WO2003085106A1 - Produits de prevention et/ou remedes pour le cancer - Google Patents

Produits de prevention et/ou remedes pour le cancer Download PDF

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Publication number
WO2003085106A1
WO2003085106A1 PCT/JP2003/004272 JP0304272W WO03085106A1 WO 2003085106 A1 WO2003085106 A1 WO 2003085106A1 JP 0304272 W JP0304272 W JP 0304272W WO 03085106 A1 WO03085106 A1 WO 03085106A1
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seq
dna
nucleotide sequence
receptor protein
present
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PCT/JP2003/004272
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English (en)
Japanese (ja)
Inventor
Yuichi Hikichi
Koji Yoshimura
Isao Kaieda
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Takeda Chemical Industries, Ltd.
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Priority to AU2003236358A priority Critical patent/AU2003236358A1/en
Publication of WO2003085106A1 publication Critical patent/WO2003085106A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to antisense oligonucleotides useful for cancer prevention and treatment, screening for cancer prevention and treatment agents, and the like.
  • Antisense oligonucleotides when introduced into cells, hybridize with RNA having a complementary sequence and induce RNA degradation by RNaseH to inhibit protein translation, or hybridize to inhibit direct protein synthesis. Also bring. 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 genes, and some antisense oligonucleotides are being developed for clinical application.
  • Protein tyrosine kinase genes form the largest family of oncogenes and are deeply involved in cell proliferation and differentiation and morphogenesis (Harvey Lecto. 94, 81-119, 1998- 1999), it has been picked up as a good target gene for developing botanicals.
  • drug development targeting the EGF receptor, HER2 receptor or VEGF receptor which is classified as receptor tyrosine kinase (RTK)
  • RTK receptor tyrosine kinase
  • RTK receptor tyrosine kinases
  • RTK is considered to be one of the subfamilies with a high probability of success as pharmaceuticals.
  • Eph erythropoietin producing human hepatocellular carcinoma cell line
  • the Ephr in gene family identified as ligands for the Eph receptor, has also been reported to consist of at least eight members.
  • extremely localized expression in embryos and the brain suggests their involvement in morphogenesis during development and proper projection of nerve axons ( Annu. Rev. Neurosci. 21, 309-345 (1998)), but its role in cancer has also attracted attention in recent years. For example, increased expression of Eph receptor in various cancers has been reported
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, found that cancer cells cause apoptosis by suppressing the expression of NEPHA gene expressed in cancer cells. Further studies based on this finding have led to the completion of the present invention.
  • an oligonucleotide having a nucleotide sequence identical or substantially identical to the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14,
  • the medicament according to the above (6) which is an agent for preventing and / or treating cancer.
  • a medicament comprising the compound according to (17) or a salt thereof, (18a) a medicament comprising the compound according to (17a) or a salt thereof, (19) the medicament according to the above (18), which is an agent for preventing and / or treating cancer; (19a) the medicament according to the above (18a), which is an agent for preventing and / or treating cancer;
  • a method for preventing and treating cancer which comprises administering to an animal an effective amount of the oligonucleotide according to (1).
  • Oligonucleotide containing a base sequence substantially identical to the base sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 (hereinafter, abbreviated as oligonucleotide of the present invention or antisense oligonucleotide of the present invention) Is an Eph receptor protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 11 (hereinafter, the receptor protein of the present invention or the Eph receptor protein of the present invention) (May be abbreviated as) and can inhibit the synthesis or function of the RNA, or through the interaction with the receptor protein-related RNA of the present invention.
  • the expression of the receptor protein gene can be regulated and / or controlled.
  • Oligonucleotides containing a base sequence substantially identical to the base sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 include nucleotides represented by SEQ ID NO: 13 or SEQ ID NO: 14. Any oligonucleotide having a nucleotide sequence that hybridizes under stringent conditions and having substantially the same activity as the oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 It may be something.
  • substantially equivalent activities include, for example, the activity of inhibiting the expression of the receptor protein of the present invention.
  • the expression inhibiting activity include transcription inhibition, splicing inhibition of precursor RNA, inhibition of mRNA translocation to the cytoplasm, and inhibition of translation into protein.
  • activities such as the above-mentioned expression inhibitory activity are equivalent (eg, about 0.01 to 100 times, preferably about (0.5 to 20 times, more preferably about 0.5 to 2 times).
  • the measurement of the above-mentioned inhibitory activity and the like can be performed according to a known method.
  • the inhibitory activity is determined by using a transformant containing the receptor protein of the present invention, an in vivo or in vitro gene expression system of the receptor protein of the present invention, or an in vivo or in vitro translation system of the receptor protein of the present invention.
  • DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14 under high stringent conditions include, for example, SEQ ID NO: 13 or SEQ ID NO: 14 A base having about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more homology with the base sequence. DNA containing a sequence is used. .
  • Hybridization can be performed by a method known per se or a method analogous thereto, for example, a method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Etc.
  • the procedure can be performed according to the method described in the attached instruction manual. More preferably, it can be carried out under high stringency 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, preferably about 60 to 60. The condition of 5 t is shown. Particularly, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
  • Oligonucleotides are polydeoxynucleotides containing 2-dexoxy-D-reports, polydeoxynucleotides containing D-reports, N-glycosides of purine or pyrimidine bases.
  • Other types of oligonucleotides or other polymers with non-nucleotide backbones eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • specialty Other polymers containing a suitable bond these polymers include base pairing as found in DNA and RNA, and contain a nucleotide having a configuration permitting base attachment).
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified oligonucleotides (or unmodified oligonucleotides). Nucleotides), as well as those with known modifications, such as labeled, capped, methylated, one or more natural nucleotides, as known in the art.
