted with other amino acids.
  • the C-terminus may be any one of a hydroxyl group (one COOH), a carboxylate (—COO—), an amide (one C ⁇ NH 2 ) and an ester (one COOR). Is also good.
  • the partial peptide used in the present invention has a lipoxyl group (or carboxylate) other than the C-terminal, and the N-terminal amino acid residue ( E.g., methionine residue) whose amino group is protected by a protecting group, N-terminal cleavage in vivo, glutamine residue generated by lipamine dynamin oxidation, on the side chain of amino acid in the molecule And the like, and those in which the substituents are protected by an appropriate protecting group, or complex peptides such as so-called glycopeptides to which sugar chains are bound.
  • the N-terminal amino acid residue E.g., methionine residue
  • the partial peptide used in the present invention can also be used as an antigen for producing an antibody.
  • a salt with a physiologically acceptable acid eg, an inorganic acid, an organic acid
  • a base eg, an alkali metal salt
  • acid addition salts that are chemically acceptable.
  • 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, succinic 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, succinic acid, benzoic
  • the protein or its partial peptide or a salt thereof used in the present invention can be produced from the cells or tissues of the above-mentioned human or warm-blooded animal by a known method for purifying a protein, or a protein encoding the protein. It can also be produced by culturing a transformant containing DNA. Also, peptide synthesis described later It can also be manufactured according to the law.
  • 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 achieved by using a combination of chromatography methods.
  • a commercially available resin for protein synthesis can usually be used.
  • resins include, for example, chloromethyl resin J3, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM Resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2,, 4'-dimethoxyphenyl Rumo Fmoc aminoethyl) phenoxy resin.
  • an amino acid appropriately protected with an ⁇ -amino group and a side chain functional group 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 carried out in a highly diluted solution to obtain a target protein or partial peptide or an amide thereof. Get the body.
  • the protected amino acid may be added directly to the resin with a racemization inhibitor additive (eg, HOB t, HO OB t), or may be pre-protected as a symmetric anhydride or HO BT ester or HO OB t ester
  • a racemization inhibitor additive eg, HOB t, HO OB t
  • the amino acid can be added to the resin after activation.
  • 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.
  • Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpiperidone; 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 the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 120 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C 1 _Z, Br—Z, and adaman.
  • Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the carboxyl group may be, for example, an alkyl esterified (eg, a linear, branched or cyclic alkyl such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl).
  • an alkyl esterified eg, a linear, branched or cyclic alkyl such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl.
  • Esterification aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarponyl hydrazide , Butoxycarbonyl hydrazide, trityl hydrazide and the like.
  • aralkyl esterification eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification
  • phenacyl esterification eg, benzyloxycarponyl hydrazide , Butoxycarbonyl hydrazide, trityl hydrazide and the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • Suitable groups for this esterification include, for example, lower groups such as an acetyl group.
  • a group derived from carbonic acid such as an arylo group such as a class (C ⁇ 6 ) alkanoyl group or a benzoyl group, a benzyloxy carbonyl group, or an ethoxycarbonyl group is used.
  • Examples of a group suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz and C 1 2 - B z 2- two Torobenjiru, B r- Z, such as t one-butyl is used.
  • Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc. Can be
  • Examples of the activated carboxylic acid group of the starting material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOB t)].
  • active ester alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOB t)
  • the activated amino group of the raw material for example, a corresponding phosphoric amide is used. .
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or trifluoromethane.
  • Acid treatment with dichloromethane, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. Is also used.
  • the elimination reaction by the above acid treatment is generally carried out at a temperature of about 120 ° C to 40 ° C.
  • 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 form of a protein or partial peptide for example, first, after amidating and protecting the ⁇ -hydroxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side with a desired chain length. And a protein or partial peptide from which only the protecting group for the ⁇ -amino group at the ⁇ -terminal of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the carboxyl group at the C-terminal has been removed. Are produced, and these proteins or peptides are condensed 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 lyophilized to obtain the desired protein or peptide amide.
  • an ester of a protein or peptide for example, after condensing an ⁇ -hydroxyl group of a carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, a desired protein is prepared in the same manner as in the amide of a protein or peptide. It is possible to obtain an ester of quality or peptide.
  • the partial peptide used in the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein used in the present invention with an appropriate peptidase.
  • a peptide synthesis method for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to thereby remove the desired peptide. Can be manufactured.
  • Examples of the known condensation method and elimination of the protecting group include the methods described in the following 1 to 1. ⁇ . Bodanszky and MA Ondet ti, Peptide Synthes is, Interscience Publ ishers, New York (1966)
  • 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. Preferably it is DNA.
  • the DNA may be any of a genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, one of the above-described cells / tissue-derived cDNA libraries, and a synthetic DNA.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like.
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • Examples of the DNA encoding the protein used in the present invention include, for example, a DNA containing the base sequence represented by SEQ ID NO: 2 or a base string represented by SEQ ID NO: 2 that is highly stringent.
  • Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 50% or more, preferably about 6%, of 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 is performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). be able to.
  • the procedure can be performed according to the method described in the attached instruction manual. More preferably, it can be performed under high stringent conditions.
  • High stringency 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 ° C, preferably about 60 to 70 ° C.
  • the conditions at 65 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
  • DNA having the base sequence represented by SEQ ID NO: 2 or the like is used.
  • the DNA encoding the partial peptide used in the present invention may be any DNA containing the nucleotide sequence encoding the partial peptide used in the present invention described above. Further, it may be any of genomic DNA, genomic DNA library, the above-described cell-tissue-derived cDNA, the above-described cell-tissue-derived cDNA library, and synthetic DNA.
  • Examples of the DNA encoding the partial peptide used in the present invention include, for example, a DNA having a part of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or the nucleotide sequence represented by SEQ ID NO: 2 Contains a nucleotide sequence that hybridizes under high stringent conditions, and has substantially the same activity as the protein of the present invention.
  • a DNA containing a part of a DNA encoding a protein or the like is used.
  • DNA capable of hybridizing with the base sequence represented by SEQ ID NO: 2 has the same significance as described above.
  • DNAs that completely encode the proteins and partial peptides used in the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply abbreviated as the proteins of the present invention)
  • the DNA of the present invention is amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA incorporated into an appropriate vector is used as the cloning means of the present invention. Selection can be carried out by hybridization with a DNA fragment coding for a part or the whole region of the protein or labeled with synthetic DNA.
  • the hybridization method can be carried out according to, 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.
  • the DNA base sequence can be converted by PCR, a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM _K (Takara Shuzo Co., Ltd.), etc., using the 0DA-LA PCR method. It can be carried out according to a known method such as the gapped duplex method or the Kimkel method or a method analogous thereto.
