WO2004000346A1

WO2004000346A1 - Preventives/remedies for cancer

Info

Publication number: WO2004000346A1
Application number: PCT/JP2003/007926
Authority: WO
Inventors: Yuichi Hikichi; Ryosuke Katsuyama; Yuichi Kakoi
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2002-06-24
Filing date: 2003-06-23
Publication date: 2003-12-31
Also published as: AU2003243951A1

Abstract

A compound or its salt controlling (inhibiting) the activity of a protein having an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:1, a compound controlling (inhibiting) the expression of the gene of the above protein, an antisense nucleotide having a base sequence or a part thereof which is complementary or substantially complementary to the base sequence of a DNA encoding the above protein or its peptide fragment, an antibody against the above protein or its peptide fragment, etc. are usable as preventives/remedies for cancer and apoptosis promoters.

Description

Cancer prevention and treatment technology

The present invention relates to an agent for preventing and treating cancer, a diagnostic agent, and the like. Background art

In cancer chemotherapy, the development of new anticancer drugs has improved life-prolonging effects and is increasing the number of cases toward cure. However, most of the currently used anticancer drugs are highly cytotoxic, damaging DNA and inhibiting cell division. Strong side effects often appear in bone marrow, where cell division is active.

In order to comprehensively analyze gene expression, a microarray method in which cDNA or oligonucleotides are immobilized has been developed. Techniques for finding disease-specific changes in gene expression have become widespread, and their usefulness has been confirmed. For example, Af fymetrix's GeneChip system is being widely used for diagnosing diseases such as cancer and finding drug discovery target genes. When introduced into cells, antisense oligonucleotides hybridize to RNAs with complementary sequences and induce RNase H degradation of RMs to inhibit protein translation, or to inhibit direct protein synthesis by hybridization. Bring. Because it is possible to specifically suppress the function of the target gene, it is frequently used as a means of analyzing the function of genes, and some antisense oligonucleotides are being developed for clinical application.

It is known that maintaining FAS-mediated homeostasis plays an important role mainly in the immune system. Receptor-a factor that binds to the FAS Death domain

Interacting Serine / Threonine Kinase 1 (hereinafter referred to as RIPK1) was discovered, but a molecule showing homology to this RIPK1, RIPK2, has recently been cloned (J. Biol. Chem. 273). , 12296-12300, 1998). Subsequently, RIPK2 binds to apoptosis-related molecules such as Caspase and c IAP, and also binds RIPK2 to MCF7 and 293T. It has been reported that apoptosis is induced by forced expression in cells (J. Biol. Chem. 273, 12296-12300, 1998 and J. Biol. Chem. 273, 16968-16975, 1998). McCarthy et al., Looking at expression in eight cancer cell lines, reported that expression was low except for Raj i (derived from B cells) (J. Biol. Chem. 273, 16968- 16975, 1998). More recently, studies on RI2 knockout mice have reported that RIPK2 is a molecule that acts downstream of Toll-like Receptor (TLR) -2, 3, 4 and is involved in innate immunity ( Nature 416, 190-194, 2002; Nature 416, 194-199, 2002).

There is a strong need for drugs that target molecules that are specifically expressed in cancer cells and that can inhibit the growth of cancer cells or induce apoptosis. In addition, most studies on the RIM2 gene relate to the immune system, and there are no reports of increased expression in cancer tissues.RIPK2 is considered to be a factor that induces apoptosis based on experiments using MCF7 and 293T cell lines. Have been. Disclosure of the invention

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found the RIM2 gene as a gene whose expression is significantly increased in cancer tissues, particularly malignant cancer tissues. Furthermore, they found that apoptosis was induced by suppressing the function of the RIPK2 gene, which has been considered to be a factor that induces apoptosis.Based on this finding, the inventors conducted further studies and completed the present invention. Reached.

That is, the present invention

(1) A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a compound that inhibits the activity of a salt thereof, or a salt thereof Prevention and treatment of cancer

(2) Contains a compound or a salt thereof that inhibits the expression of a protein or a partial peptide thereof 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. (3) a polynucleic acid encoding a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof An antisense polynucleotide containing a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of leotide or a part thereof,

(4) the antisense polynucleotide according to (3), which has the base sequence represented by SEQ ID NO: 4;

(5) a prophylactic or therapeutic agent for cancer comprising the antisense polynucleotide according to (3) above,

(6) Prevention and treatment of cancer comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide or a salt thereof Agent,

(7) Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary (1), (2), cancer, testis, thyroid, thyroid, brain or hematological

(5) A prophylactic or therapeutic agent according to (6),

(7a) The prophylactic or therapeutic agent according to (1), (2), (5) or (6) above, wherein the cancer is a hormone-independent cancer.

(7b) The cancer (1), (2), (5) or

(6) The preventive and therapeutic agents described in

(7c) The cancer is breast cancer, (1), (2), (5) or

(6) The preventive and therapeutic agents described in

(8) a diagnostic agent for cancer comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof;

(9) a diagnostic agent for cancer comprising a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof,

(10) Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary (8) or cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor

(9) The diagnostic agent described, (10a) The diagnostic agent according to (8) or (9), wherein the cancer is a hormone-independent cancer,

(10 b) The diagnostic drug according to the above (8) or (9), wherein the cancer is a HER2-expressing cancer.

(10c) The diagnostic agent according to (8) or (9), wherein the cancer is breast cancer,

(11) A prophylactic / therapeutic agent for cancer comprising a compound having a Receptor-Interacting Serine / Threonine Kinase 2 activity inhibitory activity or a salt thereof,

(12) A prophylactic / therapeutic agent for cancer comprising a compound having an inhibitory effect on the expression of Receptor-Interacting Serine / Threonine Kinase 2 or a salt thereof,

(13) a prophylactic or therapeutic agent for cancer characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof; Screening method,

(13a) a method for screening a pharmaceutical compound, which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof,

(13b) The pharmaceutical compound described in (13a) above, wherein the pharmaceutical compound is a compound for preventing or treating cancer, a compound used for preventing or treating cancer, or a compound having Z or Z preventive or therapeutic effect. The screening method of

(14) Prevention of cancer characterized by containing a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof · Kits for screening therapeutic agents,

(14a) A screen of a pharmaceutical compound characterized by containing 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. Kit for

(1b) The pharmaceutical compound described in (14a) above, wherein the pharmaceutical compound is a compound for cancer prevention / treatment, a compound used for cancer prevention / treatment, or a compound having Z or cancer prevention / treatment effects. Screening kit,

(15) a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof PC leakage 00 hire ₇₉₂₆

(5a) A method for screening a cancer prophylactic and / or therapeutic agent characterized by using a nucleotide, (15a) containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. A method for screening a pharmaceutical compound, which comprises using a polynucleotide encoding a protein or a partial peptide thereof, (15b) a compound for preventing or treating cancer, The method according to (15a) above, which is a compound used for the prevention and treatment of Z and a compound having Z or cancer prevention and treatment effects.

(16) Prevention of cancer characterized by containing a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, Kit for screening therapeutic agents,

(16a) a pharmaceutical compound characterized by containing a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof; Screening kit,

(16b) The pharmaceutical compound described in (16a) above, wherein the pharmaceutical compound is a compound for cancer prevention / treatment, a compound used for cancer prevention / treatment, or a compound having Z or cancer prevention / treatment effects. Screening kit,

(17) An agent for preventing or treating cancer, which can be obtained by using the screening method according to (13) or (15) or the screening kit according to (14) or (16).

(17a) a pharmaceutical compound obtainable by using the screening method according to (13a) or (15a) or the screening kit according to (14a) or (16a);

(17b) The pharmaceutical compound described in (17a) above, wherein the pharmaceutical compound is a compound for preventing or treating cancer, a compound used for preventing or treating cancer, and / or a compound having an effect of preventing or treating cancer. Or a salt thereof,

(17c) an agent for preventing or treating cancer comprising the compound or salt thereof according to (17b) above, (18) a protein or a partial peptide which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a compound which inhibits the activity of the salt or a salt thereof Apoptosis action promoter,

(19) a compound or a salt thereof which inhibits the expression of a protein or a partial peptide thereof 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; Apoptosis promoting agent

(20) A method for screening an apoptosis-activating agent, which comprises using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof. ,

(20a) The screening method according to (15a) above, wherein the pharmaceutical compound is a compound for promoting an apoptotic effect, a compound used for promoting an apoptotic effect, and / or a compound having an apoptotic effect promoting effect.

(20b) an apoptosis-enhancing agent comprising the compound according to (20a) or a salt thereof,

(21) an agent for promoting apoptosis, characterized by using a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding a partial peptide thereof; Screening method,

(22) an apoptosis-enhancing agent comprising the antisense polynucleotide according to (3),

(23) Promoting apoptosis effect 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 or a salt thereof Agent,

(24) a compound that inhibits the activity of a protein, or a partial peptide or a salt thereof, having the same or substantially the same amino acid sequence as that represented by SEQ ID NO: 1 in a mammal; Or a salt thereof, or a compound that inhibits the expression of a gene of the protein or an effective amount of a salt thereof, comprising the steps of: • (25) inhibits the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, or expresses a gene of the protein Cancer prevention and treatment methods characterized by inhibiting

(26) Inhibits the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, or inhibits the expression of a gene of the protein. A method of promoting apoptosis, characterized by inhibiting

(27) A protein or a part thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 for producing a prophylactic or therapeutic agent for cancer. Use of a compound or a salt thereof that inhibits the activity of a peptide or a salt thereof, or a compound or a salt thereof that inhibits expression of a gene of the protein,

(28) Activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide or a salt thereof for producing an apoptosis promoting agent And a salt thereof, or a compound inhibiting the expression of the gene of the protein or a salt thereof. BEST MODE FOR CARRYING OUT THE INVENTION

A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 used in the present invention (hereinafter sometimes referred to as the protein of the present invention or the protein used in the present invention) Are cells of human warm-blooded animals (eg, guinea pig, rat, mouse, chicken, egret, bush, hidge, horse, monkey, etc.) (eg, hepatocytes, spleen cells, nerve cells, glial cells, Glands i3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, fat cells, immune cells (eg, Macrophages, T cells, B cells, natural killer cells, obesity cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, cartilage Cells, osteocytes, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or any of these cells P2003 / 007926

8 progenitor cells, stem cells or cancer cells, etc.) or any tissue where these cells are present, such as the brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, Cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, kidney, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels , Heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joints, skeletal muscle, etc., or a synthetic protein .

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, and more preferably about 50% or more of the amino acid sequence represented by SEQ ID NO: 1. Amino acid sequences having a homology of 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.

Amino acid sequence homology can be calculated using a homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Search Tool).

Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above. However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable. Examples of substantially equivalent activities include, for example, signal transduction activity (eg, RIPK2 intracellular signal transduction activity), phosphorylation activity, and the like. Substantially identical indicates that the properties are qualitatively (eg, physiologically or pharmacologically) identical. Therefore, the signal transduction activity and the phosphorylation activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.

The signal transduction activity and phosphorylation activity can be measured by a method known per se, for example, the method described in J. Biol. Chem. 273, 16968-16975, 1998 or a method analogous thereto. . RIPK 2 is autophosphorylated in signaling Activates the NF KB signal.

For example, phosphorylation activity can be determined by introducing an expression vector for the protein of the present invention (eg, RI PK2) into cells, solubilizing the cells with an appropriate solubilization buffer, and immunoprecipitation. After obtaining a protein (eg, RI PK2), it is reacted with radiolabeled ATP in an appropriate buffer, and the reaction mixture is subjected to SDS gel electrophoresis, and the protein of the present invention is obtained by autoradiography or the like. (Eg, RI PK 2) can be measured for the degree of autophosphorylation.