  • Is substituted with an analog, modified with an intramolecular nucleotide for example, has an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidite, thiolbamate, etc.), a charged bond or sulfur
  • bonds for example, phosphorothioate, phosphorodithioate, etc.
  • proteins for example, proteins (nucleases, nucleases / inhibitors) , Toxins, antibodies, signal peptides, poly-L-lysine, etc.) or sugars (for example, monosaccharides), etc., or those having an intermolecular compound (for example, acridine, psoralen, etc.)
  • Compounds containing chelating compounds eg, metals, radioactive metals, boron, oxidizable metals, etc.
  • alkylating agents those with modified bonds
  • eg, ⁇ -anomers Type nucleic acid
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted to a base.
  • the oligonucleotide of the present invention may be a modified nucleic acid (RNA, DNA).
  • modified nucleic acid include a sulfur derivative, a thiophosphate derivative, and a nucleic acid that is resistant to degradation of polynucleoside amide or oligonucleoside amide.
  • oligonucleotide of the present invention a base represented by SEQ ID NO: 13 DNA containing a sequence, DNA containing the base sequence represented by SEQ ID NO: 14 and the like are used.
  • the oligonucleotide of the present invention is produced according to a known method, for example, a solid phase or liquid phase synthesis method (eg, an automated nucleic acid synthesizer, liquid phase technology, etc.). Alternatively, it may be produced according to known gene engineering techniques (eg, use of reverse transcriptase, PCR synthesis, etc.).
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 11 include, for example, about 55% or more, and preferably about 60% or more of the amino acid sequence represented by SEQ ID NO: 11, More preferably, an amino acid sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. .
  • Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 11 include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 11 A protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 11 is preferred.
  • Substantially the same activity includes, for example, ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity, and cell motility regulating activity. Substantially the same indicates that their activities are the same in nature. Therefore, activities such as ligand binding activity, signal transduction activity, cell death inhibitory activity, cell adhesion activity and cell motility control activity are equivalent (eg, about 0.01 to 100 times, preferably Is preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times), but the quantitative factors such as the degree of activity and the molecular weight of the protein may be different.
  • the amino acid sequence of the receptor protein is the N-terminus (amino terminus) at the left end and the C-terminus (aminopropyl end) at the right end, according to the convention of peptide notation.
  • Receptor protein of the present invention, C-terminal, the force Rupokishiru group (-C00H), Karupokishire bets (-C00-), amide (- C0NH 2) or ester (- C00R) may be either.
  • R in the ester e.g., methyl, Echiru, n- propyl, C 1H5 alkyl group such as isopropyl or n- butyl, cyclopentyl Le, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, (3 6 _ 12 Ariru groups, such as single-naphthyl shed, for example, benzyl, phenylene route C DOO 2 alkyl or ⁇ - naphthylmethyl etc.
  • ⁇ - naphthyl such as phenethyl - C, such as _ 2 alkyl groups in addition to C 7 _ 14 Ararukiru group, Viva port Iruo Kishimechiru group commonly used as an oral ester.
  • the receptor protein of the present invention When the receptor protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the receptor protein of the present invention also includes those in which the lipoxyl group is amidated or esterified. .
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the receptor protein of the present invention is the protein mentioned above, Amino group protecting groups methylate Onin residues of N-terminal (e.g., formyl group, C 2 such Asechiru - such Ashiru group such as 6 Al Kanoiru group ), The N-terminal side is cleaved in vivo, and the daryumil group formed is pyroglutamine-oxidized, the substituent on the side chain of the amino acid in the molecule (eg, -0H, -SH, amino group, imidazole group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C 2 such Asechiru - those protected by 6 etc. Ashiru groups such Arukanoiru group), a sugar chain Complex proteins such as bound so-called glycoproteins are also included.
  • Amino group protecting groups methylate Onin residues of N-terminal e.g., formyl group, C 2 such Asechir
  • receptor protein of the present invention include, for example, a receptor protein containing the amino acid sequence represented by SEQ ID NO: 11 and the like.
  • a partial peptide of the receptor protein of the present invention (hereinafter, sometimes abbreviated as the partial peptide of the present invention) can also be used.
  • the partial peptide of the present invention may be any peptide as long as it is a partial peptide of the above-described receptor protein of the present invention.
  • the receptor protein molecules of the present invention For example, a portion having a substantially identical activity or a portion located inside the cell membrane is used.
  • substantially the same activity refers to, for example, ligand binding activity, signal information transmission activity, cell death inhibitory activity, cell adhesion activity, and cell motility control activity.
  • Ligand binding activity, signal transduction activity, cell death suppressing activity, measurements, such as cell adhesion activity and cell motility control activity, the amino acid number of the partial base peptide of the c the present invention can be carried out according to known methods, Peptides having an amino acid sequence of at least 5 or more, preferably 10 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences of the above-described receptor protein of the present invention are preferable. .
  • a substantially identical amino acid sequence is at least about 50%, preferably at least about 60%, more preferably at least about 70%, still more preferably at least about 80%, and most preferably at least about 50% of these amino acid sequences.
  • An amino acid sequence having about 90% or more, most preferably about 95% or more homology is shown.
  • the partial peptide of the present invention comprises: (i) one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence; (Ii) one or two or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) (Iii) one or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the above amino acid sequence It may be substituted with an amino acid.
  • Specific examples of the partial peptide of the present invention include the 60th to 73rd or 491st to 504th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 11. Examples of such peptides include:
  • the C-terminus carboxyl group of the present invention (- C00H), Karupoki Shireto (- C00-), amide (- C0NH 2) or esters may be a (-C00R) (R is as defined above Is shown).
  • R is as defined above Is shown.