  • the DNA encoding the cloned protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme or added with 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 stop 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 of the protein of the present invention may be prepared, for example, by (i) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and It can be produced by ligating downstream of a promoter in an appropriate expression vector.
  • the vector examples include a plasmid derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast (eg, SH19, pSH15), bacteriophages such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, ⁇ 1, ⁇ c / CMV, pRc / RSV, p cDNAI / Neo is used.
  • E. coli eg, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast eg, SH19, pSH15
  • bacteriophages such as ⁇ phage
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR promoter when animal cells are used as host, SR promoter, SV40 promoter, LTR mouth motor, CMV promoter, HSV-TK promoter, etc.
  • CMV cytomegalovirus
  • SRo It is preferable to use a promoter overnight.
  • trp promoter When the host is Eshierihia genus bacterium, trp promoter, lac promoter, re cA promoter Isseki one, AP L promoter one evening one, l pp promoter, T 7 promoter one is when the host is Ru Bacillus der When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may further include an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter, sometimes abbreviated as SV40 ori), and the like, if desired.
  • SV40 ori an SV40 replication origin
  • the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [Mesotorekise Ichito (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp r) , neomycin resistance gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance).
  • dh fr dihydrofolate reductase
  • Amp r ampicillin phosphorus resistant gene
  • Ne o r neomycin resistance gene
  • the target gene can also be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, a PhoA 'signal sequence, an OmpA signal sequence, etc.
  • the host is a bacterium belonging to the genus Bacillus, an amylase signal sequence, a subtilisin, a signal sequence, etc.
  • the host is a yeast.
  • an insulin signal sequence, an interferon signal sequence, an antibody molecule, a signal sequence, etc. can be used.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia examples include, for example, Escheric hia coli K12 ⁇ DH1 [Procedures ⁇ ob ⁇ the ⁇ national ⁇ academy ⁇ ob ⁇ science ⁇ ob ⁇ the ⁇ USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal ⁇ OB ⁇ Molecular ⁇ Biology (Journal, of Molecular Biology), 120, 517 (1978)], HB101 [Journal 'OB'Molecular ⁇ Bio mouth, 41, 459 (1969)] , C 600 [Genetics, 39, 440 (1954)].
  • Bacillus spp. include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1 984)].
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12 and Schizosacclmromyces poibe NCYC 1913, NCYC2036, Pichia pastoris KM71 and the like are used.
  • Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; Sf cell); MGl cell derived from the midgut of Trichoplusia ni; Five TM cells, cells derived from Mamestra b rassicae or cells derived from Estigmena acrea are used.
  • Sf cell a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used.
  • Sf cell include Sf9 cell (ATCC CRL1711), Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like. Used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chinini—zhamster cell CHO (hereinafter CHO (dh fr ”) cell Abbreviation), mouse
  • L cells mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, etc. are used.
  • 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.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like.
  • the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • ⁇ of the medium is preferably about 5 to 8.
  • a medium for culturing the genus Escherichia for example, a medium containing glucose and casamino acid (9) (Miller, Journal of Obesperimen-in 'Molecular Genetics (Journal of Experiments in (Molecular Genetics), 431-43, Cold Spring Harbor Laboratory, New York 1972].
  • a drug such as 3j3-indolylacrylic acid can be added in order to make the promoter work efficiently.
  • culturing is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
  • the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours.
  • the culture medium When culturing a transformant in which the host is an insect cell or an insect, the culture medium is 10% immobilized in a Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). Those to which additives such as serum are appropriately added are used. It is preferable to adjust the ⁇ ⁇ of the culture medium 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.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association at Association, Vol. 199, 519 (1967) )], And 199 medium [Proceeding of the Society for the Biological Medicine], 73, 1 (1950)].
  • the pH is about 6-8.
  • Culture is usually performed at about 30t: ⁇ 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 are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme and Z or frozen. Microbial cells or cells broken by thawing, etc. Thereafter, 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-100 TM .
  • Purification of the protein contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods.
  • These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method utilizing the difference in hydrophobicity, a method utilizing the difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the protein thus obtained when obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in the form of a salt, a known method or a method analogous thereto Can be converted into a free form or another salt.
  • 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, dalicosidase and the like are used.
  • the presence of the protein of the present invention thus produced can be measured by 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, these may be simply abbreviated to the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, Can be produced according to the antibody or antisera production method described above.
  • 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.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times.
  • Examples of the 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.
  • 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.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • 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) 3.
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • PEG polyethylene glycol
  • myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP2 / 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%.
  • Cell fusion can be carried out efficiently by adding and incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes.
  • Various methods can be used to screen the monoclonal antibody-producing hybridomas.
  • the hybridoma culture supernatant is applied directly or onto a solid phase (eg, microplate) on which a protein antigen is adsorbed together with a carrier.
  • a solid phase eg, microplate
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse
  • protein A labeled with a radioactive substance or enzyme and bind to the solid phase.
  • a method for detecting a monoclonal antibody that has been isolated adding a hybridoma culture 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, etc. Examples include a method for detecting the bound monoclonal antibody.
  • the selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine).
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as hybridomas can grow.
  • RPMI 1640 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.)
  • a 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 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide gas.
  • 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 known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)).
  • immunoglobulin separation and purification methods eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)
  • an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody.
  • the polyclonal antibody of the present invention is produced according to a known method or a method analogous thereto. can do.
  • a immunizing antigen protein antigen
  • 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. It can be produced by collecting the contents and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier.
  • any kind may be cross-linked at any ratio.
  • a serum albumin, a thyroglobulin, a hemocyanin, etc. in a weight ratio of about 0.1 to hapten per 1 hapten.
  • a method of coupling at a ratio of 20 and preferably about 1 to 5 is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • daltaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like 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 in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 times, 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. '
  • DNA of the present invention is abbreviated as DNA of the present invention in the description of antisense nucleotide
  • the antisense nucleotide having a substantially complementary base sequence or a part thereof includes a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof, Any anti-protein having an action capable of suppressing the expression of the DNA can be used. It may be a sense nucleotide, but antisense DNA is preferred.
  • the nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of 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). % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more.
  • the nucleotide sequence of the portion encoding the N-terminal site of the protein of the present invention for example, , about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more.
  • the nucleotide sequence of the portion encoding the N-terminal site of the protein of the present invention for example, , about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more.
  • Mouth In the case of an antisense nucleotide that directs RNA degradation by RNaseH, it is about 70% or more, preferably about 80% or more, complementary to the entire nucleotide sequence of the DNA of the present invention including introns.
