WO2003061697A1

WO2003061697A1 - Preventives/remedies for cancer

Info

Publication number: WO2003061697A1
Application number: PCT/JP2002/013642
Authority: WO
Inventors: Yuichi Hikichi; Ryosuke Katsuyama; Yuichi Kakoi; Satoshi Nishizawa
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2001-12-27
Filing date: 2002-12-26
Publication date: 2003-07-31

Abstract

It is intended to provide preventives/remedies for cancer and so on. More specifically, it is possible to use a compound or its salt controlling 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, an antisense nucleotide having a base sequence or a part thereof complementary or substantially complementary to the base sequence of a DNA encoding a protein or its peptide fragment having an amino acid sequence which is the same or substantially the same as the amino acid sequence represented by SEQ ID NO:1, and so on are usable as preventives/remedies for cancer.

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 pile cancer drugs has improved the survival benefit and is increasing the number of cases toward cure. However, most of the currently used pile cancer agents damage DNA and are highly cytotoxic, inhibiting cell division.Therefore, they cause considerable damage to normal cells. 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, and the technology for finding disease-specific changes in gene expression has spread, and its usefulness has been confirmed. I have. For example, Aiiymetrix's GeneChip system is being used extensively for diagnosing diseases such as cancer and discovering drug discovery target genes.When introduced into cells, antisense oligonucleotides become complementary to RNA with complementary sequences. It hybridizes and induces RNAse degradation by RNAse H to inhibit protein translation, or it also results in direct inhibition of protein synthesis by hybridization. Since it is possible to specifically suppress the function of the target gene, it is frequently used as a means for analyzing the function of a gene, and some antisense oligonucleotides are being developed for clinical application.

Recently, it has been revealed that the To11 receptor functions as a receptor involved in innate immunity in insects without an adaptive immune system. A molecule showing homology to this To11 receptor, Toll-like receptors (TLRs), also exists in mammals and is also implicated in innate immunity. To date, nearly 10 TLRs have been cloned. For example, TLR 2 and TLR 6 recognize lipoproteins and glycolipids, TLR4 recognizes lipopolysaccharide, and TLR5 It has been reported that the recognition of Flagellin and that of Ding 119 are related to the recognition of 0 DNA (Nature Immunology 2 (8), 675-680, 2001). TLR 1 (Proc Natl Acad Sci USA 95 (2), 588-593, 1998) is involved in the recognition of microbial products in cooperation with TLR2 (J Immunol. 165 (12), 7125- 7132, 2000) and that it inhibits the action of the TLR 2 and TLR 6 heteroreceptors (J Inunu nol. 166 (1), 15-19, 2001). More recently, analysis of TLR 1 knockout mice have been made, TLR1, at the same time it is reconfirmed acting Oite both the TLR 2 and ligand recognition, P am ₃ CSK ₄ as a new TLR 1 ligand Trisyl peptides (J Immunol. 169 (1), 10-14, 2002) and OspA (Nat Med. 8 (8), 878-884, 2002) have been reported. Analysis of TLR 2 and TLR4 is also progressing in the study of signal transduction factors.For example, from the analysis of Receptor- interacting serine / threonine kinase-2 (RI PK2) knockout mice, Although shown to work in transmission (Nature. 416 (6877), 190-194, 2002), very little is known about the TLR1 signal.

On the other hand, paclitaxel, which is frequently required as a pile cancer drug, exhibits a lipopolysaccharide-like effect on mouse macrophages, and Kawasaki et al. Found that this effect was due to TLR4 and MD2 signals (J Biol Chem. 275 (4), 2251-2254, 2000). However, they report that both TLR4 and mouse MD2 are required, and that this MD2 is species-specific and that human MD2 does not show the LPS-like effect of paclitaxel. Most of these studies on the TLR1 gene involve cells of the immune system, and there is no report on the relationship between the TLR1 gene and cancer (solid cancer).

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. There is also a keen need for effective treatments for existing pile cancer drug-resistant cancers and drugs that increase the sensitivity to existing anticancer drugs from the viewpoint of reducing side effects. Disclosure of the invention The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found a gene whose expression is remarkably increased in cancer tissues. The invention has been completed.

That is, the present invention

(1) 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; Prevention and treatment of cancer

(2) A compound or a salt thereof that inhibits the expression of a gene of a protein or a partial peptide thereof or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Prevention and treatment of cancer, which contains

(3) a base complementary or substantially complementary to a base sequence of a DNA having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a DNA encoding a partial peptide thereof; An antisense nucleotide containing the sequence or a part thereof; (4) a prophylactic / therapeutic agent for cancer comprising the antisense nucleotide according to the above (3);

(5) 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 thereof or a salt thereof Agent,

(6) Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, Ovarian cancer, testicular cancer, thyroid cancer, thyroid cancer, brain tumor or hematological tumor, the prophylactic or therapeutic agent described in the above (1) to (5),

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

(8) a cancer diagnostic agent comprising a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, (9) Cancer is colorectal, breast, lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, The diagnostic agent according to (7) or (8), which is ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor, or blood tumor,

(10) a prophylactic / therapeutic agent for cancer comprising a compound having a To 11-like receptor activity inhibitory activity or a salt thereof,

(11) A prophylactic / therapeutic agent for cancer comprising a compound having a To 11-like receptor expression inhibitory activity or a salt thereof,

(12) A cancer characterized by using a protein or a partial peptide thereof or a salt thereof containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Screening methods for prophylactic and therapeutic agents,

(13) Prevention of cancer characterized by containing 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 · Kit for screening therapeutic agents,

(14) Prevention of cancer characterized by using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof · Screening methods for therapeutic agents,

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

(16) An agent for preventing or treating cancer, which can be obtained by using the screening method according to (12) or (14) or the screening kit according to (13) or (15).

(17) 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 Apoptosis inducer,

(18) containing a compound or a salt thereof that inhibits the expression of the gene of a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 Apoptosis inducer, (19) an apoptosis-inducing agent characterized by using a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof; Screening method,

(20) An apoptosis-inducing agent characterized by using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. Screening method,

(21) a compound or a salt thereof that inhibits the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof; Pile cancer drug sensitivity enhancer

(22) a compound or a salt thereof that inhibits the expression of a protein or a partial peptide or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1; (23) A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 to a mammal, Administering a compound or a salt thereof that inhibits the activity of the partial peptide or a salt thereof, or an effective amount of a compound or a salt thereof that inhibits the expression of the gene of the protein or the partial peptide or a salt thereof. Cancer prevention and treatment methods,

(24) Prevention of cancer A protein or a partial peptide or a partial peptide or an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 for producing a therapeutic agent. The present invention also provides use of a compound or a salt thereof that inhibits the activity of a salt thereof, or a compound or a salt thereof that inhibits gene expression of the protein or its partial peptide or a salt thereof. Brief description of the drawings.

FIG. 1 shows the results of examining the effect of paclitaxel on apoptosis induction. BEST MODE FOR CARRYING OUT THE INVENTION

An amino acid identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1 The protein containing the sequence (hereinafter, also referred to as the protein of the present invention or the protein used in the present invention) may be a human warm-blooded animal (eg, guinea pig, rat, mouse, chicken, egret, bush, hidge) Cells (eg, hepatocytes, spleen cells, nerve cells, glial cells, knee / 3 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelium) Cell, smooth muscle cell, fibroblast, fiber cell, muscle cell, fat cell, immune cell (eg, macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, Acidocytes, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, breast cells, hepatocytes or stromal cells, or these cells Progenitor cells, stem cells or cancer cells, etc.) or any tissue in which these cells are present, e.g.

, Brain, various parts of the brain (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum), spinal cord, pituitary gland, stomach, knee, kidney, liver, gonad , Thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testicle, ovary, placenta, uterus It may be a protein derived from bone, joint, skeletal muscle, or the like, or may be a synthetic protein.

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

Examples of the protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, a protein having an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 described above. However, a protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable. Examples of substantially equivalent activities include, for example, signal transduction activity (eg, intracellular signal transduction activity of a To11-like receptor (preferably TLR1)), ligand binding activity (eg, To11) Receptor (preferably TLR 1) and its ligand or small molecule-binding activity, and the like. Substantially the same means that their properties are (E.g., physiologically or pharmacologically) indicate homogeneity. Therefore, the signal transmission activity and the ligand binding activity may be equivalent (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). Although preferred, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.

Examples of the ligand include a bacterial lipopeptide (preferably a triacylated lipopeptide or the like) or N-palmitoyl-S-di palmitoylglyceryl (Pam ₃ ) Cys- Ser- (Lys) ₄ obtained by chemical synthesis. (CSK ₄ ), N-palmitolyl-S-dilaurylgryceryl (N-Pam-S-Lau ₂ ) trisyl peptides such as CSK _4, and outer-surface lipoprotein (OspA) derived from Borrelia burg dorieri.

The signal transduction activity and the ligand binding activity can be measured by a known method, for example, the method described in J. Ifflmunol. 166 (1), pp. 15-19, 2001 or a method analogous thereto. . TLR family members activate the NF-κB signal by ligand binding. Therefore, for example, the activity of the protein used in the present invention (eg, To11-like receptor (eg, TLR1)) depends on the expression vector of the protein, the NF- / B binding sequence and the promoter or the promoter. It is introduced into cells together with the reporter gene etc. contained in Enhansa, and if necessary, (a) contains ligands such as microbial cell lysate, microbial culture supernatant, eukaryotic cell lysate, and eukaryotic cell culture supernatant And (b) a substance having the same binding activity as the ligand itself or (c) the natural ligand, and the expression level of the reporter is measured by a known method. The reporter expression level can be measured, for example, by using the repo overnight protein activity using luciferase activity, alkaline phosphatase activity or the like as an index.

In addition, the apoptotic activity against a pile cancer agent (eg, paclitaxel) in the presence or absence of expression of the protein (eg, TLR1) used in the present invention is measured and compared by a known method, and the signal transduction activity is measured. Can be measured. Apoptosis activity against an anticancer agent (eg, paclitaxel, etc.) in the presence and absence of a ligand can be measured and compared by a known method, and the signal transduction activity can be measured.

Furthermore, in order to measure TLR1 signaling used in the present invention with higher sensitivity, a TLR1 signal-related molecule (eg, TLR2, MD2, etc.) is expressed. A cell having the ability may be used, or the above activity may be measured by forcibly expressing a TLR1 signal-related molecule (eg, TLR2, MD2, etc.).