The signal transduction activity can be determined by introducing an expression vector for the protein of the present invention (eg, RI PK2) into a cell together with a repo overnight gene containing an NFκB binding sequence in a promoter or an enhancer. A liquid containing a signal transduction activity or a suppressive substance such as a microorganism cell disruption solution, a microorganism culture supernatant, a eukaryotic cell disruption solution, or a eukaryotic cell culture supernatant is added, and the reporter expression level is known per se. Measure according to the method. The repo overnight expression level can be measured using, for example, a reporter protein activity using luciferase activity, alkaline phosphatase activity, or the like as an index.

Examples of the protein used in the present invention include: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 100, preferably about 1 to 30, An amino acid sequence in which about 1 to 10 amino acids have been deleted, more preferably a number (1 to 5) of amino acids, and 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, 1 to 1) An amino acid sequence to which about 00, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) of amino acids are added; ③ represented by SEQ ID NO: 1 The amino acid sequence contains one or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids. Inserted amino acid sequence, ④ 1 in the amino acid sequence represented by SEQ ID NO: 1 Or two or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably the number (1 to 5)) of other amino acids So-called muteins such as proteins containing an amino acid sequence substituted with or an amino acid sequence obtained by combining them are also included. When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.

In the present specification, the protein has a N-terminus at the left end (amino terminus) and a C-terminus at the right end (capillary end) according to the convention of peptide labeling. The protein used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group (-C00H), carboxylate (-C00-), amide (- It may be either C0NH ₂ ) or ester (-C00R).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, isopropyl, C, such as n- butyl, _ ₆ alkyl groups, such as cyclopentyl, C ₃ _ ₈ cycloalkyl group such as cyclohexyl cyclo, for example, phenyl, alpha-naphthyl of which C ₆ _ _l2 7 aryl group, e.g., benzyl, one Nafuchiru C, such as phenylene Lou C Bok ₂ § alkyl group or alpha-naphthylmethyl, such as phenethyl, such as _ ₂ alkyl groups C ₇ _ ₁₄ Ararukiru group, such as pin bar opening Iruokishimechiru group is used.

When the protein used in the present invention has a lipoxyl group (or lipoxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. . As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, the protein used in the present invention, the amino acid residue (e.g., main Chionin residues) of N-terminal Amino group protecting groups (e.g., formyl group, _ ₆ Ashiru groups such as C _Myu6 Arukanoiru such Asechiru group ), N-terminal dalluminin residue generated by cleavage in vivo, oxidized with lipamine, substituents on the side chains of amino acids in the molecule (eg-0H , -SH, amino group, imidazole group, I Ndoru group, in Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C such Asechi group, such as C WINCH ₆ Ashiru groups such as _ ₆ Arukanoiru group) Protected proteins and complex proteins such as so-called glycoproteins to which sugar chains are bound are also included.

Specific examples of the protein used in the present invention include, for example, protein containing an amino acid sequence represented by SEQ ID NO: 1.

Examples of the partial peptide of the protein used in the present invention include: It is a partial peptide of the protein used, and preferably may be any peptide having the same properties as the protein used in the present invention described above. Specifically, for the purpose of preparing the antibody of the present invention described below, a peptide having the 27th to 578th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 and the like can be mentioned. For example, at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, and most preferably 200 or more of the constituent amino acid sequences of the protein used in the present invention. Peptides having more than one amino acid sequence are used.

In the partial peptide used in the present invention, one or more (preferably about 1 to 10, more preferably a number (1 to 5)) amino acids in the amino acid sequence are deleted. Or 1 or 2 or more (preferably, about 1 to 20, more preferably, about 1 to 10, and more preferably, about 1 to 5) amino acids are added to the amino acid sequence. Or 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, 1 to 5) amino acids are inserted into the amino acid sequence. Or one or more (preferably about 1 to 10, more preferably several, and more preferably about 1 to 5) amino acids in the amino acid sequence are replaced with other amino acids. It may be. The partial peptide used in the present invention, the C-terminus force Rupokishiru group (- C00H), Karupokishire Ichito (-C00 "), amido (- C0NH ₂₎ or may be any of the ester (-C00R).

Further, similar to the protein used in the present invention, the partial peptide used in the present invention includes those having a carboxyl group (or carboxylate) in addition to the C-terminal, N-terminal amino acid residues ( (E.g., methionine residue) whose amino group is protected by a protecting group, glutamine residue generated by cleavage of N-terminal side in vivo and pyrodartamine oxidation, substitution on the side chain of amino acid in the molecule is Also included are those protected with an appropriate protecting group, and complex peptides such as so-called glycopeptides to which sugar chains are bonded.

The partial peptide used in the present invention can also be used as an antigen for producing an antibody. As the salt of the protein or partial peptide used in the present invention, salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts) are used. Are preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The protein, its partial peptide, or a salt thereof used in the present invention can be produced from the above-mentioned human or warm-blooded animal cell or tissue by a known method for purifying a protein, or a DNA encoding the protein. Can also be produced by culturing a transformant containing It can also be produced according to the peptide synthesis method described below.

When producing from human or mammalian tissues or cells, the human or mammalian tissues or cells are homogenized, then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography. Purification and isolation can be performed by combining chromatography such as chromatography.

For the synthesis of the protein or partial peptide or a salt thereof, or an amide thereof used in the present invention, a commercially available resin for protein synthesis can be usually used. Examples of such a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM. Resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2,, 4, dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2 ′, 4,1-dimethoxyphenylmethyl resin F moc aminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, a protein or partial peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial Obtain peptides or their amides.

For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carpoimides, DCC, N, N'-diisopropylcarpoimide, N-ethyl-N, 1- (3-dimethylaminoprolyl) carpoimide, and the like are used. For these activations, the protected amino acid is added directly to the resin along with a racemization inhibitor (eg, HOB t, HO OB t), or as a symmetrical anhydride or H〇B t ester or H〇OB t ester After the protected amino acid is activated in advance, it can be added to the resin.

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. For example,

Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylvinylidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; Sulfoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof are used. The reaction temperature is appropriately selected from a range that is known to be used for a protein bond formation reaction, and is usually appropriately selected from a range of about −20 ° C. to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation cannot be obtained even after repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole so that subsequent reactions are not affected. .

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isopolnyoxycarbonyl, 4-methoxybenzyloxycarbonyl, C11Z, Br-Z, a Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenyl Phosphinothio oil, Fmoc, and the like are used. '

The lipoxyl group can be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.). Alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarponyl It can be protected by hydrazide, t-butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups appropriately used for the esterification, for example, low-grade (C ,. ₆₎ Arukanoiru groups such Asechiru group, Aroiru group such Benzoiru group, Benjiruokishika Ruponiru group, and a group derived from carbonic acid such as ethoxycarbonyl group Used. Examples of a group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.

The protecting group of the phenolic hydroxyl group of tyrosine, for _{example, Bz l, C 1 2 -} Bz l, 2- two Torobenjiru, B r- Z, such as t one-butyl is used.

As the protecting group for histidine imidazole, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used. Can be

Examples of the activated carbonyl group of the raw material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (for example, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, H〇NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and ester with H'OB t). As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.

Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or anhydrous hydrogen fluoride, Acid treatment with sulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc. Reduction by a system is also used. The elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 ° C. to 40 ° C. In the acid treatment, for example, anisol, phenol, thioanisole, methacresol, paracresol It is effective to add a force thione scavenger such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol and the like. The 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributofan is 1,2-ethanedithiol, 1,4- In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.

The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.

As another method for obtaining an amide of a protein or partial peptide, for example, first, after amidating and protecting the α-hydroxyl group of the amino acid at the carboxy terminal, a peptide (protein) chain is added to the amino group side with a desired chain length. The protein or partial peptide from which only the K-amino group protecting group at the Ν-terminal of the peptide chain has been removed, and the protein or partial peptide from which only the C-terminal carboxyl group-protecting group has been removed And condensing these proteins or peptides in a mixed solvent as described above. Details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein or peptide can be obtained. The crude protein or peptide can be purified using various known purification means, and the main fraction can be freeze-dried to obtain the desired amide of the protein or peptide.

To obtain an ester of a protein or peptide, for example, After condensing the α-hydroxyl group of an amino acid with a desired alcohol to form an amino acid ester, a desired protein or peptide ester can be obtained in the same manner as in the case of a protein or peptide amide.

The partial peptide used in the present invention or a salt thereof can be produced according to a peptide synthesis method known per se, or by cleaving the protein used in the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, a partial peptide or an amino acid capable of constituting the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting peptide is removed to produce the desired peptide. can do. Known condensation methods and elimination of protecting groups include, for example, the methods described in the following ① to ⑤.

(DM. Bodanszky and M.A. Ondet ti, Peptide de Synthesis (Pepti de Synthes is), Interscience Pub lishers, New York (1966%;

② Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

(3) Nobuo Izumiya et al. Basics and experiments on peptide synthesis, Maruzen Co., Ltd. (1975)

治 Haruaki Yajima and Shunpei Sakakibara, Laboratory for Biochemical Experiments 1, Protein Chemistry IV, 205, (1977)

治 Supervised by Haruaki Yajima, Development of Continuing Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten

After the reaction, 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. . When 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. Examples of the DNA include genomic DNA, genomic DNA library, the above-described cell-tissue-derived cDNA, Any of the above-described cell / tissue-derived cDNA library and synthetic DNA may be used. The vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. In addition, reverse RNA was directly prepared using a preparation of total RNA or mRNA fraction from the above-mentioned cell 'tissue.

It can also be amplified by transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method).

Examples of the DNA encoding the protein used in the present invention include a DNA containing the base sequence represented by SEQ ID NO: 2 or the base sequence represented by SEQ ID NO: 2 and high stringency. Any DNA that contains a nucleotide sequence that hybridizes under the following conditions and encodes a protein having substantially the same properties as the protein containing the amino acid sequence represented by SEQ ID NO: 1 described above. It may be something. Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 under conditions of eight istrines include, for example, about 50% or more, preferably about 6% with the nucleotide sequence represented by SEQ ID NO: 2. 0% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more. DNA or the like is used.

Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Saibrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.

High stringent end conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70: preferably about 60 to 6 The conditions at 5 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used. The polynucleotide (eg, DNA) encoding the partial peptide used in the present invention may be any polynucleotide containing the above-described nucleotide sequence encoding the partial peptide used in the present invention. In addition, any of a genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA may be used.

Examples of the DNA encoding the partial peptide used in the present invention include, for example, a DNA containing a part of a DNA containing the base sequence represented by SEQ ID NO: 2, or a base represented by SEQ ID NO: 2. A DNA containing a base sequence that hybridizes with the sequence under high stringent conditions, and a DNA containing a part of a DNA encoding a protein having substantially the same activity as the protein of the present invention is used. The DNA that can be hybridized with the base sequence represented by SEQ ID NO: 2 has the same meaning as described above.

The same hybridization method and high stringency conditions as described above are used.

D which completely encodes the protein and partial peptide used in the present invention (hereinafter, these may be simply referred to as the protein of the present invention in the description of cloning and expression of the DNA encoding them). As a means for closing NA, DNA amplified by a PCR method using a synthetic DNA primer containing a part of the nucleotide sequence encoding the protein of the present invention, or a DNA incorporated into an appropriate vector is used as the DNA of the present invention. Can be selected by hybridization with a DNA fragment coding for a part or the entire region of the above-mentioned protein or labeled with a synthetic DNA. The method of hybridization is, for example,

Molecular Cloning 2nd (J. Sambrook 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 using the ODA-LA PCR method using PCR, a known kit, for example, Mutan ™ -super Express Km (Takara Shuzo), Mutan ^TM- K (Takara Shuzo), or the like. , Gapped duplex method, Kunkel method and the like, or a method analogous thereto. The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be used after digesting with a restriction enzyme or adding a linker, if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.