  • Antisense nucleotides having preferably at least about 90%, most preferably at least about 95% homology are each suitable.
  • an antisense having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof A polynucleotide, preferably, for example, an antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof (more preferably, A base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or an antisense polynucleotide having a part thereof.
  • An antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted by a group.
  • the sugar (doxylipose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2'-O-methylation,
  • the base pyrimidine, purine
  • an antisense nucleotide capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the nucleotide sequence information of the cloned or determined DNA encoding the protein. And combine them.
  • a polynucleotide can hybridize with the RNA of the protein gene of the present invention and can inhibit the synthesis or function of the RNA, or can interact with the protein-related RNA of the present invention. Through the action, the expression of the protein gene of the present invention can be regulated and controlled.
  • Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention can be used in vivo and in vitro. It is useful for regulating and controlling the expression of protein genes in plants and for treating or diagnosing diseases.
  • the term “corresponding” means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes.
  • nucleotide, nucleotide sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) under the direction derived from the nucleotide (nucleic acid) sequence or its complement. ing. 5 'end hairpin loop, 5' end 6—base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein code region, ORF translation stop codon, 3' end untranslated region, The 3 'end palindrome region and the 3' end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as the region of interest.
  • Antisense (poly) nucleotides are polynucleotides containing 2-deoxy D-report and polynucleotides containing D-report.
  • polynucleotides that are N-glycosides of purine or pyrimidine bases or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or special linkages And the like (provided that the polymer contains a nucleotide having a configuration that allows base pairing and base attachment as found in DNA and RNA), and the like.
  • They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides).
  • Nucleotides and also those with known modifications, such as those with a label known in the art, capped, methylated, and one or more natural nucleotides.
  • Substituted with an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramide, phorbamate, etc.), a charged bond or a sulfur-containing bond ( For example, those having phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nuclease inhibitors, ibis) Having side-chain groups such as amino acids, antibodies, signal peptides, poly-L-lysine, etc., and sugars (eg, monosaccharides), and those having an interactive compound (eg, acridine, psoralen, etc.) Those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals (
  • nucleoside may include not only those containing purine and pyrimidine bases but also those containing 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 functional groups such as ethers, amines, etc. May be converted to
  • the antisense (poly) nucleotide (nucleic acid) of the present invention includes RNA, DNA, Or modified nucleic acids (RNA, DNA).
  • modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphoate 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 enhance the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and if toxic. Reduces the toxicity of antisense nucleic acids.
  • 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.).
  • 'Other groups are cap groups specifically located at the 3' end or 5 'end of nucleic acids to prevent degradation by nucleases such as exonuclease and RNase. Is mentioned.
  • capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as dalicol such as polyethylene glycol and tetraethylene dalicol.
  • the inhibitory activity of the antisense nucleic acid is determined by the transformant of the present invention, It can be examined using an external gene expression system or an in vivo or in vitro translation system of the protein of the present invention.
  • the nucleic acid can be applied to cells by various known methods.
  • 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 Abbreviated as NA), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the antibody of the present invention), and an antisense nucleotide of the DNA of the present invention (hereinafter, referred to as an antibody). (May be abbreviated as the antisense nucleotide of the present invention).
  • a drug containing the antisense nucleotide 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 includes, 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, descending It can be used as a prophylactic / therapeutic agent for cancer such as organ cancer, brain tumor or blood tumor, and an apoptosis inducer.
  • a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention includes: 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, testis It can be used as a prophylactic / therapeutic agent for cancer such as cancer, thyroid cancer, knee cancer, brain tumor or blood tumor. It can also be used as an apoptosis action regulator, preferably as an apoptosis inducer.
  • the protein of the present invention has an activity of suppressing apoptosis induced by an anticancer agent (eg, paclitaxel, etc.), the activity of the protein of the present invention
  • an anticancer agent eg, paclitaxel, etc.
  • the compound or its salt that regulates (promotes or inhibits, preferably inhibits) can be used also as an anticancer drug sensitivity enhancer.
  • the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention.
  • the present invention provides a compound which regulates (promotes or inhibits, preferably inhibits) 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, or a compound thereof.
  • a salt screening method Provided is a salt screening method.
  • the ligand binding activity or signal transduction activity of a cell capable of producing the protein of the present invention is used.
  • the protein expression vector of the present invention when screening for a compound having a ligand binding promoting action or an intracellular signaling promoting action, it is necessary to (i) use the protein expression vector of the present invention to bind the NF- ⁇ B binding sequence to a promoter or enhancer. (Ii) the protein expression vector of the present invention containing the NF- ⁇ B binding sequence in the promoter or enhancer; When the cells are introduced into cells together with the reporter gene and cultured in the presence of the test compound, the expression levels of the reporters are compared. The expression of the reporter can be measured by using the luciferase activity, the lipophilic enzyme activity, etc. as an index.
  • a compound having an inhibitory action on ligand binding or intracellular signal transduction When screening a compound, (i) introducing the protein expression vector of the present invention into cells together with a repo overnight gene containing a NF- ⁇ B binding sequence in a promoter or an enhancer; (A) A solution containing a ligand such as a microbial cell lysate, a microbial culture supernatant, a eukaryotic cell lysate, or a eukaryotic cell culture supernatant, (b) the ligand itself or (c) equivalent to a natural ligand (Ii) culturing a protein expression vector of the present invention together with a reporter gene containing a NF- ⁇ binding sequence in a promoter or enhancer.
  • a ligand such as a microbial cell lysate, a microbial culture supernatant, a eukaryotic cell lysate, or a eukaryotic cell culture supernatant,
  • liquid containing ligands such as microbial cell lysate, microbial culture supernatant, eukaryotic cell lysate, or eukaryotic cell culture supernatant;
  • ligands such as microbial cell lysate, microbial culture supernatant, eukaryotic cell lysate, or eukaryotic cell culture supernatant;
  • ligand itself or (C) natural No After adding a substance having the same binding activity as Gand, and culturing in the presence of the test compound, measure the expression level of the repo overnight and compare.
  • the ligand examples include a lipopeptide derived from bacteria (preferably, a triacylated lipopeptide or the like), or N-palmitoyl S-dipalmitoylglyceryl (Pam 3 ) Cys-Ser- (Lys) 4 obtained by chemical synthesis. (CSK 4 ), N-palmitolyl-S-dil aurylgryceryl (N-Pam-S-Lau 2 ) Trisyl peptides such as CSK 4, and outer-surface lipoprotein (Osp A) derived from Borrelia burg dorferi Can be
  • the repo overnight expression level can be measured using a reporter protein activity using luciferase activity, alkaline phosphatase activity or the like as an index.