  • the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carbonyl group is amidated or esterified are also included in the partial peptide of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the partial peptide of the present invention has a N-terminal methionine residue in which the amino group of the methionine residue is protected with a protecting group, and the N-terminal side is cleaved in vivo to produce Dartamyl group is pyroglutamine-oxidized, amino acid side chain in the molecule is protected by a suitable protecting group, or sugar chain Conjugated peptides, such as so-called glycopeptides, to which is bound.
  • Examples of the salt of the receptor protein or its partial peptide of the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
  • 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) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like are used.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid
  • the receptor protein of the present invention or a salt thereof can be produced from the above-described human or mammalian cells or tissues by a known method for purifying a receptor protein, or encodes the receptor protein of the present invention described later. It can also be produced by culturing a transformant containing DNA. Also, the protein can be produced by the protein synthesis method described later or according to this method.
  • the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the resulting extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be carried out by combining the above chromatography methods.
  • a commercially available resin for protein synthesis can be usually used.
  • resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxy resin.
  • Methylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2', 4'dimethyloxyphenyl Fmocaminoethyl) Phenoxy resins and the like can be mentioned.
  • amino acids having appropriately protected amino groups and side chain functional groups are condensed on the resin according to various known condensation methods in accordance with the amino acid sequence of the target protein or peptide.
  • a protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain a target protein or partial peptide. Or its amide form.
  • various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • the carbopimides include DCC, ⁇ , ⁇ ′-diisopropyl carbopimide, and ⁇ -ethyl- ⁇ ′-(3-dimethylaminoprolyl) carbopimide.
  • Activation by these involves the addition of a protected amino acid directly to the resin along with a racemization inhibitor additive (eg, HOBt, HOOBt), or a symmetrical acid anhydride or HOBt ester or H ⁇ Bt ester. Can be added to the resin after activation of the protected amino acid in advance.
  • the solvent used for activating the protected amino acid or for condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, and N-methylpyrrolidone
  • 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.
  • 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 20 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the amino group of the starting material include Z, Boc, succinyl-pentyloxycarbonyl, isoporiloxycarbonyl, 4-methoxybenzyloxycarbonyl, C1-Z, Br-Z, Adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group may be, for example, an alkyl esterified (eg, methyl, ether, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Or cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-cyclobenzyl ester, benzhydryl esterification), phenacyl ester Can be protected by benzyloxycarbonyl hydrazide, short-lived butoxycarbonyl hydrazide, trityl hydrazide, etc.
  • alkyl esterified eg, methyl, ether, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl,
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarbonyl group and the like are used.
  • groups suitable for etherification include, for example, a benzyl group, a tetrahydropyranyl group, and a t-peptizole group.
  • the protecting group of the phenolic hydroxyl group of tyrosine for example, B zl, C 1 2 - B z 1, 2 twelve Torobenjiru, B r- Z, such as tertiary butyl is used.
  • Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc.
  • Activated carbonyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitro Phenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt).
  • active esters eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitro Phenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt.
  • active esters eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitro Phenol, cyanomethyl alcohol, paranitrophenol, H ⁇ NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with H
  • Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride or methanesulfonic acid. , Trifluoromethanesulfonic acid, trifluoroacetic acid or this Acid treatment with a mixed solution thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, and the like, and reduction with sodium in liquid ammonia are also used.
  • the elimination reaction by the above acid treatment generally takes about one
  • the reaction is carried out at a temperature of 20 ° C to 40 ° C.
  • the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tributofan is 1,2-ethanedithiol, 1,4 —In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, 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.
  • an amide form of a protein for example, first, after amidating and protecting the ⁇ -hydroxyl group of the carboxy-terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After the elongation, a protein was prepared by removing only the protecting group of the ⁇ -amino group at the ⁇ -terminal of the peptide chain and a protein was obtained by removing only the protecting group of the carboxy group at the C-terminus. In such a mixed solvent. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method, and a desired crude protein can be obtained. This crude protein is purified by various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • ester for example, after condensing the carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the ester of the desired protein is converted in the same manner as the amide of the protein. Obtainable.
  • the partial peptide of the receptor protein of the present invention or a salt thereof can be obtained by cleaving the receptor protein of the present invention with an appropriate peptidase according to a known peptide synthesis method. And can be manufactured by 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 objective peptide is produced by condensing a partial peptide or amino acid capable of constituting the receptor protein of the present invention with the remaining portion, and if the product has a protecting group, removing the protecting group to produce the desired peptide. Can be.
  • Known condensation methods and elimination of protecting groups include, for example, the following (i) to
  • the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, 'liquid chromatography', and recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained as a salt, it is converted to a free form by a known method. be able to.
  • the polynucleotide encoding the receptor protein of the present invention is not particularly limited as long as it contains the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. Is also good.
  • the polynucleotide is RNA such as DNA or mRNA encoding the receptor protein of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
  • Examples of the DNA encoding the receptor protein of the present invention include genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, and cells derived from the above-described cells. It may be either a conventional cDNA library or synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • DNA may be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR) using a preparation of all RNA or mRNA fractions from the above-mentioned cells' tissues. it can.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • a DNA containing the nucleotide sequence represented by SEQ ID NO: 12 or the nucleotide represented by SEQ ID NO: 12 Has DNA that hybridizes under high stringent conditions to DNA containing the sequence, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal transduction activity, cell death)
  • Any DNA may be used as long as it encodes a receptor protein having inhibitory activity, cell adhesion activity, and cell motility regulating activity.
  • Examples of the DNA which hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 12 under high stringent conditions include, for example, about 55% or more of the nucleotide sequence represented by SEQ ID NO: 12; Preferably containing a base sequence having a homology of about 60% or more, preferably about 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more. DNA or the like is used.