Examples of the protein used in the present invention include: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 100, preferably about 1 to 30; An amino acid sequence in which about 10 amino acids have been deleted, more preferably about 1 to 5 amino acids, and 2 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (for example, about 1 to 100 amino acids). Preferably about 1 to 30, more preferably about 1 to 10, and more preferably a number (1 to 5) of amino acid sequences; ③ an amino acid sequence represented by SEQ ID NO: 1 Or an amino acid sequence into which two or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably about 1 to 5) amino acids have been inserted, 1 SEQ ID NO: 1 or 1 in the amino acid sequence represented by Is an amino acid in which two or more (eg, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, and more preferably a number (1 to 5) amino acids have been substituted with other amino acids) It also includes so-called muteins, such as proteins containing a sequence or an amino acid sequence combining them.

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 protein in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (capilloxy terminus) according to the convention of peptide labeling. The protein used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group (one COOH), a carboxylate (one COO-), an amide ( — It may be either CONH ₂ ) or ester (one COOR).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, isopropyl, C WINCH ₆ alkyl group, such as n- butyl, for example, C _3, such as cyclohexyl cyclopentyl, cyclohexylene - ₈ cycloalkyl group, for example, phenyl, alpha-naphthyl C ₆ _ ₁₂ 7 aryl group, such as, for example, benzyl, phenyl, such as phenethyl - - 2 alkyl or alpha-naphthylmethyl etc. alpha-Nafuchiru § C ₇ _ E ₄ Ararukiru groups such as alkyl groups, such as pivaloyl I Ruo carboxymethyl group is found using.

When the protein used in the present invention has a lipoxyl group (or lipoxylate) at a position other than the C-terminus, a protein in which the lipoxyl group is amidated or esterified is also included in the protein used in the present invention. included. 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, such as C Bok ₆ Arukanoiru such Asechiru group (: I _6- Glycyl group), N-terminal glutamine residue generated by cleavage in vivo, pyroglutamine oxidation, Substituent on the side chain of amino acid in the molecule (for example, 1 OH, one SH, amino group, imidazole group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., protected by a formyl group, a C WINCH ₆ Ashiru group such as C ₆ Arukanoiru group such Asechiru group) Or complex proteins such as so-called glycoproteins to which sugar chains are bound.

Specific examples of the protein used in the present invention include, for example, a protein (TLR1) containing the amino acid sequence represented by SEQ ID NO: 1. The partial peptide of the protein used in the present invention is the partial peptide of the protein used in the present invention described above, and preferably has the same properties as the protein used in the present invention described above. Any one may be used. 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 one or more amino acid sequences (preferably, About 1 to 20 amino acids, more preferably about 1 to 10 amino acids, and even more preferably about 1 to 5 amino acids are added, or 1 or 2 or more amino acids (preferably , About 1 to 20 amino acids, more preferably about 1 to 10 amino acids, still more preferably a number (1 to 5) amino acids, or 1 or 2 or more (preferably (About 1 to 10, more preferably several, and still more preferably about 1 to 5) amino acids may be substituted with other amino acids.

In the partial peptide used in the present invention, the C-terminus may be any one of a hydroxyl group (one COOH), a carboxylate (—COO—), an amide (one C〇NH ₂ ) and an ester (one COOR). Is also good.

Further, similar to the protein used in the present invention, the partial peptide used in the present invention has a lipoxyl group (or carboxylate) other than the C-terminal, and the N-terminal amino acid residue ( E.g., methionine residue) whose amino group is protected by a protecting group, N-terminal cleavage in vivo, glutamine residue generated by lipamine dynamin oxidation, on the side chain of amino acid in the molecule And the like, and those in which the substituents are protected by an appropriate protecting group, or complex peptides such as so-called glycopeptides to which sugar chains are bound.

The partial peptide used in the present invention can also be used as an antigen for producing an antibody.

As the salt of a protein or partial peptide used in the present invention, a salt with a physiologically acceptable acid (eg, an inorganic acid, an organic acid) or a base (eg, an alkali metal salt) is used. Preferred are acid addition salts that are chemically acceptable. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) Succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The protein or its partial peptide or a salt thereof used in the present invention can be produced from the cells or tissues of the above-mentioned human or warm-blooded animal by a known method for purifying a protein, or a protein encoding the protein. It can also be produced by culturing a transformant containing DNA. Also, peptide synthesis described later It can also be manufactured according to the law.

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 achieved by using a combination of chromatography methods.

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 usually be used. Such resins include, for example, chloromethyl resin J3, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM Resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2,, 4'-dimethoxyphenyl Rumo Fmoc aminoethyl) phenoxy resin. Using such a resin, an amino acid appropriately protected with an α-amino group and a side chain functional group is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. 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 carried out in a highly diluted solution to obtain a target protein or partial peptide or an amide thereof. Get the body.

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'-diisopropylcarpo- imide, N-ethyl N '-(3-dimethylaminoprolyl) carbo- imide and the like are used. For these activations, the protected amino acid may be added directly to the resin with a racemization inhibitor additive (eg, HOB t, HO OB t), or may be pre-protected as a symmetric anhydride or HO BT ester or HO OB t ester The amino acid can be added to the resin after activation.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpiperidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; Sulfoxides such as dimethylsulfoxide, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, or an appropriate mixture thereof are used. The reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 120 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. 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. When a sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole to prevent the subsequent reaction from being affected.

Examples of the protecting group for the amino group of the starting material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C 1 _Z, Br—Z, and adaman. Tyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

The carboxyl group may be, for example, an alkyl esterified (eg, a linear, branched or cyclic alkyl such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). Esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarponyl hydrazide , Butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower groups such as an acetyl group. A group derived from carbonic acid such as an arylo group such as a class (C ^ ₆ ) alkanoyl group or a benzoyl group, a benzyloxy carbonyl group, or an ethoxycarbonyl group is used. Examples of a group suitable for etherification include a benzyl group, a tetrahydrobiranyl group, and a t-butyl group.

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

Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc. Can be

Examples of the activated carboxylic acid group of the starting material include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4 Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with HOB t)]. As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used. .

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

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 form of a protein or partial peptide, for example, first, after amidating and protecting the α-hydroxyl group of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side with a desired chain length. And a protein or partial peptide from which only the protecting group for the α-amino group at the Ν-terminal of the peptide chain has been removed and a protein or partial peptide from which only the protecting group for the carboxyl group at the C-terminal has been removed. Are produced, and these proteins or peptides are condensed in a mixed solvent as described above. Details of the condensation reaction are the same as described above. 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 lyophilized to obtain the desired protein or peptide amide.

In order to obtain an ester of a protein or peptide, for example, after condensing an α-hydroxyl group of a carboxy-terminal amino acid with a desired alcohol to form an amino acid ester, a desired protein is prepared in the same manner as in the amide of a protein or peptide. It is possible to obtain an ester of quality or peptide.

The partial peptide used in the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein used in the present invention with an appropriate peptidase. As a peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to thereby remove the desired peptide. Can be manufactured. Examples of the known condensation method and elimination of the protecting group include the methods described in the following ① to ①. ΦΜ. Bodanszky and MA Ondet ti, Peptide Synthes is, Interscience Publ ishers, 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, Biochemistry Laboratory Lecture 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. The DNA may be any of a genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, one of the above-described cells / tissue-derived cDNA libraries, and a synthetic DNA.

The vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. In addition, it is also possible to directly amplify by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the above-mentioned cell'tissue. it can.

Examples of the DNA encoding the protein used in the present invention include, for example, a DNA containing the base sequence represented by SEQ ID NO: 2 or a base string represented by SEQ ID NO: 2 that is highly stringent. A protein containing a base sequence that hybridizes under the conditions and substantially containing the amino acid sequence represented by SEQ ID NO: 1 described above. Any DNA may be used as long as it encodes a protein having the same properties as the above.

Examples of the DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 50% or more, preferably about 6%, of the nucleotide sequence represented by SEQ ID NO: 2. 0% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more. DNA or the like is used.

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

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

More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, DNA having the base sequence represented by SEQ ID NO: 2 or the like is used.

The DNA encoding the partial peptide used in the present invention may be any DNA containing the nucleotide sequence encoding the partial peptide used in the present invention described above. Further, it may be any of genomic DNA, genomic DNA library, the above-described cell-tissue-derived cDNA, the above-described cell-tissue-derived cDNA library, and synthetic DNA.

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

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

DNAs that completely encode the proteins and partial peptides used in the present invention (hereinafter, in the description of the cloning and expression of DNAs encoding them, these may be simply abbreviated as the proteins of the present invention) As a means for cloning, the DNA of the present invention is amplified by PCR using a synthetic DNA primer having a part of the nucleotide sequence encoding the protein of the present invention, or the DNA incorporated into an appropriate vector is used as the cloning means of the present invention. Selection can be carried out by hybridization with a DNA fragment coding for a part or the whole region of the protein or labeled with synthetic DNA. The hybridization method can be carried out according to, for example, the method described in Molecular Cloning 2nd (J. Saibrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted by PCR, a known kit, for example, Mutan ™ -super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ _K (Takara Shuzo Co., Ltd.), etc., using the 0DA-LA PCR method. It can be carried out according to a known method such as the gapped duplex method or the Kimkel method or a method analogous thereto.

The DNA encoding the cloned protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme or added with a linker if desired. The DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.

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

Examples of the vector include a plasmid derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), a plasmid derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), a plasmid derived from yeast (eg, SH19, pSH15), bacteriophages such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., pAl-11, ρΧΤ1, ρc / CMV, pRc / RSV, p cDNAI / Neo 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 animal cells are used as host, SR promoter, SV40 promoter, LTR mouth motor, CMV promoter, HSV-TK promoter, etc. Among them, CMV (cytomegalovirus) promoter, SRo It is preferable to use a promoter overnight. When the host is Eshierihia genus bacterium, trp promoter, lac promoter, re cA promoter Isseki one, AP _L promoter one evening one, l pp promoter, T 7 promoter one is when the host is Ru Bacillus der When the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. In addition to the above, the expression vector may further include an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter, sometimes abbreviated as SV40 ori), and the like, if desired. Can be used. As the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [Mesotorekise Ichito (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Amp ^r) , neomycin resistance gene (hereinafter sometimes abbreviated as Ne o ^r, G418 resistance). In particular, using Chinese hamster cells lacking the dh fr gene When is used as a selection marker, the target gene can also be selected using 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 bacterium belonging to the genus Escherichia, a PhoA 'signal sequence, an OmpA signal sequence, etc., and when the host is a bacterium belonging to the genus Bacillus, an amylase signal sequence, a subtilisin, a signal sequence, etc., and the host is a yeast. If the host is an animal cell, an insulin signal sequence, an interferon signal sequence, an antibody molecule, a signal sequence, etc. can be used.

Using the vector containing 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, Escheric hia coli K12 · DH1 [Procedures · ob · the · national · academy · ob · science · ob · the · USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal · OB · Molecular · Biology (Journal, of Molecular Biology), 120, 517 (1978)], HB101 [Journal 'OB'Molecular · Bio mouth, 41, 459 (1969)] , C 600 [Genetics, 39, 440 (1954)].