  • the apoptotic activity against a pile cancer agent eg, paclitaxel
  • the protein eg, TLR1
  • the signal transduction activity is measured.
  • Apoptosis activity against stake cancer drugs eg, paclitaxel, etc.
  • stake cancer drugs eg, paclitaxel, etc.
  • TLR1 signal transduction used in the present invention with higher sensitivity
  • cells having the ability to express TLR1 signal-related molecules eg, TLR2, MD2, etc.
  • the above activity may be measured by forcibly expressing a TLR1 signal-related molecule (eg, TLR2, MD2, etc.).
  • Examples of cells having the ability to produce the protein of the present invention include, for example, a host transformed with a vector containing a DNA encoding the protein of the present invention.
  • the main (transformant) is used.
  • a host for example, animal cells such as COS7 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.
  • Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation production. Products, cell extracts, plant extracts, animal tissue extracts, and the like.
  • the ligand binding activity or signal transduction activity in the case (ii) is reduced by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case (i).
  • the test compound to be tested is a compound that inhibits the activity of the protein of the present invention, and the ligand binding activity or signal transmission activity in the case (ii) above is preferably about 20% or more as compared with the case (i) above.
  • a test compound that increases the activity of the protein of the present invention by 30% or more, more preferably about 50% or more, can be selected as a compound that promotes the activity of the protein of the present invention.
  • the compound having the activity of inhibiting the activity of the protein of the present invention is a safe and low toxic drug for suppressing the physiological activity of the protein of the present invention, for example, a colorectal 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, knee cancer, brain It is useful as a prophylactic / therapeutic agent for cancer such as tumors or hematological tumors, an apoptosis inducer, an anticancer agent, and a sensitivity enhancer.
  • 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.
  • 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 It is a compound selected from liquids, plasma, etc.
  • the salt of the compound those similar to the aforementioned salts of the peptide of the present invention are used.
  • compounds or salts thereof that regulate the expression of the gene encoding the protein of the present invention also include, for example, breast cancer, colon cancer, breast cancer, Lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, knee It is useful as an agent for preventing and treating cancer such as cancer, brain tumor, or blood tumor, an apoptosis inducer, and an anticancer drug sensitivity enhancer.
  • the DNA of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) 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 that is characterized by making a comparison 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) 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 or ELISA method or a method analogous thereto.
  • the amount of mRNA that can be measured can be determined 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 SEQ ID NO: 2 or a primer thereof as a primer It can be measured according to a PCR method using a nucleic acid containing a part or a method analogous thereto.
  • the gene expression level in the case of the above (iv) is inhibited by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii).
  • the test compound to be tested as a compound that suppresses the expression of the gene encoding the protein of the present invention is about 20% or more as compared with the above (iii).
  • a test compound that increases by preferably 30% or more, more preferably about 50% or more, can be selected as a compound that promotes the expression of a 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 may be 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 cancer, kidney cancer, brain tumor or hematological tumor, an apoptosis inducer, and an anticancer drug sensitivity enhancer.
  • 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, etc. You.
  • Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients 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 known method, 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 liquid for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like
  • suitable dissolution aids for example, alcohol (eg, ethanol), polyalcohol ( For example, propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyetylene (5 Omol) adduct of hydrogenated castor oil)] and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol for example, propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyetylene (5 Omol) adduct of hydrogenated castor oil)
  • 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
  • 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), suppositories and the like, and usually 5 to 500 mg, especially
  • the injection preferably contains 5 to 100 mg of the above antibody, and other dosage forms contain 10 to 25 mg of the above antibody.
  • compositions may contain other active ingredients as long as the compound and the above-mentioned compound do not cause an undesirable interaction.
  • the preparations obtained in this way are safe and low toxic, such as, for example, human or warm-blooded animals (eg, mice, rats, puppies, sheep, bushy, pussi, puma, birds, cats, dogs, Orally or parenterally to monkeys, chimpanzees, etc.) Can be given.
  • human or warm-blooded animals eg, mice, rats, puppies, sheep, bushy, pussi, puma, birds, cats, dogs, Orally or parenterally to 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 100 mg, preferably about 1.0 to 50 mg per day. More preferably, about 1.0 to 2 O mg is administered.
  • the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like.
  • a compound or a compound that regulates the activity of the protein of the present invention for the purpose of treating breast cancer may be used.
  • the salt is orally administered to an adult (as body weight of 6 O kg) in the form of an injection, the compound or a salt thereof is about 0.01 to 3 O mg per day, preferably about 0.1 to 3 O mg. It is convenient to administer by injection about 2 O mg, more preferably about 0.1-1 O mg, to the cancerous lesion. In the case of other animals, the dose can be administered in terms of the body weight of 60 kg.
  • the above compounds can be used in combination with existing pile cancer drugs (eg, paclitaxel, docetaxel, etc.), and reduce the side effects of pile cancer drugs that damage normal cells.
  • the timing of administration is not limited, and they may be administered to the subject at the same time or at an interval.
  • the dose can be appropriately selected based on the dose used clinically.
  • the compounding ratio of the above compound and the anticancer agent can be appropriately selected according to the administration subject, administration route, target disease, symptom, combination, and the like.
  • 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, and therefore, the quantification of the protein of the present invention in a test solution, particularly It can be used for quantification by sandwich immunoassay.
  • the antibody of the present invention is allowed to react competitively with the test solution and the labeled protein of the present invention, and the ratio of the labeled protein of the present invention bound to the antibody is measured.
  • a method for quantifying the protein of the present invention in a test solution and
  • 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 monoclonal antibody of the present invention In addition to quantifying the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like is also possible. it can.
  • 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. . For example, nephelometry, competition method, immunometric method and sandwich method are preferably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, a lanthanide element and the like are used.
  • the radioisotope for example, [1 2 5 I], [1 3 1 I], [3 H], which need use include [1 4 C].
  • the above enzyme a stable enzyme having a large specific activity is preferable. For example, i3-galactosidase,] 3-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • binding between the antibody or antigen and the label A tin-avidin system can also be used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.
  • a test solution is allowed to react 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 preferably an antibody having a different site to which the protein of the present invention binds.
  • 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 sandwich method, for example, a competition method, an immunometric method, a nephelometry method, or the like.
  • a competition method an antigen in a test solution and a labeled antigen are applied to the antibody.
  • the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), and the labeling amount of either B or F is measured. Quantify the amount of antigen in the test solution.
  • 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.
  • An immobilized antibody is used, or an immobilized method is used in which the first antibody is soluble and an immobilized antibody is used as the second antibody.
  • 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. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase 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 measurement system of the present invention may be constructed by adding ordinary technical considerations to those skilled in the art to the ordinary conditions and procedures in each method. For details of these general technical means, reference can be made to reviews and written documents.