  • Hybridization can be performed by a known method or a method analogous thereto, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be carried out according to the method described in the attached instruction manual. More preferably, hybridization can be performed under high stringency conditions.
  • the 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 condition of 65 T: is shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.
  • the DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. Also, any of genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. DNA can also be directly amplified by the RT-PCR method using an mRNA fraction prepared from the cells and tissues described above.
  • the DNA encoding the partial peptide of the present invention includes, for example, DNA having a partial nucleotide sequence of DNA containing the nucleotide sequence represented by SEQ ID NO: 12, or DNA represented by SEQ ID NO: 12.
  • a DNA having a partial nucleotide sequence of the DNA to be used is used.
  • a DNA encoding the receptor protein of the present invention may be used.
  • Amplification by the PCR method using a synthetic DNA primer having a partial nucleotide sequence of the nucleotide sequence, or DNA incorporating the DNA into a suitable vector is performed for partial or whole region of the receptor protein of the present invention. Selection can be performed by hybridization with a DNA fragment to be coded or labeled with a synthetic DNA.
  • the DNA base sequence can be replaced by ODA-LA PCR using PCR or a known kit, for example, Mutan TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc.
  • the method can be performed according to known methods such as the gapped duplex method and the Kunkel method, or a method analogous thereto.
  • the DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end, and TAA, TGA or TAG as a translation stop codon at the 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.
  • the DNA encoding the receptor protein of the present invention includes, for example, (a) a DNA fragment encoding the receptor protein of the present invention; Can be incorporated into an expression vector by ligating the DNA downstream of the promoter in an appropriate expression vector.
  • vectors for cloning or expression include plasmids derived from E. coli (eg, pCR4, pCR2.K pBR322, pBR325, pUC12, pUC13), and plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194) ), Yeast-derived plasmids (eg, pSH19,
  • PSH15 bacteriophage
  • bacteriophage such as ⁇ phage
  • animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.
  • pAl-11 XTK pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.
  • pAl-11 XTK pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.
  • the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRo when an animal cell is used as a host, SRo; a promoter, an SV40 promoter, an LTR promoter, a CMV promoter, an HSV-TK promoter, and the like can be mentioned. Of these, it is preferable to use CMV promoter, SRa promoter and the like.
  • S S When the host is Escherichia, trp promoter, 1 ac promoter, recA promoter, ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ promoter, 1 ⁇ promoter, etc., and when the host is Bacillus, S S When the host is yeast, such as ⁇ 1 promoter, S ⁇ 2 promoter, pe ⁇ promoter, etc., ⁇ 05 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. If the host is an insect cell, the polyhedrin promoter and the P10 promoter
  • expression vectors include, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter, referred to as the
  • Examples of the c- selectable marker that can be used include a gene for dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance ], An ampicillin resistance gene (hereinafter sometimes abbreviated as Amp 1 ), a neomycin resistance gene (hereinafter sometimes abbreviated as Ne of, G418 resistance), and the like.
  • dhfr gene for dihydrofolate reductase
  • MTX metalhotrexate
  • Amp 1 An ampicillin resistance gene
  • Ne of, G418 resistance neomycin resistance gene
  • the target gene can be selected even with a thym-free medium.
  • a signal sequence suitable for the host can be added to the N-terminal side of the receptor protein of the present invention. If the host is a genus Escherichia, the Ph A signal sequence, the ⁇ mp A signal sequence, etc., if the host is a Bacillus genus, the ⁇ -amylase signal sequence, subtilisin signal sequence, etc. If the host is yeast, MF, signal sequence, SUC2, signal sequence, etc.If the host is an animal cell, insulin signal sequence, Hi-interferon, signal sequence, antibody molecule, signal sequence, etc. Are available respectively.
  • a transformant can be produced.
  • Examples of the host used in the present invention include Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like.
  • Escherichia coli K12-DH1 Escherichia coli K12-DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103.
  • Bacillus bacteria include, for example, Bacillus subtilis (Bacillus).
  • yeast examples include, for example, Saccharomyces cerevisiae AH22, AH22R-, ONA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036 Pichia pastoris (Pichia pastoris) ) Is used.
  • Insect cells include, for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell; Si cell), or the midgut of Trichoplusia ni Original MG1 cells, High Five TM cells derived from Trichoplus ia ni eggs, Mames tra
  • Sf cells derived from brass icae or cells derived from Estigmena acrea are used.
  • viruses When the virus is BmNPV, a cell line derived from silkworm (Bombyx mori N; BmN cell) is used.
  • Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells (hereinafter, In Vivo, 13, 213-217 (1977)) and the like are used.
  • insects for example, silkworm larvae are used [Nature, vol. 315, 592 (1985)].
  • animal cells examples include monkey cells COS-7, Vero, Chinese Hams Yuichi cell CH0 (hereinafter abbreviated as CH0 cells), dhfr gene-deficient Chinese Hams Yuichi cells CH0 (hereinafter abbreviated as CHO (dhfr) cells). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
  • Transformation of a bacterium belonging to the genus Escherichia can be performed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) or Gene, 17, 107 (1982). it can.
  • Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
  • Transformation of yeast can be performed, for example, according to the method described in Methods in Enzymology, Vol. 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978). Can be.
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nitrogen sources inorganic substances and others.
  • a carbon source For example, glucose, dextrin, soluble starch, sucrose, etc.Nitrogen sources include, for example, inorganic or nitric acid salts such as ammonium salts, nitrates, corn steep liquor, peptone, power zein, meat extract, soybean meal, potato extract, etc.
  • the organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added.
  • the pH of the medium is preferably about 5-8.