Examples of Bacillus spp. Include, for example, Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1 984)].

Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12 and Schizosacclmromyces poibe NCYC 1913, NCYC2036, Pichia pastoris KM71 and the like are used.

Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of night roth moth (Spodoptera frugiperda cell; Sf cell); MGl cell derived from the midgut of Trichoplusia ni; Five ™ cells, cells derived from Mamestra b rassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, a silkworm-derived cell line (Bombyx mori N cell; BmN cell) or the like is used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711), Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like. Used.

As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chinini—zhamster cell CHO (hereinafter CHO (dh fr ") cell Abbreviation), mouse

L cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells, etc. are used.

For transforming Escherichia sp., For example, Processings of the

• National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 69, 2110 (1972) and Gene

, 17, 107 (1982). For transformation of Bacillus sp., For example, Molecular & General Genetics, Vol. 168, 111 (

1979).

To transform yeast, see, for example, Methods in Enzymology, 194, 182-187 (1991), Proceedings of the National Academy of Sciences, Saienshiizu-O blanking tHE Interview one SA _{(p roc. Natl. Acad.} Sci. USA), 75 vol., by the method described, for example, 1929 (1978). In order to transform insect cells or insects, for example, to transform animal cells which can be performed according to the method described in Bio Z Technology, 6, 47-55 (1988), etc. For example, Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 263--267 (19995) (published by Shujunsha), Virology, 52, 45, 6 (1 It can be carried out according to the method described in 973). In this way, 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 the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. Ρρ of the medium is preferably about 5 to 8.

As a medium for culturing the genus Escherichia, for example, a medium containing glucose and casamino acid (9) (Miller, Journal of Obesperimen-in 'Molecular Genetics (Journal of Experiments in (Molecular Genetics), 431-43, Cold Spring Harbor Laboratory, New York 1972]. If necessary, a drug such as 3j3-indolylacrylic acid can be added in order to make the promoter work efficiently. When the host is a bacterium belonging to the genus Escherichia, culturing is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.

When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours.

When culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, KL et al., Proc. Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and 0.5% casamino acids SD medium [Bitter, GA, et al., Processing the Op. The National. Academy of Sciences of the U.S.A .— (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The cultivation is usually performed at about 20 to 35 ° C for about 24 to 72 hours, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an insect cell or an insect, the culture medium is 10% immobilized in a Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). Those to which additives such as serum are appropriately added are used. It is preferable to adjust the ρ 培 of the culture medium to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association at Association, Vol. 199, 519 (1967) )], And 199 medium [Proceeding of the Society for the Biological Medicine], 73, 1 (1950)]. Preferably, the pH is about 6-8. Culture is usually performed at about 30t: ~ 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.

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, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasonic wave, lysozyme and Z or frozen. Microbial cells or cells broken by thawing, etc. Thereafter, a method of obtaining a crude extract of the protein by centrifugation or filtration is appropriately used. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ^™ . When the protein is secreted into the culture solution, after completion of the culture, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.

Purification of the protein contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods. These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods using difference in molecular weight, methods using charge differences such as ion-exchange chromatography, methods using specific affinity such as affinity chromatography, methods such as reversed-phase high-performance liquid chromatography, etc. A method utilizing the difference in hydrophobicity, a method utilizing the difference in isoelectric point such as isoelectric focusing, and the like are used.

When the protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in the form of a salt, a known method or a method analogous thereto Can be converted into a free form or another salt.

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

The presence of the protein of the present invention thus produced can be measured by enzymatic immunoassay western blotting using a specific antibody. The antibody against the protein or partial peptide or a salt thereof used in the present invention is any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the protein or partial peptide or a salt thereof used in the present invention. You may. An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter, these may be simply abbreviated to the protein of the present invention in the description of the antibody) uses the protein of the present invention as an antigen, Can be produced according to the antibody or antisera production method described above.

[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. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of the warm-blooded animal to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, goats, and chickens, and mice and rats are preferably used.

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. By fusing the antibody-producing cells obtained with myeloma cells of the same or different species, a monoclonal antibody-producing hybridoma can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975) 3. 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, SP2 / 0, and AP-1, but P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) used and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is used at a concentration of about 10 to 80%. Cell fusion can be carried out efficiently by adding and incubating at 20 to 40 ° C, preferably 30 to 37 ° C for 1 to 10 minutes. Various methods can be used to screen the monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is applied directly or onto a solid phase (eg, microplate) on which a protein antigen is adsorbed together with a carrier. Then, add an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mouse) or protein A labeled with a radioactive substance or enzyme, and bind to the solid phase. A method for detecting a monoclonal antibody that has been isolated, adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, adding a protein labeled with a radioactive substance, an enzyme, etc. Examples include a method for detecting the bound monoclonal antibody.

The selection of the monoclonal antibody can be performed according to a known method or a method analogous thereto. Usually, it can be performed in an animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine). As a selection and breeding medium, any medium can be used as long as hybridomas can grow. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) Alternatively, a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culture temperature is usually from 20 to 40 ° C, preferably about 37 ° C. The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(b) Purification of monoclonal antibody

Monoclonal antibodies can be separated and purified by known methods, for example, immunoglobulin separation and purification methods (eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Absorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification method in which the antibody is collected using an active adsorbent such as protein A or protein G and the bond is dissociated to obtain the antibody). Can be done.

(Preparation of polyclonal antibody)

The polyclonal antibody of the present invention is produced according to a known method or a method analogous thereto. can do. For example, a 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. It can be produced by collecting the contents and separating and purifying the antibody. Regarding a complex of an immunizing antigen and a carrier protein used for immunizing a warm-blooded animal, the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier. As long as it can be efficiently performed, any kind may be cross-linked at any ratio.For example, a serum albumin, a thyroglobulin, a hemocyanin, etc., in a weight ratio of about 0.1 to hapten per 1 hapten. A method of coupling at a ratio of 20 and preferably about 1 to 5 is used.

In addition, various condensing agents can be used for force coupling between the hapten and the carrier. For example, daltaraldehyde, carbodiimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used. The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The administration is usually performed once every about 2 to 6 times, for a total of about 3 to 10 times.

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

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

Complementary to the nucleotide sequence of DNA encoding the protein or partial peptide used in the present invention (hereinafter, these DNAs may be abbreviated as DNA of the present invention in the description of antisense nucleotide), or The antisense nucleotide having a substantially complementary base sequence or a part thereof includes a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof, Any anti-protein having an action capable of suppressing the expression of the DNA can be used. It may be a sense nucleotide, but antisense DNA is preferred.

The nucleotide sequence substantially complementary to the DNA of the present invention is, for example, about 70% of the entire nucleotide sequence or a partial nucleotide sequence of the nucleotide sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). % Or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more. In particular, of the total nucleotide sequence of the complementary strand of the DNA of the present invention, (a) in the case of an antisense nucleotide directed to translation inhibition, the nucleotide sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, , About 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably about 95% or more. (Mouth) In the case of an antisense nucleotide that directs RNA degradation by RNaseH, it is about 70% or more, preferably about 80% or more, complementary to the entire nucleotide sequence of the DNA of the present invention including introns. Antisense nucleotides having preferably at least about 90%, most preferably at least about 95% homology are each suitable.

Specifically, an antisense having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof A polynucleotide, preferably, for example, an antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof (more preferably, A base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or an antisense polynucleotide having a part thereof.

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

To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are, for example, chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. It may be substituted by a group. The sugar (doxylipose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2'-O-methylation, The base (pyrimidine, purine) may also be chemically modified, and may be any of those which hybridize to DNA having the base sequence represented by SEQ ID NO: 2. These antisense nucleotides can be produced using a known DNA synthesizer or the like.

According to the present invention, an antisense nucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the nucleotide sequence information of the cloned or determined DNA encoding the protein. And combine them. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention and can inhibit the synthesis or function of the RNA, or can interact with the protein-related RNA of the present invention. Through the action, the expression of the protein gene of the present invention can be regulated and controlled. Polynucleotides complementary to the selected sequence of the protein-related RNA of the present invention, 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 protein genes in plants and for treating or diagnosing diseases. The term “corresponding” means having homology or being complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. The “correspondence” between a nucleotide, nucleotide sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) under the direction derived from the nucleotide (nucleic acid) sequence or its complement. ing. 5 'end hairpin loop, 5' end 6—base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein code region, ORF translation stop codon, 3' end untranslated region, The 3 'end palindrome region and the 3' end hairpin loop may be selected as preferred regions of interest, but any region within the protein gene may be selected as the region of interest.

Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize with the target region, the target nucleic acid is Can be said to be “antisense”. Antisense (poly) nucleotides are polynucleotides containing 2-deoxy D-report and polynucleotides containing D-report. Other types of polynucleotides that are N-glycosides of purine or pyrimidine bases or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or special linkages And the like (provided that the polymer contains a nucleotide having a configuration that allows base pairing and base attachment as found in DNA and RNA), and the like. They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides). Nucleotides) and also those with known modifications, such as those with a label known in the art, capped, methylated, and one or more natural nucleotides. , Substituted with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramide, phorbamate, etc.), a charged bond or a sulfur-containing bond ( For example, those having phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nuclease inhibitors, ibis) Having side-chain groups such as amino acids, antibodies, signal peptides, poly-L-lysine, etc., and sugars (eg, monosaccharides), and those having an interactive compound (eg, acridine, psoralen, etc.) Those containing chelating compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified bonds (eg, alpha-anomeric nucleic acids) Etc.). Here, “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those containing other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. May be converted to

The antisense (poly) nucleotide (nucleic acid) of the present invention includes RNA, DNA, Or modified nucleic acids (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphoate derivatives, and polynucleoside amides, which are resistant to degradation of oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to enhance the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and if toxic. Reduces the toxicity of antisense nucleic acids.

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; ST Crooke et al. Ed. , Ant isense Research and Applicat ions, 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.). These can be attached to the 3 'end or 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. 'Other groups are cap groups specifically located at the 3' end or 5 'end of nucleic acids to prevent degradation by nucleases such as exonuclease and RNase. Is mentioned. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as dalicol such as polyethylene glycol and tetraethylene dalicol.

The inhibitory activity of the antisense nucleic acid is determined by the transformant of the present invention, It can be examined using an external gene expression system or an in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods. 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 Abbreviated as NA), an antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the antibody of the present invention), and an antisense nucleotide of the DNA of the present invention (hereinafter, referred to as an antibody). (May be abbreviated as the antisense nucleotide of the present invention).