  • the protein of the present invention can be quantified with high sensitivity.
  • 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, and 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, genital cancer Can be diagnosed as having cancer, such as cancer, brain tumor or hematological tumors, or more likely to be affected in the future.
  • a pile cancer agent eg, paclitaxel
  • 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, in humans or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, monkeys, Abnormalities (genetic abnormalities) of DNA or mRNA encoding the protein of the present invention or its partial peptide in chimpanzees and the like, for example, damage, mutation or reduced expression of the DNA or mRNA, It is useful as a gene diagnostic agent for increasing or overexpressing the DNA or mRNA.
  • warm-blooded animals e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, monkeys
  • Abnormalities (genetic abnormalities) of DNA or mRNA encoding the protein of the present invention or its partial peptide in chimpanzees and the like for example, damage, mutation or reduced expression of
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989)), Processings Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)) Can be implemented.
  • PCR-SSCP method 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, testis cancer, thyroid cancer, knee cancer, brain tumor or It can be diagnosed that the possibility of cancer such as hematological tumors is high.
  • the antisense nucleotide of the present invention which complementarily binds to the DNA of the present invention and can 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, spleen cancer.
  • Prevention and treatment of cancers such as kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, knee cancer, brain tumor or blood tumor, apoptosis inducer, anti- It can be used as a cancer drug sensitivity enhancer.
  • the antisense nucleotide When used as the above-mentioned prophylactic or therapeutic agent, it can be formulated and administered according to known methods.
  • the antisense nucleotide after inserting the antisense nucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, a human or mammal (for example, it can be administered orally or parenterally to rats, egrets, sheep, sheep, bush, horses, cats, dogs, monkeys, etc.).
  • the antisense nucleotide 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 with a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.
  • the antisense nucleotides described above are used alone or in combination with a carrier such as ribosome for the purpose of improving pharmacokinetics, extending half-life, and improving the efficiency of cellular uptake.
  • the antisense nucleotide may be administered to vein, subcutaneous, intra-articular cavity, cancer lesion, etc.
  • the dosage of the antisense nucleotide varies depending on the target disease, the administration target, the administration route, etc. However, for example, when the antisense nucleotide of the present invention is administered for the purpose of treating breast cancer, generally in an adult (body weight 60 kg), about 0.1 to 10 O of the antisense nucleotide is administered per day. mg.
  • the antisense nucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of its expression.
  • 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.
  • cancers such as brain tumors or hematological tumors, an apoptosis inducer, and a susceptibility enhancer for pile cancer agents.
  • 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 manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, 7, 221, 2001). For example, it can be produced by linking a known lipozyme to a part of the 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.
  • the above double-stranded RNA or lipozyme When used as the above-mentioned prophylactic or therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. Similar to the antisense nucleotide, the protein of the present invention or an abtamer of DNA encoding the protein of the present invention can also suppress the expression of the gene of the present invention, and the protein or the protein used in the present invention in vivo.
  • the function of the DNA used in the present invention can be suppressed, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney
  • cancer such as cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, knee cancer, brain tumor or blood tumor, apoptosis inducer, pile It can be used as a drug sensitivity enhancer.
  • Abutama can be produced according to a known method.
  • Antibodies of the present invention having an activity of neutralizing 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, biliary tract cancer, spleen cancer , Renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor, etc. It can be used as a low-toxicity and safe drug such as a drug sensitivity enhancer.
  • the prophylactic / therapeutic agents for the above-mentioned diseases containing the antibody of the present invention have low toxicity and can be used as a night medicine or as a pharmaceutical composition of an appropriate dosage form in humans or mammals (eg, rat, rabbit, etc.). It can be given orally or parenterally (eg, intra-articular) to mice, higgies, bush, elephants, cats, dogs, monkeys, etc.).
  • the dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for treatment of adult breast cancer * prevention, the antibody of the present invention is used as a single dose.
  • 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.
  • Such compositions are provided in dosage forms suitable for oral or parenteral administration (eg, intravenous injection).
  • Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
  • the “prophylactic / therapeutic agent for cancer comprising a compound having an activity of modulating To11-like receptor or a salt thereof” of the present invention and an expression regulating effect of ⁇ 11-like receptor
  • Compounds having the activity regulating action of ⁇ ⁇ 11-like receptor” Any compound that has the compound, such as colorectal cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterus It is used as a prophylactic / therapeutic agent for cancer such as cancer, ovarian cancer, testicular cancer, thyroid cancer, knee cancer, brain tumor, or blood tumor, an apoptosis inducer, and an enhancer for cancer cell susceptibility.
  • the “compound having a To 11-like receptor expression regulating action” may be any compound having a To 11-like receptor expression regulating action, such as 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, kidney cancer, It is used as a prophylactic / therapeutic agent for cancers such as brain tumors and blood tumors, an apoptosis inducer, and a cancer susceptibility enhancer.
  • 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 (sometimes abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the same.
  • Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
  • the calcium phosphate method, the electric pulse method, and the lipofection method It can be produced by transferring the desired DNA by a coagulation method, microinjection method, particle gun method, DEAE-dextran method, or the like.
  • the target exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like.
  • the DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a known cell fusion method.
  • mice for example, porcupines, pigs, sheep, goats, porcupines, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
  • mice for example, pure strains such as C57BL / 6 strain, DBA2 strain, etc.
  • a crossing strain a BSC SFi strain, a BDF strain, a B6D 2F one strain, a BALB / c strain, an ICR strain, etc.
  • a rat eg, Wistar, SD, etc.
  • Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.
  • the exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the original DNA originally possessed by a non-human mammal.
  • DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. DNA that has been used is used, and also includes abnormal DNA.
  • the abnormal DNA refers to a DNA that expresses an abnormal protein of the present invention, and for example, a 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 from the target animal.
  • various mammals having the DNA of the present invention having high homology to the human DNA eg, egret, dog, cat, guinea pig, hampus, rat, mouse, etc.
  • a DNA construct eg, a vector, etc.
  • a DNA-transferred mammal that highly expresses the DNA of the present invention can be created.
  • 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 Animal viruses such as baculovirus are used.
  • a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.
  • Examples of the promoter that regulates the expression of DNA include, for example, a promoter of DN 1 derived from a virus (eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.).
  • a promoter of DN 1 derived from a virus eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.