The expression vector for the protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from DNA encoding the protein of the present invention, and (mouth) ligating the DNA fragment downstream of a promoter in an appropriate expression vector. It can be manufactured by

Examples of the vector include a plasmid derived from Escherichia coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast (eg, p SH19, pSH15), pacteriophage such as phage λ, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, pXTl, pRc / CMV, pRcZRSV, pcDNA I / N eo or the like is used.

The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when an animal cell is used as a host, SRo; Promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

Of these, it is preferable to use the CMV (cytomegalovirus) promoter, SRo; When the host is Eshierihia genus bacterium, trp promoter one, lac promoter, re cA promoter, AP _L promoter - ter, 1 [rho P promoter one, such as T7 promoter, if the host is a strain of the genus Bacillus, SP01 promoter When the host is yeast, such as overnight, SPO2 promoter, penP promoter, and the like, PHO5 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 contain, in addition to the above, an enhancer, a splicing signal, a poly-A addition signal, a selection promoter, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired. Can be used. Selection methods include, for example, a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter abbreviated as Amp ¹ ) If there is), the neomycin resistance gene (hereinafter, Ne o sometimes abbreviated as ^r, G418 resistance), etc. in the like. in particular, dh fr gene deficient Chinese hamster cell select dh fr gene using the marker one When used as a target gene, the target gene can also be selected on a thymidine-free medium.

If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is a genus Escherichia, the PhoA 'signal sequence and the OmpA * signal sequence are used.When the host is a Bacillus genus, the α-amylase signal sequence and subtilisin signal sequence are used. Signal sequence, SUC2 signal sequence, etc. If the host is an animal cell, use insulin 'signal sequence, Hi-I interferon signal sequence, antibody molecule, signal sequence, etc. it can.

Using the vector containing the DNA encoding the protein of the present invention thus constructed, a transformant can be produced.

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.

Specific examples of the genus Escherichia include, for example, Escherichia coli.

(Escherichia coli) K12-DH 1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA 221 [Journal of Molecular Biology] , 120, 517 (1978)], HB 101

[Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)] and the like are used.

Examples of Bacillus bacteria include, for example, Bacillus' subtilis (Bacillus

snbtilis) MI 1 14 [Gene, 24, 255 (1983)], 207-21 [Journal of 03007926

twenty one

Biochemistry, 95, 87 (1984)].

Examples of yeast include Saccharomyces cerevisiae AH22, AH22R—, NA87-11A, DKD—5D, 20B—12, Schizosaccharomyces pombe NC YC 1913, NCYC2036, and Pichia pastoris (Pichia pastor is) K M71 or the like is used.

As insect cells, for example, when the virus is Ac NPV, the cell line derived from the larvae of the night moth (Spodoptera frugiperda cell; S ； cell), the MG1 cell derived from the midgut of Trichoplusia ni, High Five ™ cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a cell line derived from silkworm (Bombyx mori N cell; BmN cell) is used. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, J.L., et al., In Vivo, 13, 213-217, (1977)) and the like are used. As insects, for example, silkworm larvae are used [Maeda et al., Nature, Vol. 315, 592 (1985)].

As animal cells, for example, monkey cell COS-7, Vero, Chinese cell, Muster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chinini-zhamster cell CHO (hereinafter, CHO (dh fr)) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.

Natl. Acad. Sci. USA, 69, 2110 (1972) and Gene, 17, 107 (1982) can be used to transform Escherichia species. .

To transform Bacillus, for example, use Molecular & General

Genetics, vol. 168, 111 (1979) can be used.

The yeast can be transformed according to the method described in, for example, Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). Can be. Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

To transform animal cells, for example, follow the method described in Cell Engineering Separate Volume 8, New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do it.

Thus, a transformant transformed with the expression vector containing the DNA encoding the protein can be obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and others. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn chip liquor, peptone, potato zein, meat extract, soybean meal, and potato extract. Examples of the inorganic or organic substance such as a liquid and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing the genus Escherichia, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] is preferable. Here, for example, a drug such as 3 / 3-indolylacrylic acid can be added to make the promoter work efficiently if necessary.

When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration or stirring may be added.

When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually performed at about 30 to 40 for about 6 to 24 hours, and if necessary, aeration and stirring may be added.

When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium (CBostian, KL et al., Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)) And a SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. Medium pH Is preferably adjusted to about 5 to 8. The culture is carried out usually at about 20 to ^35! 0 to about 24 to 72 hours, with aeration or agitation if necessary.

When culturing an insect cell or a transformant in which the host is an insect, the culture medium is supplemented with Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)). Those appropriately added are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , Vol. 8, 396 (1959)], RPMI 1640 medium [The] journal of the American Medical Association 199, 519 (1967)], 199 medium

[Proceeding of the Society for the Biological Medicine, 73, 1 (1950)]. Preferably, the pH is about 6-8. The cultivation is usually performed at about 30 to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.

As described above, 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.

When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to sonication, lysozyme and Z or freeze-thawing. After the cells or cells are destroyed by the method, a method of obtaining a crude extract of the protein by centrifugation or filtration is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-1100 ™. When the protein is secreted into the culture solution, after completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.

Purification of the protein contained in the culture supernatant or extract obtained in this manner is a separation known per se. Purification methods can be combined appropriately. As these known separation and purification methods, solubility such as salting-out and solvent precipitation is used. PT / JP2003 / 007926

24 methods, methods that mainly use differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and methods that use differences in charge, such as ion-exchange chromatography. Methods that use specific affinity such as affinity chromatography, methods that use differences in hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods that use differences in isoelectric points such as isoelectric focusing. The method used is used.

When the protein thus obtained is obtained as a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when the protein is obtained as a salt, a method known per se or It can be converted to a free form or other salts by a method analogous thereto.

The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

The presence of the protein of the present invention thus produced can be measured by, for example, enzymatic immunoassay western blotting using a specific antibody. The antibody against the protein or partial peptide or a salt thereof used in the present invention is any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. You may.

An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter sometimes simply referred to as the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, It can be produced according to a known antibody or antiserum production method.

[Preparation of monoclonal antibody]

(a) Preparation of monoclonal antibody-producing cells

The protein of the present invention is administered to a warm-blooded animal itself or together with a carrier or diluent at a site capable of producing an antibody upon administration. Antibody production ability upon administration TJP2003 / 007926

25 Complete Freund's adjuvant or incomplete Freund's adjuvant may be given to increase the level. Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. 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.

When preparing monoclonal antibody-producing cells, a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization and contained in them. The hybridoma producing the monoclonal antibody can be prepared by fusing the animal-producing cells obtained with myeloma cells of the same or different species. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of myeloma cells include myeloma cells of warm-blooded animals such as NS-1, P3U1, SP 2/0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is used at a concentration of about 10 to 80%. The cell fusion can be carried out efficiently by adding the mixture and incubating at 20 to 40 ° C., preferably 30 to 37, for 1 to 10 minutes. Various methods can be used to screen for monoclonal antibody-producing hybridomas.For example, a hybridoma culture supernatant is added to a solid phase (eg, a microplate) on which a protein antigen is adsorbed directly or together with a carrier. Next, an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse) or protein A, and a monoclonal antibody bound to the solid phase A monoclonal antibody bound to a solid phase is prepared by adding a hybridoma supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, adding a protein labeled with a radioactive substance, an enzyme, or the like. And a method for detecting antibody.

The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a medium for selection and breeding, any medium can be used as long as the hybridoma can grow. For example, RPMI 164 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd. )) Or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.). The culture temperature is usually from 20 to 40 ° C, preferably about 37. The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. Cultivation can usually be performed under 5% CO2. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(b) Purification of monoclonal antibody

Monoclonal antibodies can be separated and purified by methods known per se, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE) Absorption and desorption method, ultracentrifugation method, gel filtration method, antigen binding Solid phase or specific purification method of collecting antibody only with an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody) Can do it.

(Preparation of polyclonal antibody)

The polyclonal antibody of the present invention can be produced by a method known per se or a method analogous thereto. For example, an immunizing antigen (protein antigen) itself or a complex thereof with a carrier protein is formed, and immunization is performed on a warm-blooded animal in the same manner as in the above-described method for producing a monoclonal antibody. The antibody can be produced by collecting the antibody-containing substance of the antibody and separating and purifying the antibody.

Regarding the complex of immunizing antigen and carrier protein used to immunize warm-blooded animals, the type of carrier-protein and the mixing ratio of carrier and hapten depend on the efficiency of antibody against hapten immunized by cross-linking with carrier. Well done For example, any substance may be cross-linked at any ratio.For example, serum serum albumin, psiloculopurine, hemocyanin, etc. may be used in a weight ratio of about 0.1 to 2 with respect to 1 hapten. A method of coupling at a rate of 0, preferably about 1 to 5 is used. Further, various condensing agents can be used for force coupling between the hapten and the carrier. For example, an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.

The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant ゃ Incomplete Freund's adjuvant may be administered to enhance body production during administration. The administration is usually made once every about 2 to 6 weeks, for a total of about 3 to 10 times.

The polyclonal antibody can be collected from the blood, ascites, etc., preferably from the blood of a warm-blooded animal immunized by the above method.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulin as in the above-described separation and purification of the monoclonal antibody.

Polynucleotide encoding the protein or partial peptide used in the present invention (eg, DNA (hereinafter, in the description of antisense polynucleotides, these DNAs may be abbreviated as the DNA of the present invention). The nucleotide sequence of the polynucleotide of the present invention (eg, DNA) is complementary to the nucleotide sequence of the polynucleotide of the present invention (eg, DNA). Any antisense polynucleotide may be used as long as it contains a basic or substantially complementary base sequence or a part thereof and has an action capable of suppressing the expression of the DNA. Antisense DNA is preferred.

The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). And about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. In particular, of the total nucleotide sequence of the complementary strand of the DNA of the present invention, In the case of the antisense polynucleotide thus obtained, it is at least about 70%, preferably about 80%, complementary to the complementary sequence of the nucleotide sequence (for example, the nucleotide sequence near the start codon) of the N-terminal site of the protein of the present invention. % Or more, more preferably about 90% or more, and most preferably about 95% or more of the antisense polynucleotide which is directed to RNA degradation by RNase H. In this case, it is at least about 70%, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% with the complementary strand of the entire nucleotide sequence of the DNA of the present invention including introns. Antisense polynucleotides having the above homology are each suitable.

Specifically, an antisense polynucleotide containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or a part thereof, Preferably, for example, an antisense polynucleotide containing a nucleotide sequence complementary to the nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 2, or a part thereof (more preferably, a nucleotide sequence represented by SEQ ID NO: 2 A base sequence complementary to the base sequence of DNA containing the base sequence to be prepared, or an antisense polynucleotide containing a part thereof.

An antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.