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 symptom severity, and prediction of disease progression. Therefore, a drug containing the antisense nucleotide of the gene encoding the protein of the present invention, a compound that inhibits the activity of the protein of the present invention or a salt thereof, or an antibody against the protein of the present invention includes, for example, colon cancer, breast cancer , Lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, descending It can be used as a prophylactic / therapeutic agent for cancer such as organ cancer, brain tumor or blood tumor, and an apoptosis inducer. (1) Screening of drug candidate compounds for diseases

Since the expression of the protein of the present invention is increased in cancer tissues and has an apoptosis-suppressing action, a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention includes: For example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis It can be used as a prophylactic / therapeutic agent for cancer such as cancer, thyroid cancer, knee cancer, brain tumor or blood tumor. It can also be used as an apoptosis action regulator, preferably as an apoptosis inducer.

Further, since the protein of the present invention has an activity of suppressing apoptosis induced by an anticancer agent (eg, paclitaxel, etc.), the activity of the protein of the present invention The compound or its salt that regulates (promotes or inhibits, preferably inhibits) can be used also as an anticancer drug sensitivity enhancer.

Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention.

That is, the present invention provides a compound which regulates (promotes or inhibits, preferably inhibits) the activity (eg, ligand binding activity, signal transduction activity, etc.) of the protein of the present invention characterized by using the protein of the present invention, or a compound thereof. Provided is a salt screening method.

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, preferably inhibits) the activity of the protein of the present invention, which is characterized by comparing the ligand binding activity or the signal transduction activity of the mixture, is used.

In the above screening method, for example, in the case of (i) and (ii), the ligand binding activity or signal transduction activity is described in a known method, for example, L Immunol. 166 (1), pages 15-19, 2001. Measure and compare according to the method described above or a method equivalent thereto.

Specifically, when screening for a compound having a ligand binding promoting action or an intracellular signaling promoting action, it is necessary to (i) use the protein expression vector of the present invention to bind the NF-κB binding sequence to a promoter or enhancer. (Ii) the protein expression vector of the present invention containing the NF-κB binding sequence in the promoter or enhancer; When the cells are introduced into cells together with the reporter gene and cultured in the presence of the test compound, the expression levels of the reporters are compared. The expression of the reporter can be measured by using the luciferase activity, the lipophilic enzyme activity, etc. as an index.

Specifically, a compound having an inhibitory action on ligand binding or intracellular signal transduction. When screening a compound, (i) introducing the protein expression vector of the present invention into cells together with a repo overnight gene containing a NF-κB binding sequence in a promoter or an enhancer; (A) A solution containing a ligand such as a microbial cell lysate, a microbial culture supernatant, a eukaryotic cell lysate, or a eukaryotic cell culture supernatant, (b) the ligand itself or (c) equivalent to a natural ligand (Ii) culturing a protein expression vector of the present invention together with a reporter gene containing a NF-κΒ binding sequence in a promoter or enhancer. If necessary, (a) liquid containing ligands such as microbial cell lysate, microbial culture supernatant, eukaryotic cell lysate, or eukaryotic cell culture supernatant; (b) ligand itself or (C) natural No After adding a substance having the same binding activity as Gand, and culturing in the presence of the test compound, measure the expression level of the repo overnight and compare.

Examples of the ligand include a lipopeptide derived from bacteria (preferably, a triacylated lipopeptide or the like), or N-palmitoyl S-dipalmitoylglyceryl (Pam ₃ ) Cys-Ser- (Lys) ₄ obtained by chemical synthesis. (CSK ₄ ), N-palmitolyl-S-dil aurylgryceryl (N-Pam-S-Lau ₂ ) Trisyl peptides such as CSK _4, and outer-surface lipoprotein (Osp A) derived from Borrelia burg dorferi Can be

The repo overnight expression level can be measured using a reporter protein activity using luciferase activity, alkaline phosphatase activity or the like as an index.

In addition, the apoptotic activity against a pile cancer agent (eg, paclitaxel) in the presence or absence of the expression of the protein (eg, TLR1) used in the present invention is measured and compared by a known method, and the signal transduction activity is measured. Can be measured. Apoptosis activity against stake cancer drugs (eg, paclitaxel, etc.) in the presence and absence of ligand can be measured and compared by known methods, and the signal transduction activity can be measured.

Furthermore, in order to measure the TLR1 signal transduction used in the present invention with higher sensitivity, it is also possible to use cells having the ability to express TLR1 signal-related molecules (eg, TLR2, MD2, etc.). Alternatively, the above activity may be measured by forcibly expressing a TLR1 signal-related molecule (eg, TLR2, MD2, etc.).

Examples of cells having the ability to produce the protein of the present invention include, for example, a host transformed with a vector containing a DNA encoding the protein of the present invention. The main (transformant) is used. As a host, for example, animal cells such as COS7 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. Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation production. Products, cell extracts, plant extracts, animal tissue extracts, and the like.

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

The compound having the activity of inhibiting the activity of the protein of the present invention is a safe and low toxic drug for suppressing the physiological activity of the protein of the present invention, for example, a colorectal cancer, breast cancer, lung cancer, and prostate. Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, knee cancer, brain It is useful as a prophylactic / therapeutic agent for cancer such as tumors or hematological tumors, an apoptosis inducer, an anticancer agent, and a sensitivity enhancer.

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

Compounds or salts thereof obtained using the screening method or screening kit of the present invention include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts It is a compound selected from liquids, plasma, etc. As the salt of the compound, those similar to the aforementioned salts of the peptide of the present invention 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 the expression of the gene encoding the protein of the present invention also include, for example, breast cancer, colon cancer, breast cancer, Lung, prostate, esophagus, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary, testis, thyroid, knee It is useful as an agent for preventing and treating cancer such as cancer, brain tumor, or blood tumor, an apoptosis inducer, and an anticancer drug sensitivity enhancer.

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

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

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

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 or ELISA method or a method analogous thereto. The amount of mRNA that can be measured can be determined by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a probe, or SEQ ID NO: 2 or a primer thereof as a primer It can be measured according to a PCR method using a nucleic acid containing a part or a method analogous thereto.

For example, the gene expression level in the case of the above (iv) is inhibited by about 20% or more, preferably 30% or more, more preferably about 50% or more as compared with the case of the above (iii). The test compound to be tested as a compound that suppresses the expression of the gene encoding the protein of the present invention, the gene expression level in the above (iv) is about 20% or more as compared with the above (iii). A test compound that increases by preferably 30% or more, more preferably about 50% or more, can be selected as a compound that promotes the expression of a gene encoding the protein of the present invention.

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

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention may be a test compound as described above, for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, a cell extract, or a plant extract. 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 cancer, kidney cancer, brain tumor or hematological tumor, an apoptosis inducer, and an anticancer drug sensitivity enhancer.

When a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned medicament, it can be formulated according to a conventional method.

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, etc. You. Such a composition is produced by a known method and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

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 known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid commonly used for injections. As the aqueous liquid for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, and the like, suitable dissolution aids, for example, alcohol (eg, ethanol), polyalcohol ( For example, propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyetylene (5 Omol) adduct of hydrogenated castor oil)] and the like may be used in combination. 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 compound 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), suppositories and the like, and usually 5 to 500 mg, especially The injection preferably contains 5 to 100 mg of the above antibody, and other dosage forms contain 10 to 25 mg of the above antibody.

Each of the above-mentioned compositions may contain other active ingredients as long as the compound and the above-mentioned compound do not cause an undesirable interaction.

The preparations obtained in this way are safe and low toxic, such as, for example, human or warm-blooded animals (eg, mice, rats, puppies, sheep, bushy, pussi, puma, birds, cats, dogs, Orally or parenterally to monkeys, chimpanzees, etc.) Can be given.

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 100 mg, preferably about 1.0 to 50 mg per day. More preferably, about 1.0 to 2 O mg is administered. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, and the like.For example, a compound or a compound that regulates the activity of the protein of the present invention for the purpose of treating breast cancer may be used. When the salt is orally administered to an adult (as body weight of 6 O kg) in the form of an injection, the compound or a salt thereof is about 0.01 to 3 O mg per day, preferably about 0.1 to 3 O mg. It is convenient to administer by injection about 2 O mg, more preferably about 0.1-1 O mg, to the cancerous lesion. In the case of other animals, the dose can be administered in terms of the body weight of 60 kg.

In addition, the above compounds can be used in combination with existing pile cancer drugs (eg, paclitaxel, docetaxel, etc.), and reduce the side effects of pile cancer drugs that damage normal cells. In this case, the timing of administration is not limited, and they may be administered to the subject at the same time or at an interval. The dose can be appropriately selected based on the dose used clinically. In addition, the compounding ratio of the above compound and the anticancer agent can be appropriately selected according to the administration subject, administration route, target disease, symptom, combination, and the like.

(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, and therefore, the quantification of the protein of the present invention in a test solution, particularly It can be used for quantification by sandwich immunoassay.

That is, the present invention

(i) The antibody of the present invention is allowed to react competitively with the test solution and the labeled protein of the present invention, and the ratio of the labeled protein of the present invention bound to the antibody is measured. A method for quantifying the protein of the present invention in a test solution, and

(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 measured. And a method for quantifying the protein of the present invention in a test solution.

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 to quantifying the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like is also possible. it can. 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 it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.

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, a lanthanide element and the like are used. As the radioisotope, for example, ^{[1 2 5} I], ^{[1 3 1} I], ^[3 H], which need use include ^{[1 4} C]. As the above enzyme, a stable enzyme having a large specific activity is preferable. For example, i3-galactosidase,] 3-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiosinate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Furthermore, the binding between the antibody or antigen and the label A tin-avidin system can also be used.

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. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

In the sandwich method, a test solution is allowed to react with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with another labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the labeling agent on the insolubilized carrier, the amount of the protein of the present invention in the test solution can be determined. 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 according to the San Deutsch method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds. 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 sandwich method, for example, a competition method, an immunometric method, a nephelometry method, or the like.In a competition method, an antigen in a test solution and a labeled antigen are applied to the antibody. After the competitive reaction, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated (B / F separation), and the labeling amount of either B or F is measured. Quantify the amount of antigen in the test solution. 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 first antibody was used. An immobilized antibody is used, or an immobilized method is used in which the first antibody is soluble and an immobilized antibody is used as the second antibody.

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. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.

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 measurement system of the present invention may be constructed by adding ordinary technical considerations to those skilled in the art to the ordinary conditions and procedures in each method. For details of these general technical means, reference can be made to reviews and written documents.