  • an M9 medium containing glucose and casamino acids is preferable. If necessary, an agent such as 3-indolylacrylic acid can be added to make the promoter work efficiently.
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring may be added.
  • the cultivation is usually performed for about 30 to 4 (TC for about 6 to 24 hours, and if necessary, aeration and stirring may be applied.
  • the culture medium When culturing a transformant in which the host is yeast, the culture medium may be, for example, a Burkholder minimum medium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] or 0.5 Natl. Acad. Sci. USA, 81, 5330 (1984)]].
  • the pH of the medium is preferably adjusted to about 5-8. Cultivation is usually carried out at about 20 ° C to 35 for about 24 to 72 hours, with aeration and agitation as necessary.
  • a medium such as Grace's Insect Medium (Nature, 195, 788 (1962)) is supplemented as appropriate with an additive such as immobilized 10% serum.
  • an additive such as immobilized 10% serum.
  • the pH of the medium is adjusted to about 6.2-4.
  • Culture is usually performed at about 27 ° C. for about 3 to 5 days, and if necessary, aeration and agitation are added.
  • a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology , 8 vol., 396 (1959)], RPMI1640 medium [The Journal of the Amer i can Med i cal Assoc i ation, Volume 199, 519 (1967)], 199 medium [Proceeding of the Socie ty for or the Biologi cal Med ici ne, Volume 73, 1 (1950) ] Etc. are used.
  • the pH is about 6-8.
  • Culture is usually performed at about 30 ° C to 4 (TC for about 15 to 60 hours, and aeration and agitation are added as necessary.
  • the receptor protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • Separation and purification of the receptor protein of the present invention from the above culture can be performed, for example, by the following method.
  • the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasonication, lysozyme and / or lysozyme.
  • a method of destroying bacterial cells or cells by freeze-thawing or the like, and then obtaining a crude extract of the receptor protein by centrifugation or filtration is used as appropriate.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • Purification of the receptor protein contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation and purification methods.
  • These known separation and purification methods include methods that utilize solubility such as salting out and solvent precipitation, and methods that mainly use the difference in molecular weight, such as dialysis, ultrafiltration, and gel filtration.
  • a method that utilizes a difference in charge such as ion exchange chromatography; a method that utilizes a specific affinity such as affinity chromatography; a method that utilizes a difference in hydrophobicity such as reverse-phase high-performance liquid chromatography;
  • a method utilizing the difference between isoelectric points such as point electrophoresis is used.
  • the receptor protein thus obtained When the receptor protein thus obtained is obtained as a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the receptor protein is obtained as a salt, the known method is used. Alternatively, it can be converted to a free form or another salt by a method analogous thereto.
  • the receptor protein produced by the recombinant can be arbitrarily modified or partially modified by the action of an appropriate protein-modifying enzyme before or after purification. Can also be removed.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the activity of the receptor protein of the present invention or a salt thereof thus produced can be measured by a binding experiment with a labeled ligand, an enzymimnoassay using a specific antibody, or the like.
  • the antibody against the receptor protein or its partial peptide or its salt of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it is an antibody that can recognize the receptor protein or its partial peptide or its salt of the present invention. Is also good.
  • An antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof may be a known antibody using the receptor protein or the like of the present invention as an antigen. Alternatively, it can be produced according to a method for producing an antiserum.
  • the receptor protein or the like of the present invention is administered to a mammal at a site capable of producing an antibody by administration itself or together with a carrier or a diluent.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance antibody production upon administration.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
  • a warm-blooded animal immunized with the antigen for example, a mouse 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.
  • a monoclonal antibody-producing hybridoma By fusing the antibody-producing cells contained in the above with myeloma cells, a monoclonal antibody-producing hybridoma can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting the labeled receptor protein or the like described below with antiserum, and then measuring the activity of the labeling agent bound to the antibody. .
  • the fusion operation is performed in a known manner, for example, in the case of Keller and Milstein. Act (Nature, 256, 495, 1975).
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, and PEG is preferably used.
  • myeloma cells examples include NS-1, P3UK SP2 / 0 and the like, and P3U1 is preferably used.
  • the preferred ratio between the number of somatic cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to
  • PEG6000 is added at a concentration of about 10 to 80%, and the cell fusion can be carried out efficiently by incubating at about 20 to 40 ° (: preferably at about 30 to 37 ° C for about 1 to 10 minutes).
  • hybridomas are cultured on a solid phase (eg, microplate) onto which an antigen such as a receptor protein is adsorbed directly or together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse) or protein A, which is labeled with a radioactive substance or an enzyme, is added.
  • a solid phase eg, microplate
  • an antigen such as a receptor protein
  • an anti-immunoglobulin antibody anti-mouse immunoglobulin antibody is used if the cell used for cell fusion is a mouse
  • protein A which is labeled with a radioactive substance or an enzyme
  • a method for detecting monoclonal antibodies bound to a solid phase adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A is adsorbed, and adding a receptor protein or the like labeled with a radioactive substance, an enzyme, etc. And a method for detecting a monoclonal antibody bound to a solid phase.
  • the selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or hybridoma culture medium Serum medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used.
  • the cultivation temperature is usually 20 to 40 ° (:, preferably about 37.
  • the cultivation time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the cultivation is usually 5% CO 2 gas.
  • the antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies.
  • salting out alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or specific purification by collecting only the antibody using an active adsorbent such as protein A or protein G and dissociating the bond to obtain the antibody Act].
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as the receptor protein of the present invention) and a carrier-protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting an antibody-containing substance against the receptor protein or the like and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of carrier to hapten are determined by the ratio of hapten immunized by cross-linking to carrier protein. Any antibody may be cross-linked at any ratio as long as it can be efficiently produced.