In order to prevent degradation by nucleases and other hydrolases, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphorus such as phospho-thiolate, methylphosphonate, and phosphorodithionate. It may be substituted by an acid residue. In addition, the sugar (deoxyliposome) of each nucleotide may be substituted with a chemically modified sugar structure such as 2,1-methylation, and the base portion (pyrimidine, purine) may also be chemically modified. Any one may be used as long as it hybridizes to DNA containing the nucleotide sequence represented by SEQ ID NO: 2. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention has been cloned or T JP2003 / 007926

29 It can be designed and synthesized based on the nucleotide sequence information of the DNA encoding the determined protein. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or interact with the protein-related RNA of the present invention It can regulate and control the expression of the protein gene of the present invention. Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention, and polynucleotides capable of specifically hybridizing with the protein-related RNA of the present invention, can be used in vivo and in vitro. It is useful for regulating and controlling the expression of the protein gene of the present invention, and also useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene. The “correspondence” between a nucleotide, base sequence or nucleic acid and a peptide (protein) is a nucleotide

It usually refers to the amino acids of a peptide (protein) in a directive derived from the (nucleic acid) sequence or its complement. 5 'end hairpin loop of protein gene, 5' end 6—base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 The palindrome region at the 'end and the hairpin loop at the 3' end can be selected as preferred regions of interest, but any region within the protein gene can be selected as the region of interest. The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region is as follows: The relationship between the target nucleic acid and the polynucleotide capable of hybridizing with the target is

It can be said to be "antisense". Antisense polynucleotides are polynucleotides containing 2-dexoxy D-ribose, polynucleotides containing D-reports, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or non-polynucleotides. Other polymers having a nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that such polymers are found in DNA and RNA) Base pairing (including nucleotides having a configuration that allows base attachment)). They are double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA high. Bridges, and may also be unmodified polynucleotides (or unmodified oligonucleotides), or even those with known modifications, such as those with a label known in the art, capped, Methylated, one or more natural nucleotides replaced by analogs, modified intramolecular nucleotides, e.g., uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, Those with a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), eg, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signals) Peptides, poly-L-lysine, etc.) and sugars (eg, Compounds with side groups such as monosaccharides, etc., compounds with interactive compounds (eg, acridine, psoralen, etc.), chelating compounds (eg, metals, radioactive metals, boron, oxidizable compounds) Metal, etc.), one containing an alkylating agent, and one having a modified bond (for example, α-anomer type 1 nucleic acid). Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or ethers, It may be converted to a functional group such as amine.

The antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Sense nucleic acid toxicity Make it smaller.

Thus, many modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; S. T.

Crooke et al. Ed., Ant isense Research and Appli cations, CRC Press, 1993.

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. Such additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes and increase nucleic acid uptake (eg, , Phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include cap groups specifically arranged at the 3 'end or 5' end of nucleic acids, which prevent degradation by nucleases such as exonucleases and RNAses. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various methods known per se. Hereinafter, the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter referred to as the D NA), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter referred to as “the present invention”). The use of an antibody (sometimes abbreviated as an antibody) and the antisense polynucleotide of the DNA of the present invention (hereinafter sometimes abbreviated as the antisense polynucleotide of the present invention) will be described.

Since the expression of the protein of the present invention increases in cancer tissues, it can be used as a disease marker. In other words, it is useful as a marker for early diagnosis in cancer tissues, judgment of the severity of symptoms, and prediction of disease progression. Therefore, a medicament containing an antisense polynucleotide of the gene encoding the protein of the present invention, a compound that inhibits the activity of the protein of the present invention or a salt thereof, or an antibody against the protein of the present invention may be, for example, a large intestine. Cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid gland It can be used as a prophylactic / therapeutic agent for cancer such as cancer, brain tumor or blood tumor.

(1) Screening of drug candidate compounds for diseases

The expression of the protein of the present invention is increased in cancer tissues, and when the activity of the protein of the present invention is inhibited, cancer cells undergo apoptosis. Therefore, compounds or salts thereof that modulate (preferably inhibit) the activity of the protein of the present invention include, for example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, and biliary tract. Used as a prophylactic and therapeutic agent for cancer such as cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, brain tumor, or blood tumor can do. Preferably, it is a prophylactic / therapeutic agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen, etc.)-Independent, HER2-negative, etc.

Further, the compound or a salt thereof that inhibits the activity of the protein of the present invention can also be used, for example, as an apoptosis action regulator (preferably an apoptosis action promoter).

Therefore, the protein of the present invention is useful as a reagent for screening a compound or its salt that regulates the activity of the protein of the present invention.

That is, the present invention provides a method for screening a compound or a salt thereof that regulates the activity (eg, ligand binding activity, signal transduction activity, etc.) of the protein of the present invention, characterized by using the protein of the present invention. More specifically, for example, (i) the ligand binding activity or signal transduction activity of a cell capable of producing the protein of the present invention; A method for screening a compound or a salt thereof that regulates (promotes or inhibits) the activity of the protein of the present invention, which is characterized by comparing the ligand binding activity or the signal transmission activity of the mixture, is used. In the above screening method, for example, in the case of (i) and (U), the ligand binding activity or signal transduction activity can be determined by a method known per se, for example, J. Immunol. 166 (1), pp. 15-19, 2001 Measure and compare according to the method described in or above.

Specifically, when screening for a compound having a ligand binding promoting action or an intracellular signaling promoting action, (i) the protein expression vector of the present invention is prepared by adding an NFκB binding sequence to a promoter or enhancer. And (ii) a reporter gene containing the protein expression vector of the present invention in the promoter or enhancer of the NFκB binding sequence. When the cells are introduced into cells and cultured in the presence of the test compound, the amount of reporter expression is compared. The repo overnight expression level can be measured by using a reporter protein activity using luciferase activity, alkaline phosphatase activity or the like as an index.

When screening for a compound having an inhibitory effect on ligand binding or intracellular signal transduction, specifically, U) the protein expression vector of the present invention may be used to bind the NFκB binding sequence to a promoter or enhancer. Transfection into cells with the reporter gene contained therein. If necessary, (a) a solution containing ligands such as microbial cell disruption solution, microbial culture supernatant, eukaryotic cell disruption solution, and eukaryotic cell culture supernatant (B) culturing with the addition of a substance having the same binding activity as the ligand itself or (c) a natural ligand; and (ii) using the protein expression vector of the present invention to promote the binding sequence of NFKB. Transfected cells or microbial cell culture supernatant, eukaryotic cell lysate, eukaryotic cells Youe liquid containing the ligand, such as supernatants, by adding a substance having a (b) a ligand itself or (c) a natural ligand comparable binding activity, trial Measure and compare reporter expression levels when cultured in the presence of the test compound. The expression level of the repo overnight can be measured by using the repoprotein activity using luciferase activity, alkaline phosphatase activity and the like as an index.

As a cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing a DNA encoding the protein of the present invention described above is used. As a host, for example, animal cells such as COS 7 cells, CHO cells, and HEK293 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on a cell membrane by culturing by the method described above is preferably used. The method for culturing cells capable of expressing the protein of the present invention is the same as the above-described method for culturing the transformant of the present invention. Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.

For example, the ligand binding activity or signal transduction activity in the case of (ii) is increased by about 20% or more, preferably 30% or more, more preferably about 50% or more, as compared with the case of (i). The test compound to be used is a compound that promotes the activity of the protein of the present invention, wherein the ligand binding activity or signal transduction activity in the case (ii) above is about 20% or more, preferably A test compound that reduces the activity of the protein of the present invention by 30% or more, more preferably about 50% or more, can be selected as the compound that inhibits the activity.

The compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low-toxic drug for enhancing the action of the protein of the present invention.

The compound having an activity of inhibiting the activity of the protein of the present invention is used as a safe and low-toxicity drug for suppressing the physiological activity of the protein of the present invention, for example, a prophylactic / therapeutic agent for cancer, an apoptosis promoting agent and the like. Useful.

Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts And compounds selected from plasma and the like. The salt of the compound includes the peptide of the present invention described above. The same ones as used in the above are used.

Furthermore, since the expression of the gene encoding the protein of the present invention also increases in cancer tissues, compounds or salts thereof that regulate (preferably inhibit) the expression of the gene encoding the protein of the present invention include, for example, breast cancer and colon Cancer, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid It can be used as a prophylactic and therapeutic agent for cancer such as cancer, kidney cancer, brain tumor and blood tumor. Preferably, it is a prophylactic / therapeutic agent for cancers (eg, breast cancer, etc.) that are hormone-independent (eg, estrogen, etc.)-Independent, HER2-negative, etc. Further, the compound that inhibits the expression of the gene encoding the protein of the present invention or a salt thereof can be used, for example, as an apoptosis-controlling agent (preferably, an apoptosis-promoting agent).

Therefore, the DNA of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates the expression of a gene encoding the protein of the present invention.

The screening method includes (iii) culturing cells capable of producing the protein of the present invention, and (iv) culturing cells capable of producing the protein used in the present invention in the presence of the test compound. There is a screening method which is characterized by comparing with the case.

In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases (ii) and (iv) is measured and compared.

Examples of the test compound and cells having the ability to produce the protein of the present invention include the same cells as described above.

The amount of the protein is measured by a known method, for example, using an antibody that recognizes the protein of the present invention, and measuring the protein present in a cell extract or the like according to a method such as Western analysis, ELISA, or a method analogous thereto. can do. The mRNA amount can be measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a probe, or

'PCR method using nucleic acid containing SEQ ID NO: 2 or a part thereof as one Or it can measure according to the method according to it.

For example, a test compound that promotes gene expression in the case of the above (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii) As a compound that promotes the expression of a gene encoding the protein of the present invention, the gene expression in the case of the above (iv) is about 20% or more, preferably 30% or more, as compared with the case of the above (iii). Test conjugates that inhibit at least about 50%, more preferably at least about 50%, can be selected as compounds that inhibit the expression of the gene encoding the protein of the present invention.

The screening kit of the present invention contains cells capable of producing the protein or partial peptide used in the present invention or a salt thereof, or the protein or partial peptide used in the present invention.

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a test compound as described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract. A compound selected from animal tissue extract, plasma, or the like, or a salt thereof, which is a compound or a salt thereof that regulates the activity of the protein of the present invention (eg, ligand binding activity or signal transduction activity). .

As the salt of the compound, those similar to the aforementioned salts of the protein of the present invention are used.

A compound or a salt thereof that regulates (preferably inhibits) the activity of the protein of the present invention and a compound or a salt thereof that regulates (preferably inhibits) the expression of a gene encoding the protein of the present invention are, for example, colon cancer, Breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, It is useful as a prophylactic / therapeutic agent for cancer, such as ligament cancer, brain tumor or hematological tumor, and as an apoptosis promoter. Preferably, they are agents for preventing or treating cancers (eg, breast cancer, etc.) that are hormone-independent (eg, estrogen, etc.)-Independent, HER2-negative, etc., and for promoting apoptosis. The above-described prophylaxis of a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention. When used as a therapeutic agent, follow conventional procedures Can be formulated.

For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules). ), Syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of formulation. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and tablets for tablets.

As compositions for parenteral administration, for example, injections, suppositories, etc. are used. Injections are intravenous, subcutaneous, intradermal, intramuscular, intravenous, intraarticular. And dosage forms such as agents. Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol ( For example, propylene glycol, polyethylene glycol), nonionic surfactants [for example, polysorbate 80, HCO-50 (po lyoxye thy 1 ene (50mo 1) adduc t of

hydrogenated castoroi l)]]. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository for rectal administration is prepared by mixing the above antibody or a salt thereof with a conventional suppository base.

The above-mentioned oral or parenteral pharmaceutical composition is conveniently prepared in a unit dosage form adapted to the dose of the active ingredient. Examples of such dosage unit dosage forms include tablets, pills, capsules, injections (ampoules), and suppositories, and usually 5 to 50 mg / dosage per dosage unit dosage form. In particular, it is preferable that the injection contains 5 to 100 mg of the above-mentioned antibody, and other dosage forms contain 10 to 25 mg of the above-mentioned antibody.

In addition, each of the above-mentioned compositions may cause an undesired interaction due to the combination with the above antibody. Unless otherwise specified, other active ingredients may be contained.