For example, edited by Hiro Irie, "Radio Imuno Atsushi" (Kodansha, published in Showa 49), edited by Hiro Irie, "Radio Imuno Atssey" (Kodansha, published in 1954), Eiji Ishikawa et al. "Measurement Method" (Medical Shoin, published in 1958), Eiishi Ishikawa et al., "Enzyme Immunoassay" (Second Edition) (Medical Publishing, published in 1977), Eiji Ishikawa, et al., "Enzyme Immunoassay" (3rd edition) (Medical Shoin, published in 1962), "Methods in ENZYMOLOGYJ Vol. 70 (Immunochemical TechniQues (Part A))> Ibid. Vol. 73 (Immunoc emica 1 TechniQues (Part B)), Ibid. Vol. 74 (Immunochemical TechniQues (Part 0)), ibid.Vol. 84 (Immunochemical TechniQues (Part D: Selected Immunoassays)), ibid.Vol. 92 (I. Vol. 121 (Immunochemical Tec niues (Part I: Hybridoma Technology and Monoclonal Ant ibodies))) The above can be referred to, for example, Academic Press.

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

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, and lung cancer , Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, genital cancer Can be diagnosed as having cancer, such as cancer, brain tumor or hematological tumors, or more likely to be affected in the future. In addition, when the concentration of the protein of the present invention is high, it can be diagnosed that the administration effect of a pile cancer agent (eg, paclitaxel) is low.

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, in humans or warm-blooded animals (e.g., rats, mice, guinea pigs, egrets, birds, higgies, pigs, pigs, dogs, cats, dogs, monkeys, Abnormalities (genetic abnormalities) of DNA or mRNA encoding the protein of the present invention or its partial peptide in chimpanzees and the like, for example, damage, mutation or reduced expression of the DNA or mRNA, It is useful as a gene diagnostic agent for increasing or overexpressing the DNA or mRNA.

The above-described genetic diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989)), Processings Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)) Can be implemented. For example, if overexpression or decrease is detected by Northern hybridization or DNA mutation is detected by PCR-SSCP method, for example, colorectal cancer, breast cancer, lung cancer, prostate cancer, Esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, knee cancer, brain tumor or It can be diagnosed that the possibility of cancer such as hematological tumors is high.

(4) Drug containing antisense nucleotide

The antisense nucleotide of the present invention, which complementarily binds to the DNA of the present invention and can suppress the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo. (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, spleen cancer, Prevention and treatment of cancers such as kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, knee cancer, brain tumor or blood tumor, apoptosis inducer, anti- It can be used as a cancer drug sensitivity enhancer.

When the antisense nucleotide is used as the above-mentioned prophylactic or therapeutic agent, it can be formulated and administered according to known methods.

Further, for example, after inserting the antisense nucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector, a human or mammal ( For example, it can be administered orally or parenterally to rats, egrets, sheep, sheep, bush, horses, cats, dogs, monkeys, etc.). The antisense nucleotide can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an adjuvant for promoting uptake, and administered with a gene gun or a catheter such as a hydrogel catheter. Alternatively, they can be aerosolized and administered topically into the trachea as an inhalant.

Furthermore, the antisense nucleotides described above are used alone or in combination with a carrier such as ribosome for the purpose of improving pharmacokinetics, extending half-life, and improving the efficiency of cellular uptake. The antisense nucleotide may be administered to vein, subcutaneous, intra-articular cavity, cancer lesion, etc. The dosage of the antisense nucleotide varies depending on the target disease, the administration target, the administration route, etc. However, for example, when the antisense nucleotide of the present invention is administered for the purpose of treating breast cancer, generally in an adult (body weight 60 kg), about 0.1 to 10 O of the antisense nucleotide is administered per day. mg.

Furthermore, the antisense nucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the DNA of the present invention in tissues or cells and the state of its expression.

Similarly to the above-mentioned antisense nucleotide, 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. Can suppress the function of the protein used in the present invention or the DNA used in the present invention in vivo, for example, colorectal cancer, breast cancer, lung cancer, prostate gland Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, knee cancer, It can be used as a prophylactic / therapeutic agent for cancers such as brain tumors or hematological tumors, an apoptosis inducer, and a susceptibility enhancer for pile cancer agents.

The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001). The lipozyme can be designed and manufactured based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, 7, 221, 2001). For example, it can be produced by linking a known lipozyme to a part of the RNA encoding the protein of the present invention. A part of the RNA encoding the protein of the present invention includes a portion (RNA fragment) close to the cleavage site on the RNA of the present invention, which can be cleaved by a known lipozyme.

When the above double-stranded RNA or lipozyme is used as the above-mentioned prophylactic or therapeutic agent, it can be formulated and administered in the same manner as an antisense polynucleotide. Similar to the antisense nucleotide, the protein of the present invention or an abtamer of DNA encoding the protein of the present invention can also suppress the expression of the gene of the present invention, and the protein or the protein used in the present invention in vivo. Since the function of the DNA used in the present invention can be suppressed, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney Prevention and treatment of cancer such as cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, knee cancer, brain tumor or blood tumor, apoptosis inducer, pile It can be used as a drug sensitivity enhancer.

Abutama can be produced according to a known method.

(5) a drug containing the antibody of the present invention

Antibodies of the present invention having an activity of neutralizing the activity of the protein of the present invention include, for example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer , Renal cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor, etc. It can be used as a low-toxicity and safe drug such as a drug sensitivity enhancer.

The prophylactic / therapeutic agents for the above-mentioned diseases containing the antibody of the present invention have low toxicity and can be used as a night medicine or as a pharmaceutical composition of an appropriate dosage form in humans or mammals (eg, rat, rabbit, etc.). It can be given orally or parenterally (eg, intra-articular) to mice, higgies, bush, elephants, cats, dogs, monkeys, etc.). The dosage varies depending on the administration subject, target disease, symptoms, administration route, and the like.For example, when used for treatment of adult breast cancer * prevention, the antibody of the present invention is used as a single dose. Usually, about 0.01 to 2 Omg / kg body weight, preferably about 0.1 to 1 Omg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, 1 to 5 days a day It is convenient to administer the compound by inhalation about once, 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 condition is particularly severe, the dose may be increased depending on the condition.

The antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition. You. The pharmaceutical composition used for the administration contains the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided in dosage forms suitable for oral or parenteral administration (eg, intravenous injection). Each of the above-mentioned compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.

(6) The “prophylactic / therapeutic agent for cancer comprising a compound having an activity of modulating To11-like receptor or a salt thereof” of the present invention and an expression regulating effect of Το11-like receptor "Prophylactic / Therapeutic Agents for Cancer Containing Compounds Having Salts or Their Salts" "Compounds having the activity regulating action of の ο11-like receptor” Any compound that has the compound, such as colorectal cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterus It is used as a prophylactic / therapeutic agent for cancer such as cancer, ovarian cancer, testicular cancer, thyroid cancer, knee cancer, brain tumor, or blood tumor, an apoptosis inducer, and an enhancer for cancer cell susceptibility.

The “compound having a To 11-like receptor expression regulating action” may be any compound having a To 11-like receptor expression regulating action, such as colon cancer, breast cancer, lung cancer, and prostate. Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testis cancer, thyroid cancer, kidney cancer, It is used as a prophylactic / therapeutic agent for cancers such as brain tumors and blood tumors, an apoptosis inducer, and a cancer susceptibility enhancer.

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

(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 (sometimes abbreviated as the exogenous mutant DNA of the present invention). And a non-human mammal having the same.

That is, the present invention

(1) a non-human mammal having an 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 which contains the exogenous DNA of the present invention or its mutant DNA and 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-transferred animal of the present invention) include unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like. In contrast, preferably, during the stage of embryonic development in non-human mammal development (more preferably, at the stage of a single cell or a fertilized egg cell and generally before the 8-cell stage), the calcium phosphate method, the electric pulse method, and the lipofection method It can be produced by transferring the desired DNA by a coagulation method, microinjection method, particle gun method, DEAE-dextran method, or the like. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, organs of living organisms, tissue cells, and the like, and used for cell culture, tissue culture, and the like. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned germ cells with a known cell fusion method.

As non-human mammals, for example, porcupines, pigs, sheep, goats, porcupines, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used. Among them, rodents with relatively short ontogeny and biological cycle in terms of the creation of disease animal models, and easy to breed, especially mice (for example, pure strains such as C57BL / 6 strain, DBA2 strain, etc.) As the crossing strain, a BSC SFi strain, a BDF strain, a B6D 2F _one strain, a BALB / c strain, an ICR strain, etc.) or a rat (eg, Wistar, SD, etc.) are preferable.

Examples of the “mammal” in the recombinant vector that can be expressed in mammals include humans and the like in addition to the above-mentioned non-human mammals.

The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the original DNA originally possessed by a non-human mammal. As the mutant DNA of the present invention, DNA having a mutation (for example, mutation) in the base sequence of the original DNA of the present invention, specifically, base addition, deletion, substitution with another base, etc. DNA that has been used is used, and also includes abnormal DNA. The abnormal DNA refers to a DNA that expresses an abnormal protein of the present invention, and for example, a DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.

The exogenous DNA of the present invention may be derived from a mammal of the same species or a different species from the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct downstream of a promoter capable of being expressed in animal cells. For example, when the human DNA of the present invention is transferred, various mammals having the DNA of the present invention having high homology to the human DNA (eg, egret, dog, cat, guinea pig, hampus, rat, mouse, etc.) By microinjecting a DNA construct (eg, a vector, etc.) to which the human DNA of the present invention is bound downstream of various promoters capable of expressing DNA of origin from a fertilized egg of a target mammal, for example, a mouse fertilized egg. A DNA-transferred mammal that highly expresses the DNA of the present invention can be created.

Examples of the expression vector of the protein of the present invention include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as λ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or Animal viruses such as baculovirus 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 the promoter that regulates the expression of DNA include, for example, a promoter of DN ① derived from a virus (eg, simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, poliovirus, etc.). , ② Promoters derived from various mammals (humans, ゥ egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), such as albumin, insulin II, ゥ brabrakin II, elastase, erythropoietin, endothelin, muscle creatine kinase , Glial fibrillary acidic protein, daryuthion S-transferase, platelet-derived growth factor] 3, keratin Kl, 1 ^ 10 114, collagen type I and type II, cyclic AM P-dependent protein kinase i3 I-subunit, dystro Fin, tartrate-resistant alkaline phosphatase Ichize, atrial sodium Dum diuretic factor, endothelial receptor thymic synthase (generally abbreviated as Tie 2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, meta-oral thionein I and IIA , Meta-oral proteinase 1 tissue inhibitor, MHC class I antigen (H-2L), Hras, renin, dopamine] 3-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1 (EF -1), β-actin, spider and] 3 myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component , Myoglobin, troponin (:, smooth muscle a-actin, prebub-enkephalin A, vasopressin and other promoters are used. A cytomegalovirus promoter capable of high expression throughout the body, a human peptide chain elongation factor 1 (EF-1) promoter, a human and a chicken / 3 actin promoter, and the like are suitable.