  • serum serum alpmin, shishiro glopurin, keyhole, limpet, hemocyanin, etc. are weight ratios.
  • a method of coupling the hapten 1 at a ratio of about 0.1 to 20 and preferably about 1 to 5 is used.
  • 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 can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal 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 serum described above.
  • Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.
  • non-human mammals e.g., mouse, rat, rabbit, sheep, pigeon, pig, cat, dog, monkey, etc., more specifically, dementia rat, obese mouse, atherosclerotic rabbit, For cancer-bearing mice
  • human cells e.g., human cells
  • a specific organ eg., brain, lung, large intestine, etc.
  • a tissue or cell isolated from the organ is obtained.
  • the mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells is quantified by, for example, extracting mRNA from cells or the like by a usual method, and using, for example, a technique such as TaqMan PCR. It can also be analyzed by performing Northern blotting by known means.
  • a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the mRNA of the receptor protein of the present invention or the partial peptide thereof contained in the transformant is prepared. It can be quantified and analyzed in the same way.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention and the like. Can be used for quantification of the receptor protein and the like, particularly for quantification by sandwich immunoassay. For example,
  • one antibody is an antibody that recognizes the N-terminal of the receptor protein or the like of the present invention
  • the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention. Is preferred.
  • the receptor protein and the like of the present invention can be measured using a monoclonal antibody against the receptor protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and detection by tissue staining and the like can be performed. You can do it too.
  • the antibody molecule itself may be used, or F (ab ') 2 , Fab', or Fab fraction of the antibody molecule may be used.
  • the measurement method using an antibody against the receptor protein or the like 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 an antigen (for example, the amount of a receptor protein) in a liquid to be measured.
  • any measurement method may be used as long as the amount of the body is detected by chemical or physical means, and the amount is calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • nephrometry, a competitive method, an immunometric method, and a sandwich method are suitably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes, if example embodiment, [125 1], 131 1], 3 ⁇ 4], "c], etc.
  • the enzyme large preferably stable and specific activity, e.g., beta -.
  • Galactosidase one ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate, etc., is used.
  • a biotin-avidin system may be used for binding an antibody or antigen to a labeling agent.
  • insolubilization of the antigen or antibody physical adsorption may be used.
  • a method using a chemical bond used for insolubilizing or immobilizing white matter or an enzyme may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass are used.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent and the method of insolubilization can be in accordance with those described above.
  • the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having a different binding site to the receptor protein or the like.
  • 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 receptor protein, the antibody used in the primary reaction is preferably the C-terminal.
  • an antibody that recognizes other than the N-terminal part is used.
  • the monoclonal body of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • a competition method the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. Then (BZF separation), the amount of labeling of either B or F is measured, and the amount of Bobara in the test solution is quantified.
  • a soluble antibody is used as the antibody
  • B / F separation is performed using polyethylene glycol
  • a liquid phase method using a second antibody against the above antibody or a solid phase antibody is used as the first antibody.
  • a solid phase method using a soluble first antibody and a solid phase antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen After a competitive reaction with the body, the solid phase and the liquid phase are separated, or the antigen in the test solution is allowed to react with an excessive amount of the labeled antibody, and then the immobilized antigen is added and unreacted After binding the labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
  • nephelometry the amount of insoluble sediment generated as a result of an 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 precipitate is obtained, laser nephrometry utilizing laser scattering is preferably used.
  • the receptor protein of the present invention or a salt thereof can be quantified with high sensitivity.
  • a drug containing an antisense oligonucleotide The oligonucleotide of the present invention is stable in cells, has high cell permeability, has high affinity for the target sense strand, and has low toxicity.
  • the polynucleotide of the present invention may be, for example, a disease associated with overexpression of the receptor protein of the present invention, such as cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix) Cancer, colon cancer, rectal cancer, etc.), and can be used as a medicament such as a prophylactic or Z or therapeutic agent.
  • cancer eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervix
  • Cancer colon cancer
  • rectal cancer etc.
  • the antisense oligonucleotides of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes, microspheres, or may be applied by gene therapy. , Can be provided in an added form. Additional forms include polycations such as polylysine, which acts to neutralize the charge on the phosphate backbone, and lipids that enhance interaction with cell membranes or increase nucleic acid uptake (eg, Hydrophobic substances such as phospholipids and cholesterol). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.).
  • nucleic acids 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 include capping groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase. Examples of such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the antisense oligonucleotide of the present invention When used as the above medicine, it can be formulated and administered according to a known method.
  • the antisense oligonucleotide when used, the antisense oligonucleotide is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like.
  • an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like.
  • Oral or non-human administration to human or non-human mammals eg, rats, gray egrets, sheep, higgs, bush, red sea lions, cats, dogs, monkeys, etc.
  • human or non-human mammals eg, rats, gray egrets, sheep, higgs, bush, red sea lions, cats, dogs, monkeys, etc.
  • the antisense oligonucleotide 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 then administered using a gene gun or a catheter such as a hydrogel catheter.
  • a physiologically acceptable carrier such as an adjuvant for promoting uptake
  • the dose of the antisense oligonucleotide varies depending on the target disease, the subject of administration, the route of administration, and the like.
  • the antisense oligonucleotide of the present invention may be administered to an organ (eg, liver, lung, When topically administered to the heart, kidney, etc., generally, for an adult (body weight 60 kg), about 0.1 to 100 mg of the antisense oligonucleotide is administered per day.
  • the antisense oligonucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence of the polynucleotide (eg, DNA) of the present invention in tissues or cells and the state of expression thereof.
  • the polynucleotide eg, DNA
  • the present invention provides a transformant transformed with a recombinant DNA in which a reporter gene is ligated downstream (under expression control) of the transcriptional regulatory region of the gene of the receptor protein of the present invention in the presence of a test compound.