The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or in warm-blooded animals (eg mice, rats, puppies, sheep, bush, pussi, puma, birds, cats, dogs, Monkeys, chimpanzees, etc.) orally or parenterally.

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.For example, a compound or a salt thereof that regulates the activity of the protein of the present invention for the purpose of treating breast cancer. When the compound is orally administered, generally in an adult (assuming a body weight of 60 kg), the compound or a salt thereof is used in an amount of about 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1 to 10 Omg per day. Administer 0 to 20 mg. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like, but, for example, regulates the activity of the protein of the present invention for the purpose of treating breast cancer. When a compound or a salt thereof is administered to an adult (with a body weight of 6 O kg) usually in the form of an injection, the compound or a salt thereof is about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg per day. More preferably, about 0.1 to 1 Omg is administered by injection to the cancerous lesion. In the case of other animals, the dose can be administered in terms of the weight per 6 O kg.

(2) Quantification of the protein of the present invention, its partial peptide or its salt

An antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention. In particular, it can be used for quantification by sandwich immunoassay.

That is, the present invention

(i) reacting the antibody of the present invention with a test solution and the labeled protein of the present invention competitively, and measuring the ratio of the labeled protein of the present invention bound to the antibody. A method for quantifying the protein of the present invention in a test solution characterized by:

(ii) After reacting the test solution with the antibody of the present invention insoluble on the carrier and another labeled antibody of the present invention simultaneously or continuously, the activity of the labeling agent on the insoluble carrier is determined. The present invention provides a method for quantifying the protein of the present invention in a test wave, characterized by measuring In the quantification method (ii) above, it is desirable that one antibody is an antibody that recognizes the N-terminal of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal of the protein of the present invention. .

In addition, the protein of the present invention can be quantified using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like. For these purposes, 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 in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, ^[125 iota], ^[131 1], [¾], and ^[14 c] used. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, 3-galactosidase,) 3-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Carriers such as agarose, dextran, and cellulose Examples include soluble polysaccharides, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass.

In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test wave can be quantified. 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. Also, in the immunoassay by the sandwich method, 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.

In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is an antibody having different binding sites to the protein of the present invention. Is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the protein of the present invention, the antibody used in the primary reaction is Preferably, an antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.

The monoclonal antibody of the present invention can be used in a measurement system other than the San Deutsch method, for example, a competition method, an immunometric method, or a nephrometry. In the competition method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen (F) and the recognized antigen (B) bound to the antibody are separated. I

(BZF separation) Measure the amount of labeling of either B or F, and quantify the amount of Bobara in the test wave. In this reaction method, 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, and a solid-phased antibody was used as the first antibody. Alternatively, a solid phase method using a soluble first antibody and a solid phase antibody as the second antibody is used.

In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. Reaction with an excess amount of the labeled antibody, and then adding immobilized antigen to the unreacted labeled antibody. After binding the body to the solid phase, the solid and liquid phases are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.

In nephelometry, 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.

In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, and the like need to be set. The protein measuring system of the present invention may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, reference can be made to reviews, documents, etc.

For example, edited by Hiro Irie, "Radio Imuno Atsushi" (Kodansha, published in 1974), edited by Hiro Irie, "Radio Imuno Atssey" (Kodansha, published in 1974), edited by Eiji Ishikawa et al. "The Enzyme Immunoassay Method" (2nd edition) (Medical Shoin, published in 1982), "Enzyme Immunoassay Method" (ed. 3rd Edition), edited by Eiji Ishikawa et al. Edition) (Medical Shoin, published in 1987), "Methods in ENZYMOLOGYJ Vol.

70 (Immunochemical Techniques (Part A)), ibid.Vol. 73 (Immunochemical Techniques (Part B)), ibid.Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part d: Selected Immunoassays)), ibid.Vol. 92 (Iimunochemical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)) ibid.Vol. 121 (Immunochemical

Techniques (Part I: Hybrid Technology and Monoclonal Ant ibodies)) (above, published by Academic Press) can be referred to.

As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.

Furthermore, when an increase or decrease in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It can be diagnosed as having cancer, such as kidney cancer, brain tumor or hematological tumor, or is more likely to be affected in the future.

Further, 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. In addition, for the preparation of an antibody column used for purifying the protein of the present invention, 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.

(3) Genetic diagnostics

The DNA of the present invention can be used, for example, as a probe to produce human or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, Abnormalities (genetic abnormalities) in the DNA or mRNA encoding the protein of the present invention or its partial peptide in monkeys, chimpanzees, etc.). Alternatively, it is useful as a diagnostic agent for genes such as decreased expression, increased DNA or mRNA or excessive expression.

The above-described genetic diagnosis using the DNA of the present invention includes, for example, the known Northern Hybridization ゃ PCR-SSCP method (Genomics, Vol. 5, pp. 874-879, Q989), Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)).

For example, if overexpression or decrease is detected by Northern hybridization or DNA mutation is detected by PCR-SSCP method, for example, colon cancer, breast cancer, lung cancer, and prostate Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, visceral cancer, It can be diagnosed as having a high possibility of cancer such as a brain tumor or a blood tumor.

(4) a drug containing an antisense polynucleotide The antisense polynucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppress the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo. (Eg, ligand binding activity or signal transduction activity), such as colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, and spleen Can be used as a prophylactic / therapeutic agent for cancer such as cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor . Preferably, it is a preventive / therapeutic agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen, etc.)-Independent, HER2 expression-negative, etc. In addition, the antisense polynucleotide of the present invention can be used, for example, as an apoptotic action regulator (preferably an apoptotic action promoter).

When the antisense polynucleotide is used as the above-mentioned prophylactic / therapeutic agent or modulator, it can be formulated and administered according to a method known per se.

In addition, for example, the above-mentioned antisense polynucleotide is inserted alone or into a suitable vector such as a retrovirus vector, an adenovirus vector, an adenovirus association virus vector, etc. It can be administered orally or parenterally to mammals (eg, rats, egrets, sheep, sheep, bush, birds, cats, dogs, monkeys, etc.). The antisense polynucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be made into an aerosol and administered topically into the trachea as an inhalant.

Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the above-mentioned antisense polynucleotide is formulated alone or together with a carrier such as liposome (injection), and is intravenously or subcutaneously. Etc. may be administered.

The dose of the antisense polynucleotide varies depending on the disease to be treated, the subject of administration, the route of administration, and the like.For example, when the antisense polynucleotide of the present invention is administered for the purpose of treating breast cancer, the dose is generally used for adults. (Weight 60 kg), the antisense polynucleotide is administered in an amount of about 0.1 to 10 O mg per day. 03 007926

44 Further, the antisense polynucleotide can be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells.

Similarly to the above-mentioned antisense polynucleotide, a double-stranded RNA containing a part of the RNA encoding the protein of the present invention, a lipozyme containing a part of the RNA encoding the protein of the present invention, etc. Since the expression of the gene of the present invention can be suppressed and the function of the protein used in the present invention or the DNA used in the present invention in vivo can be suppressed, for example, for example, colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, thyroid cancer Prevention and treatment of cancer such as cancer, brain tumor or blood tumor, preferably prevention of cancer (eg, breast cancer, etc.) independent of hormones (eg, estrogen), HER2 negative, etc.Furthermore, it can be used as an apoptosis action regulator or the like.

The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).

The lipozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by linking a known lipozyme to a part of RNA encoding the protein of the present invention. A part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known lipozyme.

Prevention of the above double-stranded RNA or lipozyme. When used as a therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. (5) a drug containing the antibody of the present invention

The antibody of the present invention, which has the activity of neutralizing the activity of the protein of the present invention, includes, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer It can be used as a prophylactic or therapeutic agent for cancers such as renal, bladder, uterine, ovarian, testicular, thyroid, knee, brain or blood tumors it can. Preferably, it is a prophylactic / therapeutic agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen, etc.)-Independent, HER2-expressing negative, etc. Further, the antibody of the present invention can also be used, for example, as an apoptosis action regulator (preferably, an apoptosis action promoter).

The prophylactic or therapeutic agent for the above-mentioned diseases, which comprises the antibody of the present invention, has low toxicity, and is used as it is as a liquid or as a pharmaceutical composition in an appropriate dosage form, in humans or mammals (eg, rat, rabbit, etc.). It can be given orally or parenterally (eg, intravenously, subcutaneously, etc.) to cattle, higgins, bushes, puppies, cats, dogs, monkeys, etc.).

The antibody of the present invention may be administered as it is, or may be administered as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the antibody of the present invention or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.

Examples of compositions for parenteral administration include injections, suppositories, etc., and injections include intravenous, subcutaneous, intradermal, intramuscular, and intravenous injections. Shapes may be included. Such an injection can be prepared according to a known method. Injections can be prepared, for example, by dissolving, suspending or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid commonly used for injections. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable solubilizing agents, for example, alcohol (e.g., ethanol), polyalcohol (e.g., , Propylene glycol, polyethylene glycol), nonionic surfactants [eg, Polysorbate 80, HC0-50

(polyoxye thy 1 ene (50mo 1) adduc tof hydrogenated cas tor oil)]. As the 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 preferably filled in a suitable ampoule. A suppository for rectal administration may be prepared by mixing the antibody or a salt thereof with a usual suppository base. Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such compositions are prepared by known methods and may contain carriers, diluents or excipients commonly used in the field of formulation. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used. The above-mentioned parenteral or oral pharmaceutical composition is conveniently prepared in a unit dosage form so as to be compatible with the dosage of the active ingredient. Such dosage unit forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The content of the antibody is preferably 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injection, and 10 to 25 Omg for other dosage forms.

The dosage of the prophylactic / therapeutic or diagnostic agent (pharmaceutical) containing the antibody of the present invention varies depending on the administration target, target disease, symptoms, administration route, and the like. When used, the antibody of the present invention is used in a single dose, usually about 0.01 to 2 OmgZkg body weight, preferably about 0 :! to about 1 OmgZkg body weight, and more preferably about 0.1 to 5 mgZkg body weight. Is administered by intravenous injection about 1 to 5 times a day, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.

The antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such a composition is provided as a dosage form suitable for oral or parenteral administration (eg, intravascular injection, subcutaneous injection, etc.).

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.

(6) The compound having the activity of regulating (preferably inhibiting) the activity of RI PK2 of the present invention Or a prophylactic / therapeutic agent for cancer containing a salt thereof or a "preventive / therapeutic agent for cancer containing a compound having an effect of regulating (preferably inhibiting) the expression of RI PK2 or a salt thereof". about

The “compound having an activity of regulating (preferably inhibiting) the activity of RI PK2” may be any compound having an activity of regulating (preferably inhibiting) the activity of RIPK2, such as colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, hepatic cancer It is used as a prophylactic / therapeutic agent for cancer such as cancer, brain tumor, or blood tumor.

“Compound having an action of regulating (preferably inhibiting) the expression of R I PK2”

Any compound can be used as long as it has a K2 expression regulating (preferably inhibiting) action, such as colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, Used as a prophylactic and therapeutic agent for cancer such as spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor, or blood tumor Can be

The prophylactic / therapeutic agent is produced in the same manner as described above.

(7) DNA transfer animal

The present invention relates to a DNA encoding the exogenous protein of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention) or a mutant DNA thereof (may be abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the formula:

That is, the present invention

(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,

(2) the animal according to (1), wherein the non-human mammal is a rodent;

(3) The animal according to (2), wherein the rodent is a mouse or a rat, and

(4) It is intended to provide a recombinant vector containing the exogenous DNA of the present invention or its mutant DNA, which can be expressed in mammals.