The vector preferably has a sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal (generally referred to as terminator). A DNA sequence can be used, and preferably, Simian virus SV40 terminator 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, between the promoter region and the translation region. Alternatively, it can be linked to the 3 'downstream of the translation region depending on the purpose.

The normal translation region of the protein of the present invention is DNA derived from liver, kidney, thyroid cells, fibroblasts derived from humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.). And all or part of genomic DNA from various commercially available genomic DNA libraries, or complementary DNA prepared by known methods from liver, kidney, thyroid cell, or fibroblast-derived RNA as a raw material. . In addition, exogenous abnormal DNA alters the translated region of the normal protein obtained from the above cells or tissues by point mutagenesis. Different translation regions can be created.

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

Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the exogenous DNA of the present invention in the germinal cells of the transgenic animal after transfer of the DNA indicates that the progeny of the transgenic animal has the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to keep. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has the exogenous DNA of the present invention in all of its germinal and somatic cells.

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

Transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be present in excess in all germ cells and somatic cells of the target mammal. Excessive presence of the exogenous DNA of the present invention in the germinal cells of the produced animal after DNA transfer indicates that all the offspring of the produced animal contain the exogenous DNA of the present invention in all of its germ cells and somatic cells. Means to have. The progeny of this type of animal that has inherited the exogenous DNA of the present invention has an excess of the exogenous DNA of the present invention in all of its germ cells and somatic cells.

By obtaining a homozygous animal 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 has a high level of expression of the normal DNA of the present invention, and eventually promotes the function of endogenous normal DNA, thereby finally achieving the function of the present invention. It may develop protein hyperactivity and can be used as a model animal for the disease. For example, using the normal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the hyperactivity of the protein of the present invention and diseases associated with the protein of the present invention and to examine a method for treating these diseases. It is possible. 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, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid gland It can also be used for screening tests for prophylactic and therapeutic agents for cancer such as cancer of the knee, knee, brain or blood.

On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention is confirmed to stably maintain the exogenous DNA by mating, and is reared as an animal having the DNA in a normal breeding environment. I can do it. Furthermore, the desired exogenous DNA can be incorporated into the above-mentioned plasmid and used as a raw material. The DNA construct with the Promo Youichi can be made by ordinary DNA engineering techniques. Transfer of the abnormal DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the germinal cells of the animal after the transfer of DNA means that all the offspring of the animal have the abnormal DNA of the present invention in all of the germinal and somatic cells. The progeny of this type of animal that inherits the exogenous DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. A homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing the male and female animals, it is possible to breed so that all offspring have the DNA.

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 a normal protein by the abnormal protein of the present invention in the inactive refractory disease of the protein of the present invention (dominant negative activity). Action). In addition, the mammal to which the foreign abnormal DNA of the present invention has been transferred, Because it has increased symptoms of protein, it is a prophylactic / therapeutic agent for the protein or function-inactive refractory of the present invention, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, Agents for the prevention and treatment of cancers such as biliary tract, spleen, kidney, bladder, uterine, ovarian, testicular, thyroid, knee, brain or blood tumors It can also be used for screening tests.

Further, other possible uses of the above two types of DNA transgenic animals of the present invention include, for example,

① Use as a cell source for tissue culture,

(2) Specific 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 analyzing the peptide tissue expressed by the 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 producing cell of the present invention can be specified, apoptosis, its relationship with differentiation or proliferation, or its signal transduction mechanism can be examined, and its abnormality can be examined. It is an effective research material for elucidating its action.

Furthermore, using the DNA-transferred animal of the present invention, the functional inactivity of the protein of the present invention In order to develop a therapeutic agent for a disease associated with the protein of the present invention including type refractory disease, an effective and rapid screening method for the therapeutic agent for the disease is carried out by using the above-mentioned detection method and quantitative method. Can be provided. Further, using the DNA transgenic animal of the present invention or the exogenous DNA expression vector of the present invention, it is possible to examine and develop a DNA treatment method for a disease associated with the protein of the present invention.

(8) Knockout animal

The present invention provides a non-human mammalian embryonic stem cell in which the DNA of the present invention has been inactivated, and a non-human mammal deficient in expression of the DNA of the present invention.

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 gene derived from E. 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 repo overnight gene (eg, a / 3-galactosidase gene derived from Escherichia coli), and the repotase gene is Non-human mammal according to item (6), which can be expressed under the control of a promoter.

(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 Activity of the protein of the present invention encoded by DNA , The DNA does not substantially have the ability to express the protein of the present invention (hereinafter, may be referred to as the knockout DNA of the present invention). (Abbreviated as ES cell).

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 and inserting or substituting another DNA by a genetic engineering technique. By these mutations, the knockout DNA of the present invention may be prepared, for example, by shifting the codon reading frame or disrupting the function of the promoter or exon.

Specific examples of the non-human mammalian embryonic stem cells in which the DNA of the present invention is 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 to be isolated is isolated and its exon portion is a drug resistance gene typified by a neomycin resistance gene, a hygromycin resistance gene, or lacZ (j3-galactosidase gene), cat (clo DNA sequence that disrupts the function of exon by inserting a repo overnight gene represented by the ramphenicol acetyltransferase gene) or terminates gene transcription in the intron between exons For example, by adding a po 1 yA additional signal) to prevent synthesis of the complete messenger RNA. A DNA chain having a DNA sequence constructed so as to eventually disrupt the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cells A DNA sequence on or near the DNA of the present invention used as a probe for Southern hybridization analysis or a DNA sequence on the targeting vector and a DNA sequence in a neighboring region other than the DNA of the present invention used for the production of the targeting vector Can be obtained by analyzing by a PCR method using as a primer and selecting the knockout ES cells of the present invention.

As the ES cells from which the DNA of the present invention is inactivated by the homologous recombination method or the like, for example, those already established as described above may be used, and those of known Evans and Kaufman may be used. It may be newly established according to the method. For example, mouse E In the case of S cells, currently, 129 ES cells are generally used, but since the immunological background is not clear, a pure ES system with an immunologically clear genetic background is available. For the purpose of obtaining cells, for example, a BDF mouse (C57BLZ6 and DBA / 2 using a C57BL / 6 mouse or C57BLZ6 and BDFi mice can be used satisfactorily because they have a high number of eggs collected and their eggs are robust, and they have C57BLZ6 mice as their background. ES cells can be advantageously used when generating pathological model mice in that the genetic background can be replaced by C57BL / 6 mice by backcrossing with C57BL / 6 mice.

In addition, when ES cells are established, blastocysts 3.5 days after fertilization are generally used. Many early embryos can be obtained.

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

An example of a method for determining the sex of ES cells is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. Using this method, conventionally, for example G-banding method, requires about 10 ⁶ cells for karyotype analysis, since suffices ES cell number of about 1 colony (about 50), culture The primary selection of ES cells in the early stage can be performed by discriminating between males and females, and the early stages of culture can be greatly reduced by enabling the selection of male cells at an early stage.

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

Embryonic stem cell lines obtained in this way usually have very good growth potential, Careful subculturing is necessary, as it tends to lose the ability to grow. For example, on a suitable feeder cell, such as STO fibroblasts, in a CO2 incubator (preferably 5% CO2, 95% air or 5%) in the presence of LIF (1-1000 OU / ml). Culture at about 37% oxygen, 5% carbon dioxide, 90% air, etc. At the time of subculture, for example, trypsin ZEDTA solution (usually 0.001-0.5% trypsin / 0.1-5mM EDTA Preferably, about 0.1% trypsin / "ImM EDTA) treatment is performed to form single cells, and the cells are seeded on a freshly prepared feeder cell. Such subculture is usually performed every 13 days. However, at this time, it is desirable to observe the cells and, if any morphologically abnormal cells are found, discard the cultured cells.

ES cells can be cultured in monolayers at high densities or in suspension cultures to form cell clumps under appropriate conditions to produce various types of cells such as parietal, visceral, and cardiac muscles. [MJ Evans and MH Kaufman, Nature, 292, 154, 1981; GR Martin Proceedings of Ob. 'National' Academy 'Ob. (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal 'ob embrology' and 'experimental morphology', 87, 27, 1985], the DNA-deficient cells of the present invention obtained by differentiating the ES cells of the present invention are useful in the cell biology of the protein of the present invention in the mouth of in vivo.

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

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

In the non-human mammal deficient in DNA expression of the present invention, for example, the targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of one of the targeting vectors of the present invention becomes inactive upon introduction. The homologous recombination of the transformed DNA sequence with the DNA sequence of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination, Can be knocked out.

A cell in which the DNA of the present invention has been knocked out has a DNA sequence on a Southern hybridization analysis or a gettering vector using the DNA sequence on or near the DNA of the present invention as a probe, It can be determined by PCR analysis using as primers a DNA sequence of a neighboring region other than the mouse-derived DNA of the present invention, which was used for the evening getter vector. 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 pseudopregnant non-human mammal. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially altered DNA locus of the present invention. When a part of the germ cells of the chimeric animal has a 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. It can be obtained by selecting individuals composed of cells having the added DNA locus of the present invention, for example, by judging coat color or the like. The individuals obtained in this manner are usually individuals deficient in the hetero-expression of the protein of the present invention, and mated with individuals deficient in the hetero-expression of the protein of the present invention. A homozygous deficient individual can be obtained.

When an egg cell is used, for example, a transgenic non-human mammal having a getter vector introduced into a chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by microinjection. In comparison with the transgenic non-human mammal of the present invention, by selecting those having a mutation at the DNA locus of the present invention by homologous recombination.

In this way, in the individual in which the DNA of the present invention has been knocked out, it is possible to confirm that the DNA of the animal individual obtained by the crossing is also knocked out, and to carry out rearing in an ordinary rearing environment. it can.

Furthermore, the germline can be obtained and maintained according to a standard method. That is, by crossing male and female animals having the inactivated DNA, homozygous animals having the inactivated DNA on both homologous chromosomes can be obtained. Homozygote obtained Automata can be efficiently obtained by rearing mother animals in such a way that one normal individual and one or more homozygote are obtained. By mating male and female heterozygous animals, homozygous and heterozygous animals having the inactivated DNA are bred and subcultured.

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

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.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 and compared with an untreated control animal, and the change in each organ, tissue, disease symptoms, etc. of the animal is used as an index. As a test, the therapeutic and prophylactic effects of the test compound can be tested. 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, 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 When screening for a compound having a therapeutic or prophylactic effect against cancers such as thyroid cancer, knee cancer, brain tumor, or hematological tumor, a test compound is administered to a non-human mammal deficient in DNA expression of the present invention. After the administration, a difference in the degree of onset of cancer and a difference in the degree of healing of the cancer are observed over time in the above tissues.