  • Promoters derived from various mammals such as albumin, insulin II, ⁇ brabrakin II, elastase, erythropoietin, endothelin, muscle creatine kinase , Glial fibrillary acidic protein, daryuthion S-transferase, platelet-derived growth factor] 3, keratin Kl, 1 ⁇ 10 114, collagen type I and type II, cyclic AM P-dependent protein kinase i3 I-subunit, dystro Fin, tartrate-resistant alkaline phosphatase Ichize, atrial sodium Dum diuretic factor, endothelial receptor thymic synthase (generally abbreviated as Tie 2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light
  • the vector preferably has a sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal (generally referred to as terminator).
  • terminator a DNA sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal.
  • a DNA sequence can be used, and preferably, Simian virus SV40 terminator 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, between the promoter region and the translation region. Alternatively, it can be linked to the 3 'downstream of the translation region depending on the purpose.
  • the normal translation region of the protein of the present invention is DNA derived from liver, kidney, thyroid cells, fibroblasts derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). And all or part of genomic DNA from various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cell, or fibroblast-derived RNA as a raw material. . In addition, exogenous abnormal DNA alters the translated region of the normal protein obtained from the above cells or tissues by point mutagenesis. Different translation regions can be created.
  • 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 it is ligated downstream of the above 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.
  • the presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after transfer of the DNA indicates that the progeny of the transgenic animal has the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to keep.
  • the progeny of this type of animal that has inherited the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its 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 retain the exogenous DNA by mating, and is subcultured in a normal breeding environment as the DNA-bearing animal. You 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 produced animal after DNA transfer indicates that all the offspring of the produced animal contain the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to have.
  • 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 has a high level of expression of the normal DNA of the present invention, and eventually promotes the function of endogenous normal DNA, thereby finally achieving the function of the present invention. It may develop protein hyperactivity 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, esophagus 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 of the knee, knee, brain or blood.
  • the non-human mammal having the exogenous abnormal DNA of the present invention is confirmed to stably maintain the exogenous DNA by mating, and is reared as an animal having the DNA in a normal breeding environment. I can do it.
  • the desired exogenous DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
  • the DNA construct with the Promo Youichi can be made by ordinary DNA engineering techniques. 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 animal after the transfer of DNA means that all the offspring of the animal have the abnormal DNA of the present invention in all of the germinal and somatic cells.
  • the progeny of this type of animal that inherits the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells.
  • a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing the 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 a normal protein by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention (dominant negative activity). Action).
  • the mammal to which the foreign abnormal DNA of the present invention has been transferred Because it has increased symptoms of protein, it is a prophylactic / therapeutic agent for the protein or function-inactive refractory of the present invention, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, Agents for the prevention and treatment of cancers such as biliary tract, spleen, kidney, bladder, uterine, ovarian, testicular, thyroid, knee, brain or blood tumors It can also be used for screening tests.
  • cancers such as biliary tract, spleen, kidney, bladder, uterine, ovarian, testicular, thyroid, knee, brain or blood tumors It can also be used for screening tests.
  • ⁇ ⁇ 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 producing cell of the present invention can be specified, apoptosis, its relationship with differentiation or proliferation, or its signal transduction mechanism can be examined, and its abnormality can be examined. It is an effective research material for elucidating its action.
  • the functional inactivity of the protein of the present invention In order to develop a therapeutic agent for a disease associated with the protein of the present invention including type refractory disease, an effective and rapid screening method for the therapeutic agent for the disease is carried out by using the above-mentioned detection method and quantitative method. Can be provided. Further, 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 DNA treatment method for a disease 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 has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.
  • the DNA is inactivated by introducing a repo overnight gene (eg, a / 3-galactosidase gene derived from Escherichia coli), and the repotase gene is Non-human mammal according to item (6), which can be expressed under the control of a promoter.
  • a repo overnight gene eg, a / 3-galactosidase gene derived from Escherichia coli
  • the repotase gene is Non-human mammal according to item (6), which can be expressed under the control of a promoter.
  • 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 Activity of the protein of the present invention encoded by DNA ,
  • the DNA does not substantially have the ability to express the protein of the present invention (hereinafter, may be referred to as the knockout DNA of the present invention). (Abbreviated as ES cell).
  • 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 and inserting or substituting another DNA by a genetic engineering technique.
  • the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated include, for example, The DNA of the present invention possessed by a non-human mammal to be isolated is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (j3-galactosidase gene), cat (clo DNA sequence that disrupts the function of exon by inserting a repo overnight gene represented by the ramphenicol acetyltransferase gene) or terminates gene transcription in the intron between exons For example, by adding a po 1 yA additional signal) to prevent synthesis of the complete messenger RNA.
  • a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (j3-galactosidase gene), cat (clo DNA sequence that disrupts the function of exon by inserting a repo overnight gene represented by the ramp
  • a DNA chain having a DNA sequence constructed so as to eventually disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells
  • a DNA sequence on or near the DNA of the present invention used as a probe for Southern hybridization analysis or a DNA sequence on the targeting vector and a DNA sequence in a neighboring region other than the DNA of the present invention used for the production of the targeting vector Can be obtained by analyzing by a PCR method using as a primer and selecting the knockout ES cells of the present invention.
  • ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like for example, those already established as described above may be used, and those of known Evans and Kaufman may be used. It may be newly established according to the method. For example, mouse E In the case of S cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, a pure ES system with an immunologically clear genetic background is available.
  • a BDF mouse C57BLZ6 and DBA / 2 using a C57BL / 6 mouse or C57BLZ6 and BDFi mice can be used satisfactorily because they have a high number of eggs collected and their eggs are robust, and they have C57BLZ6 mice as their background.
  • ES cells can be advantageously used when generating pathological model mice in that the genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BL / 6 mice.
  • blastocysts 3.5 days after fertilization are generally used. Many early embryos can be obtained.
  • 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.
  • An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR.
  • this method conventionally, for example G-banding method, requires about 10 6 cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture
  • the primary selection of ES cells in the early stage can be performed by discriminating between males and females, and the early stages of culture can be greatly reduced by enabling the selection of male cells at an early stage.
  • Embryonic stem cell lines obtained in this way usually have very good growth potential, Careful subculturing is necessary, as it tends to lose the ability to grow.
  • a suitable feeder cell such as STO fibroblasts
  • a CO2 incubator preferably 5% CO2, 95% air or 5%
  • LIF 1-1000 OU / ml
  • trypsin ZEDTA solution usually 0.001-0.5% trypsin / 0.1-5mM EDTA
  • trypsin ZEDTA solution usually 0.001-0.5% trypsin / 0.1-5mM EDTA
  • ImM EDTA 0.1% trypsin / "ImM EDTA
  • ES cells can be cultured in monolayers at high densities or in suspension cultures to form cell clumps under appropriate conditions to produce various types of cells such as parietal, visceral, and cardiac muscles.