Non-human mammals having the exogenous DNA of the present invention or the mutant DNA thereof (hereinafter abbreviated as the DNA transgenic animal of the present invention) include unfertilized eggs, fertilized eggs, spermatozoa, For germ cells and the like including progenitor cells, calcium phosphate is preferably used at the stage of embryonic development in non-human mammal development (more preferably, at the stage of single cells or fertilized egg cells and generally before the 8-cell stage). It can be produced by transferring the desired DNA by the method, electric pulse method, lipofection method, coagulation method, microinjection method, particle gun method, DEAE-dextran method, etc. Further, by the DNA transfer method, the exogenous DNA of the present invention intended for somatic cells, organs of living organisms, tissue cells, and the like can be transferred and used for cell culture, tissue culture, and the like. Can be fused with the above-mentioned germ cells by a cell fusion method known per se to produce the DNA-transferred animal of the present invention.

As non-human mammals, for example, porcupines, pigs, higgins, goats, magpies, dogs, cats, guinea pigs, eight-muster, ma., Males, rats and the like are used. Above all, rodents with relatively short ontogeny and biological cycles in terms of the creation of disease animal model systems, and easily breeding rodents, especially mice (for example, C57B LZ6 strains, DBA2 strains as pure strains, etc.) As a cross line, a B6C3F line, a BDFi line, a B6D2F line, a BALBZc line, an ICR line, etc., or a rat (for example, Wistar, SD, etc.) are preferable.

As the “mammal” in the recombinant vector that can be expressed in mammals, human and the like can be mentioned in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers not to the DNA of the present invention originally possessed by a non-human mammal, but to the DNA of the present invention once isolated and extracted from a mammal.

As the mutant DNA of the present invention, those having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, addition or deletion of a base, substitution with another base, etc. DNA that has been used is used, and also includes abnormal DNA.

The abnormal DNA means a DNA that expresses an abnormal protein of the present invention, and for example, DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.

The exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the target animal. In transferring the DNA of the present invention to a target animal, a DNA linked to a downstream of a promoter capable of expressing the DNA in animal cells is used. It is generally advantageous to use it as an A construct. For example, when the human DNA of the present invention is transferred, various mammals having the DNA of the present invention having a high homology to the human DNA (eg, egret, dog, cat, guinea pig, hamster, rat, mouse, etc.) By microinjecting the DNA construct (eg, vector 1) of the present invention bound to the human DNA into a fertilized egg of a target mammal, for example, a mouse fertilized egg, downstream of various promoters capable of expressing the derived DNA. It is possible to create a DNA transgenic mammal that highly expresses the DNA of the present invention.

Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as λ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or a baculovirus. For example, animal viruses such as E. coli are used. Among them, a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis or a plasmid derived from yeast are preferably used.

Examples of promoters that regulate DNA expression include: (1) a promoter of DNA derived from a virus (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.). Yuichi, ② Promoters derived from various mammals (humans, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), for example, albumin, insulin II, ゥ roplakin II, Erasuyuze, erythropoietin, Endothelin, muscle creatine kinase, glial fibrillary acidic protein, daryuthion S-transferase, platelet-derived growth factor / 3, keratin Kl, 1 ^ 10 and 14, collagen I and II , Cyclic AMP-dependent protein kinase / 3 I-subunit, dystrophin, Tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor, endothelial receptor ostial synthase (generally abbreviated as Tie 2), sodium potassium adenosine 3 kinase (Na, K-ATPase), Monofilament light chain, metamouth thionine I and I IA, metalloproteinase 1 tissue in situ, MHC class I antigen (H-2L), H-ras, renin, dopamine / 3-hydroxylase, thyroid peroxidase (TPO) , Peptide chain elongation factor la (EF-1α), β-actin, 03007926

50 Myosin light chains 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α-actin, prebub enkephalin Α, A promoter such as vasopressin is used. Among them, cytomegalovirus promoter, which can be highly expressed in the whole body, human peptide chain elongation factor 1 (EF-1) promoter, human and chicken j3 actin promoter, and the like are preferable.

The vector preferably has a sequence (generally called terminator) that terminates transcription of the target messenger RNA in the DNA-transferred mammal. For example, it is derived from viruses and various mammals. The sequence of each DNA can be used, and preferably, SV40 terminator of Simian virus or the like is used.

In addition, the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are used to further express the target exogenous DNA at 5 'upstream of the promoter region, the promoter region and the translation region. It is also possible to ligate between them or at the 3 'downstream of the translation region.

The normal translation region of the protein of the present invention is derived from a liver, a kidney, a thyroid cell, a fibroblast derived from a human or various mammals (for example,-ゥ egret, dog, cat, guinea pig, hamster, rat, mouse, etc.). All or part of genomic DNA from DNA and various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cell, and fibroblast-derived RNA can be used as raw materials. I can do it. In addition, a foreign abnormal DNA can produce a translation region obtained by mutating a normal protein translation region obtained from the above cells or tissues by a point mutagenesis method.

The translation region can be prepared as a DNA construct that can be expressed in a transgenic animal by a conventional DNA engineering technique in which the translation region is ligated downstream of the promoter and, if desired, upstream of the transcription termination site.

Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. Production after DNA transfer The presence of the exogenous DNA of the present invention in the germ cell of the product means that all the progeny of the produced animal retain the exogenous DNA of the present invention in all of the germinal and somatic cells . The offspring of such animals that have inherited the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of their germinal and somatic cells.

The non-human mammal to which the exogenous normal DNA of the present invention has been transferred is confirmed to stably maintain the exogenous DNA by mating, and is subcultured as an animal having the DNA in a normal breeding environment. I can do it.

Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the animal after transfer of DNA indicates that all of the offspring of the animal produced have the extraneous DNA of the present invention in all of their germinal and somatic cells. 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. By obtaining homozygous animals having the introduced DNA on both homologous chromosomes and mating the male and female animals, all offspring can be bred and passaged so as to have the DNA in excess.

The non-human mammal having the normal DNA of the present invention expresses the normal DNA of the present invention at a high level, and finally promotes the function of endogenous normal DNA, thereby finally obtaining the protein of the present invention. May develop hyperfunction, and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention and to examine a method for treating these diseases. It is possible.

In addition, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, a prophylactic / therapeutic agent for a disease associated with the protein of the present invention, Cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid gland It can also be used for screening tests for prophylactic and therapeutic agents for cancer such as cancer, kidney cancer, brain tumor, and blood tumor.

On the other hand, the non-human mammal having the foreign abnormal DNA of the present invention After confirming that sex DNA is stably maintained, the animal can be subcultured as a DNA-bearing animal in a normal breeding environment. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material. The DNA construct with the promoter can be prepared by ordinary DNA engineering techniques. The transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all of its germ cells and somatic cells. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germinal and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing these male and female animals, it is possible to breed so that all offspring have the DNA.

In the non-human mammal having the abnormal DNA of the present invention, 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. For example, 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.

Further, as a specific possibility, the abnormal DNA highly expressing animal of the present invention can be used to inhibit the function of the normal protein by the abnormal protein of the present invention in the function-inactive refractory disease of the protein of the present invention (dominant negative activity). Action). Further, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, the agent for preventing or treating the protein of the present invention or a functionally inactive refractory agent, for example, Colorectal, breast, lung, prostate, esophageal, stomach, liver, biliary, spleen, kidney, bladder, uterine, ovarian, testicular, It can also be used for screening tests for prophylactic and therapeutic agents for cancers such as thyroid cancer, knee cancer, brain tumor, and blood tumor.

Further, other possible uses of the above two DNA-transferred animals of the present invention include, for example, ① Use as a cell source for tissue culture,

②Specific expression or activation by the protein of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing the peptide tissue expressed by DNA Analysis of the relationship with the peptide

(3) Cells from tissues having DNA are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture.

薬剤 Screening of drugs that enhance cell function by using the cells described in ③ above, and

単離 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.

In addition, 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. Furthermore, the protein of the present invention can be identified, its relationship with apoptosis, differentiation or proliferation, or its signal transduction mechanism can be examined, and its abnormality can be examined. It is an effective research material for elucidation.

Furthermore, in order to use the DNA transgenic animal of the present invention to develop a therapeutic agent for a disease associated with the protein of the present invention, including a refractory inactive type of the protein of the present invention, Using a quantitative method or the like, it is possible to provide an effective and rapid screening method for the therapeutic agent for the disease. Also, using the DNA transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a method for treating a DNA associated with the protein of the present invention.

(8) Knockout animal The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated and a non-human mammal deficient in expression of the DNA of the present invention.

That is, the present invention

(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated,

(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a repo overnight gene (eg, a 3-galactosidase enzyme gene derived from Escherichia coli).

(3) The embryonic stem cell according to (1), which is neomycin-resistant,

(4) the embryonic stem cell according to (1), wherein the non-human mammal is a rodent;

(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,

(6) a DNA-deficient non-human mammal in which the DNA of the present invention has been inactivated,

(7) The DNA is inactivated by introducing a reporter gene (eg, a / 3-galactosidase enzyme gene derived from Escherichia coli), and the repo allele gene is a promoter of the promoter of the present invention. The non-human mammal according to (6), which can be expressed under control,

(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent, (9) the non-human mammal according to (8), wherein the rodent is a mouse, and

(10) A test compound is administered to the animal described in (7), and the expression of a reporter gene is detected. A screening method is provided.

A non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated is an artificially mutated DNA of the present invention possessed by the non-human mammal, which suppresses the expression ability of the DNA, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA substantially does not have the ability to express the protein of the present invention (hereinafter referred to as the knockout DNA of the present invention). Non-human mammalian embryonic stem cells (hereinafter abbreviated as ES cells).

As the non-human mammal, the same one as described above is used.

The method of artificially mutating the DNA of the present invention can be carried out, for example, by deleting a part or all of the DNA sequence or inserting or substituting another DNA by a genetic engineering technique. . These mutations can, for example, shift the reading frame of codons or disrupt the function of Promote or Exon. What is necessary is just to prepare the knockout DNA of the present invention.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated (hereinafter abbreviated as the DNA-inactivated ES cells of the present invention or the knockout ES cells of the present invention) include, for example, The DNA of the present invention possessed by a non-human mammal is isolated, and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or 1acZ (j3-galactosidase gene), cat Inserting a reporter gene or the like represented by (chloramphenicylacetyltransferase gene) disrupts exon function, or terminates transcription of the gene in the intron portion between exons. For example, by introducing a po 1 yA additional signal) and preventing the synthesis of a complete messenger RNA, the resulting A DNA chain having a DNA sequence constructed so as to disrupt the gene (hereinafter, abbreviated as “gathering vector 1”) is introduced into the chromosome of the animal by, for example, a homologous recombination method. A DNA sequence on or near the DNA of the present invention used for Southern hybridization analysis or a targeting vector and a DNA sequence on a targeting vector other than the DNA of the present invention used for preparing a targeting vector Can be obtained by analyzing by the PCR method using as primers and selecting the knockout ES cells of the present invention.

As the original ES cells for inactivating the DNA of the present invention by the homologous recombination method or the like, for example, those already established as described above may be used.

It may be newly established according to the method of Evans and Kaufma. For example, in the case of mouse ES cells, currently, 129 ES cells are generally used, but since their immunological background has not been fixed, a pure line that substitutes them has been used for immunological inheritance. For example, for the purpose of obtaining ES cells with a clear background, BDFi mice (C57BL / 6 and DBAZ2 and C57BL / 6 and DBAZ2) BDFi mice have the advantage of high number of eggs collected and robust eggs, and have the background of 'C57BL / 6 mice, The ES cells obtained using this were used to generate C57 It can be used to advantage in that loss can replace its genetic background with C57BL / 6 mice.