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

The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / preventive effect on a disease caused by a deficiency or damage of the protein of the present invention. It can be used as a medicament such as a safe and low toxic prophylactic or therapeutic agent. Further, a compound derived from the compound obtained by the above-mentioned screening can also be used. The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and a physiologically acceptable acid addition salt is particularly preferable. 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, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).

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

The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like. 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 10011 ^, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg of the compound is administered per day. When administered parenterally, the dosage of the compound varies depending on the subject of administration, the target disease, and the like. For example, the compound is usually administered in the form of an injection to normal adult (60 kg) breast cancer patients. When administered, about 0.01 to 3 Omg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg of the compound per day is conveniently administered by intravenous injection. is there. In the case of other animals, the amount converted per 60 kg can be administered. (8b) Method for screening for a compound that promotes or inhibits the activity of a 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 or inhibits the expression of a reporter gene. A method for screening a compound or a salt thereof is provided.

In the above-mentioned screening method, the non-human mammal deficient in expression of DNA of the present invention may be a non-human mammal deficient in expression of DNA of the present invention in which the DNA of the present invention is inactivated by introducing a reporter gene. The reporter gene can be used under the control of a promoter for the DNA of the present invention.

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

As the repo overnight gene, the same one as described above is used, and a / 3-galactosidase gene (1 ac Z), a soluble alkaline phosphatase gene or a luciferase gene is preferable. In a non-human mammal deficient in DNA expression of the present invention in which the DNA of the present invention has been replaced with the reporter overnight gene, the reporter gene exists under the control of the promoter for the DNA of the present invention. The activity of the promoter can be detected by tracing the expression of the substance encoded by.

For example, when a part of the DNA region encoding the protein of the present invention is replaced with a galactosidase gene (1 ac Z) derived from Escherichia coli, the tissue expressing the protein of the present invention originally has 6-galactosidase is expressed instead of the protein. Therefore, the present invention can be easily performed by staining with a reagent serving as a substrate for 3-galactosidase, such as 5-promo 4-chloro-3-indolyl-j3-galactopyranoside (X-gal). It is possible to observe the expression state of the protein in the animal body. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-ga1 at room temperature or at 37 ° C. After reacting at about 30 ° C for about 30 minutes to 1 hour, the jS-galactosidase reaction can be stopped by washing the tissue specimen with an ImM EDTA / PBS solution, and the coloration can be observed. . Further, mRNA encoding 1acZ may be detected according to a conventional method.

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

The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids, etc.). In particular, physiologically acceptable acid addition salts are preferred. Such salts include, for example, inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.) And organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.) And the like. The compound of the present invention which promotes or inhibits the promoter activity on DNA, or a salt thereof, regulates the expression of the protein of the present invention and regulates the function of the protein. 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, It is useful as a prophylactic / therapeutic agent for cancers such as ovarian, testicular, thyroid, knee, brain or blood tumors.

Furthermore, compounds derived from the compounds obtained by the above screening can be used in the same manner.

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

The preparations obtained in this way are safe and low toxic and can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, egrets, sheep, sheep, bushus, dogs, dogs, cats, dogs). , Monkeys, etc.).

The dose of the compound or a salt thereof varies depending on the target disease, the administration subject, the administration route, and the like.For example, when the compound of the present invention that inhibits promoter overnight activity against DNA is orally administered, Generally, in adult (assuming a body weight of 60 kg) breast cancer patients, about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg of the compound is administered per day. . When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, and the like.For example, the compound of the present invention that inhibits the activity of the promoter against DNA is usually administered in the form of an injection to an adult. When administered to breast cancer patients (as 6 O kg), the compound is administered in an amount of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. It is convenient to administer about 1 Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 6 O kg. 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 of the DNA of the present invention. Investigating or preventing the causes of various diseases caused by insufficient expression of DNA can greatly contribute to the development of therapeutic agents.

In addition, using a DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream of the DNA and injected into animal egg cells. If a so-called transgenic animal (gene-transfected animal) is created by transfection, it will be possible to specifically synthesize the protein and examine its action in the living body. Furthermore, by binding an appropriate reporter gene to the above promoter portion and establishing a cell line in which this is expressed, the action of specifically promoting or suppressing the ability of the protein itself of the present invention to produce in the body can be achieved. It can be used as a search system for low molecular weight compounds with. In the present specification, bases, amino acids and the like are indicated by abbreviations based on the abbreviations by IUPAC-IUB Comission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When amino acids may have optical isomers, L-form shall be indicated unless otherwise specified.

DNA deoxylipo nucleic acid

cDNA complementary deoxylipo nucleic acid

A adenine

T thymine

G Guanin

C

RNA liponucleic acid

mRNA-liponucleic acid

dATP phosphate

dTTP Deoxythymidine triphosphate

dGTP Deoxyguanosine triphosphate

d CTP Deoxycytidine triphosphate

ATP Adenosine triphosphate

EDTA ethylenediaminetetraacetic acid

SDS sodium dodecyl sulfate

G 1 y Glycine

A 1 a Alanin

Va 1 Valine Le u

I 1 e

S e r serine

Th r threonine

Cy s

Me t Methionin

G 1 u Dalminic acid

As p Aspartic acid

Lys lysine

A r g Arginine

H is histidine

P h e feniralanin

Ty r tyrosine

T r p Tribute fan

Pro proline

A s n asparagine

G 1 n Dal Yu Min

p G 1 u pyroglutamic acid

S ec (selenocysteine) The 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-1 (R) — lipoxamide group

To s: p-toluenesulfonyl

CHO: Formyl B z 1

Cl ₂ -Bzl 2, 6—Dichrobenzil

Bom benzyloxymethyl

Z benzyloxycarponyl

C 1 -z 2—Brozen benzyloxycarponyl

B r -Z 2 bromobenzyloxycarponyl

B oc t-butoxycarbonyl

DNP dinitrophenyl

T r t 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-norbornene-2,3-dicarboxyimide DCC N, N'-dicyclohexylcarposimide The sequence numbers in the sequence listing in the present specification show the following sequences.

[SEQ ID NO: 1]

1 shows the amino acid sequence of TLR1.

[SEQ ID NO: 2]

This shows the nucleotide sequence of DNA encoding TLR1 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 TLR1.

[SEQ ID NO: 4]

3 shows the base sequence of the antisense used in Example 2.

[SEQ ID NO: 5]

3 shows the nucleotide sequence of a primer 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]

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

[SEQ ID NO: 9]

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

[SEQ ID NO: 10]

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

[SEQ ID NO: 11]

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

[SEQ ID NO: 1 2]

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

[SEQ ID NO: 13]

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

[SEQ ID NO: 14]

3 shows the nucleotide sequence of a DNA fragment inserted into a vector in Example 3. Hereinafter, the present invention will be described in more detail with reference to examples, but the invention is not limited thereto. Example

Example 1

To identify genes that are specifically up-regulated in breast cancer tissues, we used total RNA (Table 1) extracted from two cases of breast cancer tissues and two or three other normal tissues as a material, and used ol igonucleotide microarray ( Gene expression analysis was performed using Huian Genome U95A, U95B, U95C, U95D, U95E (Aifymetrix).

The experiment method is described in the Aifymetrix Experiment Guide (Expression analysis technical manual). As a result, TLR1 gene expression was detected only in breast cancer tissues. It was below the detection limit in all normal tissues analyzed (Table 2). Table 1 RNA samples analyzed for gene expression

Tissue from which RNA was extracted Publisher Breast cancer tissue (Patient number 9) BioClinical Partners

Breast cancer tissue (patient number 11) BioClinical Partners

Fat BioChain Institute

Skeletal muscle Clontech

Clontech

Adrenal gland Clontech

Liver Clontech

Knee Cloirtech

Spleen Clontech

Clontech

Lung Cloirtech

Whole brain Clontech

Cerebellum Cloirtech

Thymus Clorvtech

Mammary gland Clontech

Salivary gland Clontech

Stomach Clorvtech

BioChain Institute

Large intestine BioChain Institute

Uterus Clontech

Cervical BioChain Institute Table 2 Comparison of TLR1 gene expression level between tissues Tissue gene expression level Breast cancer tissue (Patient number 9) 1.6

Breast cancer tissue (patient number 11) 0.9

Fat ND

Skeletal muscle ND

ND

Kidney ND

Adrenal gland ND

Liver ND

Knee ND

Spleen ND

Tracheal ND

Lung ND

Whole brain ND

Cerebellar ND

Thymus ND

Mammary gland ND

Salivary gland ND

ND ND

Large intestine ND

Uterus ND

Cervical ND ^a gene expression amount, expressed in Oligonucleotide microarray were normalized to the median of the expression amount of all genes detected as 1.

ND; not detected Example 2

To analyze the effect of the TLR1 gene, whose expression was specifically observed in breast cancer, on apoptosis, a TLR1 antisense oligonucleotide transfection experiment was performed. First, after designing an antisense (SEQ ID NO: 4) against the nucleotide 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, a 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) ₀

Using the breast cancer cell line MDA-MB-231 (In Vitro, vol. 14 (11), 91 pp. 915, purchased from ATCC, 1978, 1978) as a test cell, 18 × 10 ³ cells were used 24 days before the introduction of the oligonucleotide.播 Seed on an L plate (Falcon). OligofectAMINE (manufactured by Invitrogen) was used for oligonucleotide introduction, and the protocol was followed. Seven hours after introduction, RNA was extracted according to the protocol of R easy mini kit (manufactured by Qiagen), and cDNA was prepared using ThermoScript RT-PCR system (manufactured by GIBCO BRL). In the RCR reaction, two primers (SEQ ID NO: 6 and SEQ ID NO: 7), a primer for 18 S rRNA of QuantumRNA 18S Internal Standards (Ambion) and a competitor were simultaneously added according to the manual. Multiplex PCR was performed with no heat. After the PCR reaction, the reaction solution was subjected to electrophoresis, and the intensity of the band stained with ethidium bromide was corrected with the band intensity of 18 S rRNA using BioImagelQ software (manufactured by Genomic Solutions) and compared.

On the other hand, the effect on apoptosis was compared with the control oligo-introduced sample using Cell death detection ELISA (Roche) 3 days after the introduction of the antisense oligonucleotide.

As a result, the expression (RNA) of TLR1 was reduced to 55% at 7 hours after the introduction of antisense, compared to the time when the control (reverse) oligonucleotide was introduced (100%). On the third day after transduction, apoptosis increased by 186% compared to control (100%).

Thus, TLR1 is not only expressed in a cancer-specific manner, but also And apoptosis. Example 3

(1) Preparation of TLR1 and MD2 expression vectors

In order to construct a TLR1 expression system, the full-length TLR1 gene was cloned.