  • MJ Evans and MH Kaufman Nature, 292, 154, 1981; GR Martin Proceedings of Ob. 'National' Academy 'Ob. (Proc. Natl. Acad. Sci.
  • DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cell biology of the protein of the present invention in the mouth of in vivo.
  • the non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA amount of the animal using a known method and indirectly comparing the expression levels. .
  • non-human mammal those similar to the aforementioned can be used.
  • the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of one of the targeting vectors of the present invention becomes inactive upon introduction.
  • the homologous recombination of the transformed DNA sequence with the DNA sequence of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination, Can be knocked out.
  • a cell in which the DNA of the present invention has been knocked out has a DNA sequence on a Southern hybridization analysis or a gettering vector using the DNA sequence on or near the DNA of the present invention as a probe, It can be determined by PCR analysis using as primers a DNA sequence of a neighboring region other than the mouse-derived DNA of the present invention, which was used for the evening getter 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 pseudopregnant non-human mammal.
  • the produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially altered DNA locus of the present invention.
  • all tissues are artificially mutated from a population obtained by crossing such a chimeric individual with a normal individual. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like.
  • 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 getter vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by microinjection.
  • a transgenic non-human mammal of the present invention by selecting those having a mutation at the DNA locus of the present invention by homologous recombination.
  • the germline can be obtained and maintained according to a standard method. That is, by crossing male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. Homozygote obtained Automata can be efficiently obtained by rearing mother animals in such a way that one normal individual and one or more homozygote are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.
  • non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are very useful for producing a 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.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 and compared with an untreated control animal, and the change in each organ, tissue, disease symptoms, etc. of the animal is used as an index.
  • the therapeutic and prophylactic effects of the test compound can be tested.
  • a method for treating a test animal with a 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.
  • testicular cancer 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
  • a test compound is administered to a non-human mammal deficient in DNA expression of the present invention. After the administration, a difference in the degree of onset of cancer and a difference in the degree of healing of the cancer are observed over time in the above tissues.
  • 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 can be selected as a compound having a therapeutic / preventive effect on the above-mentioned diseases.
  • 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-mentioned screening can also be used.
  • the compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is particularly preferable.
  • 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, 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, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic 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. In a patient, about 0.1 to 10011 ⁇ , preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg of the compound is administered per day.
  • the dosage of the compound when administered parenterally, the dosage of the compound varies depending on the subject of administration, the target disease, and the like.
  • the compound is usually administered in the form of an injection to normal adult (60 kg) breast cancer patients.
  • 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 or inhibits the expression of a reporter gene.
  • a method for screening a compound or a salt thereof is provided.
  • the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene.
  • the reporter gene can be used under the control of a promoter for the DNA of the present invention.
  • test compound examples include the same compounds as described above.
  • the same one as described above is used, and a / 3-galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is preferable.
  • the reporter gene exists under the control of the promoter for the DNA of the present invention. The activity of the promoter can be detected by tracing the expression of the substance encoded by.
  • the tissue expressing the protein of the present invention originally has 6-galactosidase is expressed instead of the protein. Therefore, the present invention can be easily performed by staining with a reagent serving as a substrate for 3-galactosidase, such as 5-promo 4-chloro-3-indolyl-j3-galactopyranoside (X-gal). It is possible to observe the expression state of the protein in the animal body.
  • a reagent serving as a substrate for 3-galactosidase such as 5-promo 4-chloro-3-indolyl-j3-galactopyranoside (X-gal). It is possible to observe the expression state of the protein in the animal body.
  • the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at 37 ° C. After reacting at about 30 ° C for about 30 minutes to 1 hour, the jS-galactosidase reaction can be stopped by washing the tissue specimen with an ImM EDTA / PBS solution, and the coloration can be observed. . Further, mRNA encoding 1acZ may be detected according to a conventional method.
  • the compound or a salt thereof obtained by the above screening method is a compound selected from the test compounds described above, and is a compound that promotes or inhibits the promoter activity of the DNA of the present invention.
  • the compound obtained by the screening method may form a salt.
  • the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids, etc.).
  • physiologically acceptable acid addition salts are preferred.
  • Such salts include, for example, 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, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) And the like.
  • 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, tart
  • the compound of the present invention which promotes or inhibits the promoter activity on DNA, or a salt thereof, regulates the expression of the protein of the present invention and regulates the function of the protein.
  • 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 It is useful as a prophylactic / therapeutic agent for cancers such as ovarian, testicular, thyroid, knee, brain or blood tumors.
  • 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 administration subject, the administration route, and the like.
  • the compound of the present invention that inhibits promoter overnight activity against DNA is orally administered, Generally, in adult (assuming a body weight of 60 kg) breast cancer patients, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg of the compound is administered per day.
  • the single dose of the compound varies depending on the administration subject, target disease, and the like.
  • the compound of the present invention that inhibits the activity of the promoter against DNA is usually administered in the form of an injection to an adult.
  • the compound When administered to breast cancer patients (as 6 O kg), the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. It is convenient to administer about 1 Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 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 of the DNA of the present invention. Investigating or preventing the causes of various diseases caused by insufficient expression of DNA can greatly contribute to the development of therapeutic agents.
  • genes encoding various proteins are ligated downstream of the DNA and injected into animal egg cells. If a so-called transgenic animal (gene-transfected animal) is created by transfection, it will be possible to specifically synthesize the protein and examine its action in the living body. Furthermore, by binding an appropriate reporter gene to the above promoter portion and establishing a cell line in which this is expressed, the action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body can be achieved. It can be used as a search system for low molecular weight compounds with.
  • bases, amino acids and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Comission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below.
  • amino acids may have optical isomers, L-form shall be indicated unless otherwise specified.
  • TLR1 shows the amino acid sequence of TLR1.
  • FIG. 1 shows the nucleotide sequence of DNA containing the full length gene encoding TLR1.
  • Example 3 shows the base sequence of the antisense 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 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a primer used in Example 3.
  • Example 3 shows the nucleotide sequence of a DNA fragment inserted into a vector in Example 3.
  • Example 3 shows the nucleotide sequence of a DNA fragment inserted into a vector in Example 3.
  • RNA total RNA extracted from two cases of breast cancer tissues and two or three other normal tissues as a material, and used ol igonucleotide microarray ( Gene expression analysis was performed using Huian Genome U95A, U95B, U95C, U95D, U95E (Aifymetrix).
  • a TLR1 antisense oligonucleotide transfection experiment was performed.
  • an antisense SEQ ID NO: 4
  • a phosphorothioated oligonucleotide was synthesized, purified by HPLC, and used for an introduction experiment (Amersham Pharmacia Biotech).