In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used.However, it is more efficient to collect 8-cell embryos and culture them up to blastocysts. Many early embryos can be obtained.

Either male or female ES cells may be used, but male ES cells are generally more convenient for producing a germline chimera. It is also desirable to discriminate between males and females as soon as possible in order to reduce the complexity of culturing.

As an example of a method for determining the sex of ES cells, a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR can be given as an example. Using this method, conventionally, for example G-banding method, it requires about 1 0 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 5 0) However, the primary selection of ES cells at the initial stage of culture can be performed by gender discrimination, and the early selection of male cells can greatly reduce the labor required at the initial stage of culture.

The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes in the obtained ES cells is preferably 100% of the normal number. However, if it is difficult due to physical operations at the time of establishment, knock out the gene of the ES cells, and then use normal cells (for example, It is desirable to clone again into the mouse (cells with 2 n = 40 chromosomes).

Embryonic stem cell lines obtained in this way usually have very good proliferative properties, but they must be carefully subcultured because they tend to lose their ability to generate individuals. For example, on a suitable feeder cell, such as STO fibroblasts, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, in the presence of LIF (1 to 10,000 U / ml)) Culture at about 37 ° C with 5% carbon dioxide gas and 90% air. At the time of subculture, for example, trypsin / EDTA solution (usually 0.001 to 0.5% trypsin / 0.1 l A 5 mM EDTA (preferably, about 0.1% tris / L M EDTA) treatment may be used to form a single cell and inoculate it on a newly prepared feeder cell. Such subculture is usually performed every 1 to 3 days. At this time, cells are observed, and morphologically abnormal cells are found. If so, the cultured cells should be discarded.

ES cells can be transformed into various types of cells, such as parietal, visceral, and cardiac muscle, by monolayer culture up to high density or suspension culture until cell clumps are formed under appropriate conditions. It is possible to differentiate [M.]. Evans and MH

Kaufman, Nature 292, 154, 1981; GR Mart in, Proc. Natl. Acad. Sci. USA 78, 7634, 1981; TC Doetschman et al., Journal of Ob. And experimental morphology, Vol. 87, p. 27, 1985), and the DNA-incompletely expressed cells of the present invention obtained by differentiating the ES cells of the present invention Useful in cell biology studies.

The non-human mammal deficient in DNA expression of the present invention can be distinguished from a normal animal by measuring the mRNA level of the animal using a known method and indirectly comparing the expression level.

As the non-human mammal, those similar to the aforementioned can be used.

The non-human mammal deficient in DNA expression of the present invention can be obtained, for example, by introducing the evening-getting vector prepared as described above into a mouse embryonic stem cell or a mouse egg cell and introducing the evening-getting vector into a non-human mammal. The DNA of the present invention is inactivated by homologous recombination to replace the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination, thereby obtaining the DNA of the present invention. Can be knocked out.

Cells in which the DNA of the present invention has been knocked out can be analyzed by Southern hybridization analysis or DNA analysis using a DNA sequence on or near the DNA of the present invention as a probe. It can be determined by analysis by PCR using, as a primer, the DNA sequence of a neighboring region other than the DNA of the present invention derived from the mouse used for the targeting vector. When a non-human mammalian embryonic stem cell is used, the cell line in which the DNA of the present invention has been inactivated is cloned by homologous gene recombination, and the cell line is cloned at an appropriate time, for example, at the 8-cell stage. The chimeric embryo is injected into a human mammalian embryo or blastocyst, and the resulting chimeric embryo is transplanted into the uterus of the pseudo-pregnant non-human mammal. The resulting animal will be artificially altered from cells having a normal DNA locus of the present invention. It is a chimeric animal composed of both cells having different DNA loci of the present invention. When a part of the germ cells of the chimeric animal has the mutated 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. Can be obtained by, for example, selecting an individual composed of cells having the DNA locus of the present invention to which the DNA is added by, for example, determining the coat color. 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.

In the case of using an egg cell, for example, a transgenic non-human mammal having a chromosome into which a gettering vector has been introduced can be obtained by injecting a DNA solution into a nucleus of an egg by a microinjection method. Compared to a transgenic non-human mammal, it can be obtained by selecting a gene having a mutation in the DNA locus of the present invention by homologous recombination.

In the individual knocked out of the DNA of the present invention in this way, the animal individuals obtained by mating should also be confirmed to be knocked out of the DNA, and reared in a normal breeding environment. Can be.

Furthermore, the germline can be obtained and maintained according to a standard method. That is, by mating male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in such a manner that one normal individual and plural homozygotes are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and passaged.

The non-human mammalian embryonic stem cells in which the DNA of the present invention has been inactivated are extremely useful for producing the non-human mammal deficient in expression of the DNA of the present invention.

In addition, since 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. (8a) A method for screening a compound having a therapeutic / preventive effect on diseases caused by DNA deficiency or damage, etc. of the present invention

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.

That is, 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. To provide a method for screening a compound or a salt thereof having a therapeutic / preventive effect against diseases caused by, for example, cancer.

The non-human mammal deficient in DNA expression of the present invention used in the screening method includes the same ones as described above.

Test compounds include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds are novel compounds. Or a known compound.

Specifically, a non-human mammal deficient in expression of the DNA of the present invention is treated with a test compound, compared with a non-treated control animal, and changes in the organs, tissues, disease symptoms, etc. of the animal are used as indicators. The test compound can be tested for its therapeutic and prophylactic effects.

As 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.

For example, colon, breast, lung, prostate, esophagus, stomach, liver, biliary tract, spleen, kidney, bladder, uterus, ovary, testis When screening for a compound having a therapeutic or preventive effect against cancers such as thyroid cancer, kidney cancer, brain tumor, or blood tumor, a test compound is administered to a non-human mammal deficient in DNA expression of the present invention. Then, the difference in the degree of onset of cancer and the degree of cure of cancer in the non-administered test compound group are observed over time in the above tissues.

In the screening method, when a test compound is administered to a test animal, When the disease symptom of the test animal is improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more, the test compound has a therapeutic / preventive effect on the above-mentioned diseases. It can be selected as a compound.

The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect on a disease caused by a deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic or therapeutic agent. Further, a compound derived from the compound obtained by the above screening can also be used.

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkalis). Salts with metals and the like are used, and especially preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).

A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the drug containing the protein of the present invention described above. The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs). , Monkeys, etc.).

The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. For example, 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 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg of the compound is administered per day. In the case of parenteral administration, the single dose of the compound varies depending on the administration subject, the target disease and the like. For example, the compound is usually in the form of an injection and usually used for adult (with a body weight of 6 O kg) breast cancer. When administered to a patient, the compound is administered in a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 mg. It is convenient to administer about 1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of weight per 60 kg.

(8b) A method for screening for a compound that promotes or inhibits the overnight activity of the promoter for DNA of the present invention

The present invention provides a test compound administered to a non-human mammal deficient in expression of a DNA of the present invention, and detects the expression of a reporter gene. A method for screening a compound or a salt thereof.

In the above-described screening method, the non-human mammal deficient in expression of DNA of the present invention may be the non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactive by introducing a reporter gene. A gene which can be expressed under the control of a motor for the DNA of the present invention is used.

Examples of the test compound include the same compounds as described above.

As the repo overnight gene, the same gene as described above is used, and a / 3-galactosidase gene (1 acZ), a soluble alkaline phosphatase gene or a luciferase gene is preferable.

In a non-human mammal deficient in expression of the DNA of the present invention in which the DNA of the present invention has been replaced with the repo overnight gene, the repo overnight gene is under the control of the promoter for the DNA of the present invention. By tracing the expression of a substance encoded by one gene, the activity of the promoter can be detected.

For example, when a part of the DNA region encoding the protein of the present invention is replaced by a) 3 monogalactosidase gene (1 ac Z) derived from Escherichia coli, the tissue that expresses the protein of the present invention originally Instead of the protein of the invention,; 3-galactosidase is expressed. Therefore, by staining with a reagent that serves as a substrate for 0-galactosidase such as, for example, 5-promo 4-chloro-3-indolyl; 8-galactobilanoside (X-gal), it is easy to carry out The expression state of the protein of the present invention in an animal body can be observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with datalaldehyde or the like, and phosphate buffered physiological After washing with saline solution (PBS), react with a staining solution containing X-ga 1 at room temperature or around 37 ° C for about 30 minutes to 1 hour, and then tissue samples are washed with ImM ED TAZ PBS solution. By washing, the jS-galactosidase reaction is stopped, and coloration may be observed. In addition, mRNA encoding 1 ac Z may be detected according to a conventional method.

The compound or a salt thereof obtained by using the above-mentioned screening method is a compound selected from the above-mentioned test compounds, and is a compound that promotes or inhibits the DNA promoter activity of the present invention.

The compound obtained by the screening method may form a salt, and the salt of the compound may be a physiologically acceptable acid (eg, an inorganic acid) or a base (eg, an alkali metal). And the like, and particularly preferably a physiologically acceptable acid addition salt. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, Salts with succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used. The compound of the present invention, which promotes or inhibits the activity of the promoter against DNA, or a salt thereof can regulate the expression of the protein of the present invention and regulate the function of the protein, such as colon cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, knee It is useful as a prophylactic and / or therapeutic agent for cancer such as cancer, brain tumor, or blood tumor.

Further, a compound derived from the compound obtained by the above screening can also be used.

A drug containing the compound or a salt thereof obtained by the screening method can be produced in the same manner as the above-mentioned drug containing the protein of the present invention or a salt thereof.

The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs). , Monkeys, etc.). The dose of the compound or a salt thereof varies depending on the target disease, the subject of the administration, the administration route, and the like.For example, when the compound of the present invention that inhibits the promoter activity for DNA is orally administered, generally the adult For a breast cancer patient weighing 60 kg, the compound is administered at about 0.1-100 mg, preferably about 1.0-5 Omg, more preferably about 1.0-2 Omg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, a compound that inhibits the promoter activity of DNA of the present invention is usually administered in the form of an injection to an adult. When administered to a breast cancer patient weighing 6 O kg, the compound is administered in an amount of about 0.01 to 3 Omg per day, preferably about 0.:! To 20 mg, more preferably about 0.1 to 1 mg per day. Omg is conveniently administered by intravenous injection. In the case of other animals, the dose can be administered in terms of the weight per 6 O kg.

As described above, the non-human mammal deficient in expression of the DNA of the present invention is extremely useful for screening a compound or a salt thereof that promotes or inhibits the activity of the promoter for the DNA of the present invention. It can greatly contribute to investigating or preventing the causes of various diseases caused by insufficient expression of DNA.

In addition, using a DNA containing a promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof and injected into egg cells of an animal to produce a so-called transgenic animal (gene). Creating an introduced animal) will allow the specific synthesis of the protein and the study of its effects in living organisms. Furthermore, by binding an appropriate repo overnight gene to a portion of the above promoter and establishing a cell line in which this is expressed, the ability of the protein of the present invention itself to be produced in the body can be specifically promoted or suppressed. It can be used as a search system for low molecular compounds that have an action. In the present specification, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations of the IUPAC- IUB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When there is an optical isomer for an amino acid, the L-form is indicated unless otherwise specified.