Using two types of primers (SEQ ID NO: 8 and SEQ ID NO: 9), PCR was performed using a Marathon ready cDNA library (Clontech) as type III according to the protocol attached to Pfu polymerase (Straragene). went. PCR was performed using 20 μl of lOxPfu buffer 2 μl, lOmM each dNTP mixture (manufactured by Clontech) 0.41, 20 μM each of the above two primers prepared at 20 mM, 0.8 μl each, 铸 type cDNA solution 0.5 ^ 1, and Pfu polymerase 0.1 μl. After the pretreatment of the reaction solution at 94 ° C for 2 minutes, 94 ° C · 30 seconds, 55 ° C · 30 seconds, 72 ° C • After performing the reaction for 42 cycles of 3 minutes, 72 ° C · 7 The reaction was carried out by reacting for one minute.

After the PCR reaction, the product was separated by agarose gel electrophoresis, the desired band was cut out, and cloned into pCR4Blunt-TOP0 vector (Invitrogen). The nucleotide sequence of the inserted fragment was confirmed to be consistent with the human TLR 1 nucleotide sequence. Therefore, the vector obtained above using two additional primers (SEQ ID NO: 10 and SEQ ID NO: 11) was A CRC was performed as type 錶. PCR contains 2 μl of lOxPfu buffer, 0.4 μl of lOmM each dNTP mixture (Clontech), 0.2 μl of each of the above two primers prepared at 50 mM, 0.2 μl each of 錄 -type cDNA solution, and 0.5 μl of PfU polymerase. After pretreatment of 20 μ 液 of the reaction solution at 94 ° C for 2 minutes, the reaction was performed at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 3 minutes, and then 25 cycles of 72 ° C for 7 minutes. The reaction was carried out by reacting for one minute. After PCR reaction with Pfu polymerase (Straragene), A was added to both ends of the PCR product according to the protocol attached to pCR4-TOP vector (Invitrogen), and the product was separated by agarose scale electrophoresis. The target band was cut out and cloned into pCR4-T0P0 vector (Invitrogen). After confirming that the sequence is correct, digest it with the restriction enzyme Belli (Fujitsu Gene) and use the Ligation kit ver.2 (Takara) for P3XFLAG-CMV-14 (SIGMA) similarly treated with Belli. Import did. It was confirmed again that there was no error in the sequence, and the obtained TLR1 expression vector was used in the following experiments.

On the other hand, using two types of primers (SEQ ID NO: 12 and SEQ ID NO: 13), a human spleen-derived cDNA library (Clontech) was used as a type II, and PCR was performed using KOD polymerase (Toyobo) Was done. PCR contains 2 μl of lOxKOD buffer, 2.5 mM each dNTP mixture (manufactured by Clontech) 1.6 / zl, each of the above two primers prepared at 50 mM, 0.21 each, 铸 type cDNA solution 11, and K0D polymerase 0.2 // 1 After pre-treating the reaction solution of 20μ1 at 94 ° C for 2 minutes, 94 ° C30 seconds, 55 ° C30 seconds, 72 ° C The reaction was performed by reacting for 7 minutes.

After the PCR reaction, the product was separated by agarose gel electrophoresis, the target band was cut out, digested with the restriction enzymes Kpnl and Notl, and then digested with the same restriction enzymes pcDNA3.1 / Hyg (+) (Invitrogen) To obtain an MD2 expression vector. The nucleotide sequence of the inserted fragment of this vector is shown in SEQ ID NO: 14. The nucleotide sequence of the inserted fragment was confirmed to be consistent with the nucleotide sequence of human MD2.

(2) Effect of TLR1 on reporter activity

Next, in order to examine the activity of TLR1 in cancer cells, the expression vector obtained in (1) above was introduced into NCI-H1299 cells (purchased from ATCC) to examine the effect on various reporter activities. Was.

On the day before transfection of the following expression vectors, seed ² × 10 ⁴ NCI-H1299 strains in 24 ゥ L plates (Falcon) and inoculate the transfection using Fugene6 (Roche) according to the attached protocol. went. The TLR1 expression vector obtained in Example 3 (1) and the pcDNA3.1 vector used as a control were used in 0.19 μg wells. As a reporter, a vector having an AP1 site or an NF-κB site upstream of a secreted alkaline phosphatase gene (both from Clontech) or a vector having an ISRE site upstream of a firefly luciferase gene (Clontech) was 0.19. gZ Ueru, _P RL- TK (manufactured by Toyo ink) was used to ToTadashi reporter activity using 0.13I gZ Weru. The reporter secretory alkaline phosphatase activity or firefly luciferase activity After the second measurement, the measurement was performed according to the protocols of the Great EscAPe SEAP Fluorescence Detection Kit (Clontech) and Pitka Gene Dual Sea Pansy Dubno Relucif Erase Atsay System (Toyo Ink), respectively. The sea luciferase activity expressed by pRL-TK was measured according to the protocol of the pikki genedu alcypansy double luciferase atsushi system (manufactured by Toyo Ink), and the reporter activity value was corrected.

As a result, due to the expression of TLR1, the AP1 reporter activity was used as a control. The NF_κB reporter activity was increased 3.3-fold and the ISRE reporter activity was increased 1.7-fold, respectively, 5.6-fold compared to the 3.1 vector.

Thus, it was found that expression of TLR1 activates the AP1 signal, NF-κB signal, and ISERE signal in the lung cancer-derived NCI-H1299 strain without any additional ligand.

Therefore, a compound having a TLR1 signal-modulating action can be screened by conducting the promoter- 11 reporter accession.

(3) Effect of paclitaxel on apoptosis induction

Next, in order to see the effect of TLR1 signal activation on cancer, the effect of paclitaxel on apoptosis induction was observed.

On the day before the expression vector transfection, 5 × 10 ⁵ HCT116 (purchased from ATCC) were seeded on a 10 cm dish (Falcon), and transfection was performed according to the protocol of Fugene6 (Roche). The TLR1 expression vector and MD2 expression vector obtained in Example 3 (1) above or the pcDNA3.1 vector (manufactured by Invitrogen) used as a control are shown in Table 3 so that the number of each becomes 11 per dish. Used in the indicated combinations. Cells were harvested by trypsin at 4 hours post transflector Ekushi Yon 3 were seeded into X10 ³ pieces each 96 © El plate (manufactured by Falcon), N-palmitoyl-S -dilauryl- glyceryl CSK 4 , a ligand for TLR 1

Add (Pam ₃ CSK ₄ ) (manufactured by Peptide Research Laboratories) (1 βg / ml) and add Z or paclitaxel (Sigma), an anticancer agent (0.125 μm), and observe the effect on apoptosis I decided that. After 2 days of culture in a 96 ゥ L plate, Cell Death Detection ELISA plus kit

(Roche) was used to detect apoptosis according to the attached protocol. Figure

As shown in 1, the presence of paclitaxel has a higher absorbance than the absence of paclitaxel. It was confirmed that apoptosis was induced by clitaxel (white bar vs. gray par). In contrast, the addition of Pam ₃ CSK _4, from Rukoto been reduced absorbance, the TLR 1 ligand addition, apoptosis was found to be reduced (gray bars pair hatched par). Furthermore, TLR can be added without adding Pam ₃ CSK _4.

Transfection of 1 and MD2 was found to reduce paclitaxel-induced apoptosis (grey vs. black bars).

This clearly showed that the TLR1 signal has the effect of reducing apoptosis caused by anticancer drugs. Industrial applicability

The protein used in the present invention is a diagnostic marker for cancer, and therefore, regulates (preferably inhibits) a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein, or regulates (preferably inhibits) the expression of the protein gene. Or a salt thereof, the antibody of the present invention, the antisense nucleotide of the present invention, etc., for example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer Safe as a prophylactic / therapeutic agent for apoptosis, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, victory cancer, brain tumor or blood tumor, etc. It can be used as a novel drug, and can also be used as a therapeutic agent for cancer that is resistant to anticancer drugs. It can also reduce the dose of existing anticancer drugs and reduce side effects to increase sensitivity to anticancer drugs (eg, paclitaxel).

Claims

The scope of the claims

1. Cancer containing a compound or a salt thereof that inhibits the activity of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, or a salt thereof Prevention · Therapeutic agent.

2. It comprises 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. Cancer prevention and treatment.

3. A nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or the partial nucleotide-encoding DNA thereof, or Antisense nucleotide containing a part.

4. An agent for preventing or treating cancer comprising the antisense nucleotide according to claim 3.

5. 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.

6. Cancer is colorectal, breast, lung, prostate, esophageal, stomach, liver, biliary, spleen, kidney, bladder, uterus, ovary The preventive or therapeutic agent according to any one of claims 1 to 5, wherein the agent is cancer, testicular cancer, thyroid cancer, knee cancer, brain tumor or hematological tumor.

7. 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.

8. A diagnostic agent for cancer comprising DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof.

9. Cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophagus cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovary Claim 7 or Claim 8 which is cancer, testicular cancer, thyroid cancer, kidney cancer, brain tumor or blood tumor. The described diagnostic agent.

10. A prophylactic / therapeutic agent for cancer comprising a compound having a To 11-like receptor activity inhibitory activity or a salt thereof.

11. A prophylactic / therapeutic agent for cancer comprising a compound having a To 11-like receptor expression inhibitory activity or a salt thereof.

12. 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.

13. Prevention of cancer 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 screening therapeutic agents.

14. Prevention and treatment of cancer, characterized by using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. Agent screening method.

15. Prevention and treatment of cancer characterized by containing DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. Agent screening kit.

16. An agent for preventing or treating cancer, which can be obtained by using the screening method according to claim 12 or claim 14 or the screening kit according to claim 13 or claim 15.

17. It comprises a protein 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. Apoptosis inducer.

18. Contains a compound or a salt thereof that inhibits the expression of a protein or a partial peptide or a salt thereof, which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Apoptosis inducer.

19. A method for screening an apoptosis-inducing 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, a partial peptide thereof, or a salt thereof.

20. A method for screening an apoptosis-inducing agent, comprising using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. .

21. It comprises a compound or a salt thereof that inhibits the activity of a protein or a partial peptide thereof or a salt thereof having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Anticancer drug sensitivity enhancer.

22. It contains a compound or a salt thereof that inhibits the expression of a protein or a partial peptide thereof or a gene thereof which has the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Stake cancer drug sensitivity enhancer.

23. A compound that inhibits the activity of a protein, 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, or a mammal thereof. A method for preventing or treating cancer, which comprises administering an effective amount of a salt, or a compound or a salt thereof that inhibits the expression of the gene for the protein or its partial peptide or a salt thereof.

24. For the manufacture of a prophylactic or therapeutic agent for cancer, 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, a partial peptide thereof or a salt thereof is measured. Use of a compound that inhibits or a salt thereof, or a compound or a salt thereof that inhibits the gene expression of the protein or its partial peptide or a salt thereof.