  • a method for screening for a compound that promotes or inhibits the transcriptional regulatory activity comprising measuring and comparing each repo overnight activity when cultured in the absence and presence of the same, and a script for this method.
  • a kit for single use is also provided.
  • Examples of the transcription regulatory region of the gene of the receptor protein of the present invention include a DNA having the same or substantially the same nucleotide sequence as the nucleotide sequence represented by SEQ ID NO: 20.
  • the DNA containing a base sequence substantially identical to the base sequence represented by SEQ ID NO: 20 has a base sequence that hybridizes under high stringency end conditions, and has substantially the same sequence as SEQ ID NO: 20. Any DNA may be used as long as it has the same transcriptional regulatory activity (eg, promoter-one activity).
  • DNA that can hybridize with the nucleotide sequence represented by SEQ ID NO: 20 under high stringent conditions include, for example, the nucleotide sequence represented by SEQ ID NO: 20 A base having a homology of about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • a DNA containing the sequence is used.
  • Hybridization is performed according to a method known per se 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, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringency conditions.
  • the high stringent end conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2 OmM, and a temperature of about 50 to 70: preferably about 60 to 65.
  • a sodium concentration of about 19 to 40 mM, preferably about 19 to 2 OmM, and a temperature of about 50 to 70: preferably about 60 to 65.
  • the sodium concentration is about 19 mM and the temperature is about 65 ° C.
  • the transcription regulatory region eg, DNA containing a nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 20
  • the transcription regulatory region is substantially the same as the nucleotide sequence represented by SEQ ID NO: 20
  • a sequence obtained by further adding a genomic DNA sequence at the 5 ′ upstream to the same base sequence (preferably a promoter region) is also included.
  • the number of bases to be added is, for example, about 10 Kb or less, preferably about 5 Kb or less, more preferably about 2 Kb or less, and most preferably about 1 Kb or less.
  • reporter genes include, for example, lacZ (yogalactosidase gene), chloramphenicol acetyltransferase (CAT), luciferase, growth factor,] 3-glucuronidase, alphospholiphosphatase, Green fluorescent protein (GFP), / 3-lacquerase and the like are used.
  • a test compound that increases the amount of the repo overnight gene product can be used as a test compound for the receptor protein of the present invention.
  • a compound having an action of controlling (particularly promoting) transcriptional regulatory activity preferably promoter activity
  • ie, promoting expression of the receptor protein of the present invention can be selected as a compound having a certain activity.
  • a test compound that reduces the amount of a reporter gene product is a compound that has the activity of controlling (particularly inhibiting) the transcriptional regulatory activity (preferably, overnight promoter) of the receptor protein of the present invention, that is, the compound of the present invention.
  • the compound can be selected as a compound having an activity of inhibiting the expression of the receptor protein.
  • the test compound is, for example, a compound selected from peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like.
  • the salt of the compound those similar to the aforementioned salts of the receptor protein of the present invention are used.
  • the transformant can be cultured in the same manner as the above-described transformant containing the receptor protein of the present invention.
  • the vector construction of the repo overnight gene and the Atsey method can be performed according to known techniques (eg, Molecular Biotechnology 13, 29-43, 1999).
  • the compound having an activity of promoting the expression of the receptor protein of the present invention or a salt thereof is useful as a safe and low-toxic drug.
  • a compound having an activity of inhibiting the expression of the peptide of the present invention or a salt thereof is a safe and low-toxic drug for suppressing the physiological activity of the receptor protein of the present invention, for example, cancer (eg, non-small cell lung cancer) Ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.).
  • cancer eg, non-small cell lung cancer
  • salt those similar to the aforementioned salts of the receptor protein of the present invention are used.
  • the above compound when used as a medicine, it can be formulated according to conventional means.
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • prophylactic and therapeutic agents examples include, but are not limited to, a buffer (eg, a phosphate buffer, a sodium acetate buffer), a soothing agent (eg, benzalkonidum chloride, proforce hydrochloride, etc.), a stabilizer (eg, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • a buffer eg, a phosphate buffer, a sodium acetate buffer
  • a soothing agent eg, benzalkonidum chloride, proforce hydrochloride, etc.
  • a stabilizer eg, Human serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants and the like examples include, but are not limited to, a buffer (eg, a phosphate buffer, a sodium acetate buffer), a soothing agent (eg
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and mammals (eg, rats, mice, egrets, higgs, bushes, cats, cats, dogs, dogs, etc.). Can be administered.
  • mammals eg, rats, mice, egrets, higgs, bushes, cats, cats, dogs, dogs, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • oral administration for example, in a cancer patient (with a body weight of 60 kg), About 0.1 to 100 nig per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 nig.
  • parenteral administration the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc.
  • injection it is usually used, for example, in cancer patients (with a body weight of 60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection.
  • the dose can be administered in terms of weight per 60 kg.
  • a base, an amino acid, or the like is indicated by an abbreviation, the indication is based on an abbreviation by IUPAC-IUB Communication on Biochemical Nomenclature or a conventional abbreviation in the field. An example is shown below.
  • an amino acid can have optical isomers, the L-form is indicated unless otherwise specified.
  • Trp Tryptophan
  • FIG. 9 shows a part of the nucleotide sequence (3 ′ side) of cDNA obtained in Reference Example 3 below.
  • the nucleotide sequence of cDNA which encodes a novel receptor protein NEPHA derived from human brain is shown.
  • Example 1 shows the base sequence of the oligonucleotide used in Example 1 below.
  • Example 1 shows the base sequence of the oligonucleotide used in Example 1 below.