  • a reverse sequence SEQ ID NO: 5
  • SEQ ID NO: 5 a reverse sequence of the base sequence represented by SEQ ID NO: 4 was similarly phosphorothioated, purified by HPLC, and used (Amersham Pharmacia Biotech) 0
  • RNA expression of TLR1 was reduced to 55% at 7 hours after the introduction of antisense, compared to the time when the control (reverse) oligonucleotide was introduced (100%).
  • apoptosis increased by 186% compared to control (100%).
  • TLR1 is not only expressed in a cancer-specific manner, but also And apoptosis.
  • TLR1 expression system In order to construct a TLR1 expression system, the full-length TLR1 gene was cloned.
  • PCR was performed using a Marathon ready cDNA library (Clontech) as type III according to the protocol attached to Pfu polymerase (Straragene). went. PCR was performed using 20 ⁇ l of lOxPfu buffer 2 ⁇ l, lOmM each dNTP mixture (manufactured by Clontech) 0.41, 20 ⁇ M each of the above two primers prepared at 20 mM, 0.8 ⁇ l each, ⁇ type cDNA solution 0.5 ⁇ 1, and Pfu polymerase 0.1 ⁇ l.
  • the product was separated by agarose gel electrophoresis, the desired band was cut out, and cloned into pCR4Blunt-TOP0 vector (Invitrogen).
  • the nucleotide sequence of the inserted fragment was confirmed to be consistent with the human TLR 1 nucleotide sequence. Therefore, the vector obtained above using two additional primers (SEQ ID NO: 10 and SEQ ID NO: 11) was A CRC was performed as type ⁇ .
  • PCR contains 2 ⁇ l of lOxPfu buffer, 0.4 ⁇ l of lOmM each dNTP mixture (Clontech), 0.2 ⁇ l of each of the above two primers prepared at 50 mM, 0.2 ⁇ l each of ⁇ -type cDNA solution, and 0.5 ⁇ l of PfU polymerase. After pretreatment of 20 ⁇ ⁇ of the reaction solution at 94 ° C for 2 minutes, the reaction was performed at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 3 minutes, and then 25 cycles of 72 ° C for 7 minutes. The reaction was carried out by reacting for one minute.
  • PCR contains 2 ⁇ l of lOxKOD buffer, 2.5 mM each dNTP mixture (manufactured by Clontech) 1.6 / zl, each of the above two primers prepared at 50 mM, 0.21 each, ⁇ type cDNA solution 11, and K0D polymerase 0.2 // 1 After pre-treating the reaction solution of 20 ⁇ 1 at 94 ° C for 2 minutes, 94 ° C30 seconds, 55 ° C30 seconds, 72 ° C The reaction was performed by reacting for 7 minutes.
  • the product was separated by agarose gel electrophoresis, the target band was cut out, digested with the restriction enzymes Kpnl and Notl, and then digested with the same restriction enzymes pcDNA3.1 / Hyg (+) (Invitrogen) To obtain an MD2 expression vector.
  • the nucleotide sequence of the inserted fragment of this vector is shown in SEQ ID NO: 14. The nucleotide sequence of the inserted fragment was confirmed to be consistent with the nucleotide sequence of human MD2.
  • the expression vector obtained in (1) above was introduced into NCI-H1299 cells (purchased from ATCC) to examine the effect on various reporter activities.
  • NCI-H1299 cells purchased from ATCC
  • TLR1 expression vector obtained in Example 3 (1) and the pcDNA3.1 vector used as a control were used in 0.19 ⁇ g wells.
  • a reporter a vector having an AP1 site or an NF- ⁇ B site upstream of a secreted alkaline phosphatase gene (both from Clontech) or a vector having an ISRE site upstream of a firefly luciferase gene (Clontech) was 0.19.
  • the sea luciferase activity expressed by pRL-TK was measured according to the protocol of the pikki genedu alcypansy double luciferase atsushi system (manufactured by Toyo Ink), and the reporter activity value was corrected.
  • the AP1 reporter activity was used as a control.
  • the NF_ ⁇ B reporter activity was increased 3.3-fold and the ISRE reporter activity was increased 1.7-fold, respectively, 5.6-fold compared to the 3.1 vector.
  • TLR1 activates the AP1 signal, NF- ⁇ B signal, and ISERE signal in the lung cancer-derived NCI-H1299 strain without any additional ligand.
  • a compound having a TLR1 signal-modulating action can be screened by conducting the promoter- 11 reporter accession.
  • the presence of paclitaxel has a higher absorbance than the absence of paclitaxel. It was confirmed that apoptosis was induced by clitaxel (white bar vs. gray par). In contrast, the addition of Pam 3 CSK 4, from Rukoto been reduced absorbance, the TLR 1 ligand addition, apoptosis was found to be reduced (gray bars pair hatched par). Furthermore, TLR can be added without adding Pam 3 CSK 4.
  • the protein used in the present invention is a diagnostic marker for cancer, and therefore, regulates (preferably inhibits) a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein, or regulates (preferably inhibits) the expression of the protein gene.
  • a salt thereof, the antibody of the present invention, the antisense nucleotide of the present invention, etc. for example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer Safe as a prophylactic / therapeutic agent for apoptosis, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, victory cancer, brain tumor or blood tumor, etc.
  • It can be used as a novel drug, and can also be used as a therapeutic agent for cancer that is resistant to anticancer drugs. It can also reduce the dose of existing anticancer drugs and reduce side effects to increase sensitivity to anticancer drugs (eg, paclitaxel).
  • anticancer drugs eg, paclitaxel

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Abstract

L'invention concerne des agents préventifs/remèdes contre le cancer, notamment. Plus précisément, on peut utiliser un composé ou son sel régulant l'activité d'une protéine ayant une séquence d'acides aminés identique ou sensiblement identique à la séquence d'acides aminés représentée par SEQ ID NO:1, un nucléotide antisens ayant une séquence de base ou une partie de celle-ci complémentaire ou sensiblement complémentaire de la séquence de base d'un ADN codant pour une protéine ou son fragment peptidique ayant une séquence d'acides aminés identique ou sensiblement identique à la séquence d'acides aminés représentée par SEQ ID NO:1, et ainsi de suite en tant qu'agents préventifs/remèdes contre le cancer.
PCT/JP2002/013642 2001-12-27 2002-12-26 Agents preventifs/remedes contre le cancer WO2003061697A1 (fr)

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WO2002035236A1 (fr) * 2000-10-27 2002-05-02 Pierre Fabre Medicament Procede d'identification de nouvelles molecules se liant aux recepteurs scavengers et signalees via un recepteur toll

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