DNA: Deoxylipo nucleic acid cDNA complementary deoxyliponucleic acid A adenine

T thymine

G Guanin

C

RNA liponucleic acid

mRNA Messe, Nja lipo nucleic acid dATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate dGTP Deoxyguanosine triphosphate dCTP Deoxycytidine triphosphate

ATP Adenosine triphosphate

EDTA ethylenediaminetetraacetic acid

SD S Sodium dodecyl sulfate G 1 y

A 1 a Alanin

Va 1 Valine

Le u leucine

I 1 e

S e r serine

Th r threonine

Cy s

Me t Methionin

G 1 u Glutamic acid

As p Aspartic acid

Lys lysine

Ar g Arginine

H is histidine

Ph e feniralanin Ty r: Tyrosine

Tr p: Tribute fan

Pro: Proline

As n: Asparagine

G in: glutamine

pG 1 u: pyroglutamic acid

S e c: selenocysteine

■ In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

Me: methyl group

E t: ethyl group

B u: butyl group

Ph: phenyl group

TC: thiazolidine-4 (R) -carboxamide group

To s: P-toluenesulfonyl

CHO: Formyl

B z 1: benzyl

Cl ₂ -Bzl: 2,6-dichlorobenzene

Bom: benzyloxymethyl

Z: benzyloxycarponyl

C 1-Z: 2-clozen benzyloxycarponyl

B r— Z: 2-bromobenzyloxycarbonyl

Bo c: t-butoxycarponyl

DNP: dinitrophenyl

Trt: Trityl

Bum: t-butoxymethyl

Fmo c: N-9-Fluorenylmethoxycarbonyl

HOB t: 1-hydroxybenztriazole

HOOB t: 3, 4-dihydro-3-hydroxy-1 4-oxo-1 1, 2, 3-benzotriazine

HONB: Trihydroxy-5-norporene-2,3-dicarboximide

DCC: N, N, dicyclohexylcarposimide The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

2 shows the amino acid sequence of R I PK2.

[SEQ ID NO: 2]

This shows the base sequence of DNA encoding RIPK2 having the amino acid sequence represented by SEQ ID NO: 1.

[SEQ ID NO: 3]

1 shows the nucleotide sequence of DNA containing the full length gene encoding RIPK2.

[SEQ ID NO: 4]-This shows the base sequence of the antisense oligonucleotide used in Example 2. [SEQ ID NO: 5]

3 shows the nucleotide sequence of the oligonucleotide used in Example 2.

[SEQ ID NO: 6]

3 shows the nucleotide sequence of a primer used in Example 2.

[SEQ ID NO: 7]

3 shows the nucleotide sequence of a primer used in Example 2.

[SEQ ID NO: 8]

4 shows the nucleotide sequence of a probe used in Example 2. Hereinafter, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto. Example 1

To identify genes that are specifically upregulated in breast cancer tissue, total RNA (Table 1) extracted from two breast cancer tissues and 23 other normal tissues was used as a material. Gene expression analysis was performed using an oligonucleotide microarray (Human Genome U95A, U95B, U95C, U95D, U95E; Affymetrix).

The experimental method followed Affymetrix's experiment guide (Expression analysis technical manual). As a result, increased expression of the RIM2 gene was detected in breast cancer tissues (Table 2).

〔table 1〕

RNA extracted tissue

Breast cancer tissue (patient number 9) BioClinical Partners

Fat BioChain Institute

Skeletal muscle CI on tech

Heart Clontechi

CI on tech

Adrenal gland Clontechfh

Liver Clontech

Win Jfe Clontech

Clontech

CI on tech

Lung CI on tech

Whole brain CI on tech

Cerebellum Clontech

Thymus CI on tech

Mammary gland Clontech

Clontech

Rectal BioChain Institute

Large intestine BioChain Institute

Uterus CI on tech

Cervical BioChain Institute- (Table 2) Tissue gene expression:

Breast cancer tissue (patient number 9) 1.9

Fat ND

Skeletal muscle ND

, ND

Kidney ND

Adrenal gland ND

Liver ND

Monkey ND

Spleen ND

Tracheal ND

Lung ND

Whole brain ND

Cerebellar ND

Thymus ND

Mammary gland ND

Sleeping gland ND

ND

Rectal ND

Large intestine ND

Uterus .ND

Cervical ND

aThe gene expression level was normalized with the median expression level of all genes whose expression was detected by the oligonucleotide microarray as 1.

ND; not detected

Example 2 In order to analyze the effect of RIPK2 gene on the apoptosis observed in breast cancer tissues, RI PK2 antisense oligonucleotide transfection experiments were performed. First, after designing an antisense (SEQ ID NO: 4) for the base sequence represented by SEQ ID NO: 3, a phosphorothioated oligonucleotide was synthesized, purified by HPLC, and used for an introduction experiment (Amersham Pharmacia Biotech). As a control oligonucleotide, the reverse sequence (SEQ ID NO: 5) of the base sequence represented by SEQ ID NO: 4 was similarly phosphorothioated, purified by HPLC and used (Amersham Pharmacia Biotech).

Using the breast cancer cell line MDA-MB-231 (In Vitro, vol. 14 (11), 911-915, pp. 911-915, 1978., purchased from ATCC) as test cells, 4 × 10 ⁴ cells were obtained on the day before the introduction of the oligonucleotide. The seeds were seeded on a 24-plate (Falcon). Oligonucleotides (Invitrogen) were used for oligonucleotide introduction, and the protocol was followed. Twenty hours after the introduction, RNA was extracted according to the protocol of RNeasy mini kit (Qiagen), and cDNA was prepared using TaqManRT kit (Perkin-Elmer). At the same time, a similar reaction was carried out without adding reverse transcriptase, and used as a non-reverse transcription control. The expression level of the RIM2 gene was determined using two types of primers (SEQ ID NO: 6 and SEQ ID NO: 7) and a probe (SEQ ID NO: 8), and the ABI7700 (Perkin-Elmer) manual was used. PCR was performed according to the procedure. The expression level of RIPK2 was subtracted from the data obtained using the non-reverse transcription control from the data obtained using the cDNA, and then corrected with the total RNA amount used for comparison.

On the other hand, on the third day after the introduction of the antisense oligonucleotide, a cell death detection ELISA (Roche Co., Ltd.) was conducted to observe the effect on apoptosis when the expression of the RIPK2 gene was suppressed by introducing the antisense oligo. Was compared with the control oligo-introduced sample.

As a result, when the antisense (SEQ ID NO: 4) for the RIM2 gene was used, the expression level of the RIPK2 gene was reduced to 78% at 20 hours after the introduction compared to the control oligonucleotide (SEQ ID NO: 5). Was. On the 3rd day after the introduction, apoptosis increased to 154% when the antisense oligonucleotide (SEQ ID NO: 4) was introduced, assuming that the control oligonucleotide (SEQ ID NO: 5) was 100%. These results confirm that suppressing the expression of the RIPK2 gene, which is upregulated in cancer tissues, causes cancer cells to undergo apoptosis, and that RIPK2 gene promotes cancer cell growth and apoptosis. It was clear that they were involved in Industrial applicability

The expression of the protein of the present invention is increased in cancer tissues, and when the activity of the protein of the present invention is inhibited, cancer cells undergo apoptosis. Therefore, the protein of the present invention and a polynucleotide encoding the protein are diagnostic markers for cancer. Compounds or salts thereof that regulate (preferably inhibit) the activity of the protein, compounds or salts thereof that regulate (preferably inhibit) the expression of the protein gene include, for example, colorectal cancer, breast cancer, lung cancer, Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, sarcoma It can be used safely as a prophylactic or therapeutic agent for cancer such as cancer, brain tumors or hematological tumors. Preferably, it is a prophylactic / therapeutic agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen, etc.)-Independent, HER2 expression negative, etc. Further, the compound that inhibits the activity of the protein or a salt thereof, or the compound that inhibits the expression of the protein gene or a salt thereof can be safely used, for example, as an apoptosis-regulating agent (preferably, an apoptosis-action promoting agent). You can do that too.

In addition, the antisense polynucleotide or antibody of the present invention can inhibit the expression of the protein used in the present invention. For example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver Prevention of cancers such as cancer, biliary tract, spleen, kidney, bladder, uterus, ovary, testis, thyroid, kidney, brain or blood tumors · Therapeutic agent, preferably as a preventive agent for cancer (eg, breast cancer, etc.) that is hormone-independent (eg, estrogen) -independent, HER2-negative, etc., or an apoptotic agent (preferably It can be used safely as an apoptosis action promoter).

Claims

The scope of the claims

1. It comprises a compound or a salt thereof which 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. Cancer prevention and treatment.

2. It contains a compound or a salt thereof that inhibits the expression of a protein or a partial peptide thereof or a salt thereof that has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Prevention and treatment of cancer.

3. Complementary or substantially complementary to the nucleotide sequence of a polynucleotide encoding a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof Antisense polynucleotide containing a base sequence or a part thereof.

4. The antisense polynucleotide according to claim 3, which has a base sequence represented by SEQ ID NO: 4.

'5. An agent for preventing or treating cancer, comprising the antisense polynucleotide according to claim 3.

6. A prophylactic / therapeutic agent for cancer comprising an antibody against a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof. .

7. Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary 7. The prophylactic or therapeutic agent according to claim 1, claim 2, claim 5, or claim 6 which is a cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor.

8. A diagnostic agent for cancer comprising an antibody to 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.

9. A cancer comprising a protein containing an amino acid sequence identical to or substantially identical to the amino acid sequence represented by SEQ ID NO: i, or a polynucleotide encoding a partial peptide thereof. Diagnostics. '

10 .Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary 10. The diagnostic agent according to claim 8 or 9, which is a cancer, testicular cancer, thyroid cancer, Teng's cancer, brain tumor or hematological tumor.

1 1. A prophylactic / therapeutic agent for cancer comprising a compound having an inhibitory activity on Receptor-Interacting Serine / Threonine Kinase 2 or a salt thereof.

1 2. Receptor-Interacting Serine / Threonine Kinase 2 A compound for inhibiting or inhibiting the expression of Kinase 2 or a salt thereof, which is a prophylactic / therapeutic agent for cancer.

13 3. Prevention of cancer characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof · A method for screening a therapeutic agent.

14. Prevention of cancer characterized by containing a protein having an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof · Kit for screening therapeutic agents.

15 5. Prevention of cancer characterized by using a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof · A method for screening a therapeutic agent.

16. Prevention of cancer characterized by containing a polynucleotide encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof · Kit for screening therapeutic agents.

17. An agent for preventing or treating cancer obtainable by using the screening method according to claim 13 or claim 15 or the screening kit according to claim 14 or claim 16.

18. A protein containing 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 compound which inhibits the activity of a salt thereof or a salt thereof Apoptotic action promoter.

1 9. Inheritance of a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof An apoptosis-promoting agent comprising a compound that inhibits the expression of an offspring or a salt thereof.

20. Screening for an apoptosis-activator 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 Method.

21. An agent for promoting apoptosis, 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 polynucleotide encoding a partial peptide thereof. Screening method.

22. An apoptosis-activating agent comprising the antisense polynucleotide according to claim 3.

23. An agent for promoting apoptosis 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.

24. A compound that inhibits the activity of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, A method for preventing and treating cancer, which comprises administering an effective amount of a salt or a compound that inhibits the expression of a gene of the protein or a salt thereof.

25. Inhibits the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, or expresses a gene of the protein A method for preventing and treating cancer characterized by inhibiting cancer.

26. Inhibits the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof, or expresses a gene of the protein A method for promoting an apoptosis effect, which comprises:

27. A protein or partial peptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 for producing a prophylactic or therapeutic agent for cancer A compound that inhibits the activity of the salt or a salt thereof, or Use of a compound or a salt thereof that inhibits expression of the protein gene.

28. A protein or a partial peptide or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 for producing an apoptosis-activating agent Use of a compound or a salt thereof that inhibits the activity of the compound, or a compound or a salt thereof that inhibits the expression of a gene of the protein.