  • the human brain cDNA (CL0NTECH) contained in MTC Panel I was type III, and PCR was carried out using two primers, primer 1 (SEQ ID NO: 2) and primer 2 (SEQ ID NO: 3). .
  • the composition of the reaction solution used in the reaction was the above cDNA 0.51 as type III, 1 U of Pfu Turbo DNA Polymerase (STRATAGENE), primer 1 (SEQ ID NO: 2) and primer 1 (SEQ ID NO: 3) 1 M each, 200 l of dNTPs, and 101 of 2XGC Buf ferl (Takara Shuzo) were added to give a liquid volume of 201.
  • the PCR reaction was repeated 96 times at 96X3 for 1 minute, followed by a cycle of 96 ° C, 1 minute, 60 ° C, 1 minute, 72 ° C, and 1 minute 40 times.
  • the PCR reaction product was purified by agarose gel electrophoresis, and then purified using the plasmid vector pCR-Bluntil-T0P0 (Invitrogen) according to the prescription of Zero Blunt T0P0 PCR Cloning Kit (Invitrogen). ).
  • This was introduced into E. coli TOP10 and selected on LB agar medium containing kanamycin.
  • SEQ ID NO: 1 nucleotide sequence of cDNA encoding a part of the novel receptor protein was obtained.
  • PCR was carried out using two primers, primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6).
  • the composition of the reaction solution used in the reaction was the above-mentioned cDNA 11 as type III, and 2.5 U of Pfu Turbo DNA Polymerase (STRATAGENE), primer 3 (SEQ ID NO: 5) and primer 4 (SEQ ID NO: 6).
  • STRATAGENE Pfu Turbo DNA Polymerase
  • primer 3 SEQ ID NO: 5
  • primer 4 SEQ ID NO: 6
  • PCR was performed using two primers, Primer 5 (SEQ ID NO: 8) and Primer 1 (SEQ ID NO: 9).
  • the composition of the reaction solution used in the reaction was the above cDNA II as type III, and 2.5 U of Pfu Turbo DNA Polymerase (STRATAGENE), Primer 5 (SEQ ID NO: 8) and Primer 6 (SEQ ID NO: 9) 1 M, dNTPs were added to 200 XM, and 2XGC Buffer1 (Takara Shuzo) to make a liquid volume of 501.
  • the PCR reaction was repeated 40 times at 96, 1 minute, and 60 times for 1 minute, 72 ° C, and 2 minutes. Next, 5 l of the PCR reaction product was used as type ⁇ , and Pfu Turbo DNA Polymerase was used.
  • SEQ ID NO: 12 Based on the nucleotide sequence of the partial cDNA encoding the novel receptor protein obtained in Reference Example 1, Reference Example 2 and Reference Example 3 (SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7), The sequence (SEQ ID NO: 12) was determined. SEQ ID NO: 12 corresponds to the 71st to 789th sequence of SEQ ID NO: 1, the 284th to 1134th sequence of SEQ ID NO: 4 and the 473th to 1926th sequence of SEQ ID NO: 7 . The amino acid sequence of the protein predicted from the nucleotide sequence of the full-length cDNA (SEQ ID NO: 12) is shown in SEQ ID NO: 11.
  • NEPHA novel EphA
  • NEPHA and the human Ephr in receptor EphA7 have 50.7% homology at the amino acid level.
  • the gene encoding the full length is obtained by cleaving the cDNA fragments obtained in Reference Examples 1, 2 and 3 with an appropriate restriction enzyme and ligating them with T4 ligase or the like.
  • a primer is designed to amplify the cDNA encoding the full length based on the nucleotide sequences identified in Reference Examples 1 and 3, and the primers are obtained by PCR.
  • Reference example 5
  • a plasmid containing the nucleotide sequences of the cDNAs obtained in Reference Examples 2 and 3 was digested with restriction enzymes.
  • the composition of the reaction mixture was lg, Xol (Takara Shuzo) 5U, and ⁇ ⁇ Buf, containing a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 4 or a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 7.
  • fer (Takara Shuzo) 5 x I was added to make a liquid volume of 501.
  • the restriction enzyme reaction was performed at 37 ° C.
  • the nucleotide sequence of cDNA represented by SEQ ID NO: 13 was obtained. Further, a plasmid containing the nucleotide sequence of the cDNA obtained in Reference Example 1 (SEQ ID NO: 1) and a plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 13 were digested with restriction enzymes.
  • the composition of the reaction solution is lfig, Pvul (Takara Shuzo) 10 U, and plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 1 or plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 13.
  • the plasmid containing the nucleotide sequence of the cDNA represented by SEQ ID NO: 1 contains 743 bp and the nucleotide sequence of the cDNA represented by SEQ ID NO: 13
  • the reaction product corresponding to 5977 bp was purified from the plasmid by agarose gel electrophoresis, and these were ligated according to the prescription of DNA Ligation Kit Ver.2 (Takara Shuzo).
  • breast cancer cell line ZR-75-1 (Cancer Res, 38 (10), 3352-3364, purchased from ATCC, 1978) was used. Falcon).
  • OligofectAMINE (Invitrogen) was used and the protocol was followed. Twenty hours after the introduction, RNA was extracted according to the protocol of the RNeasy mini kit (Qiagen), and cDNA was prepared using the TaqManRT kit (Perkin-Elmer). At the same time, the same reaction was carried out without adding reverse transcriptase to obtain a non-reverse transcription control.
  • a Cell death detection ELISA (Roche) was used to examine the effect on the apoptosis when the expression of the NEPHA gene was suppressed by introducing the antisense oligonucleotide.
  • the control oligonucleotide was used to compare with the sample introduced with the oligonucleotide.
  • the expression of the NEPHA gene was 20 hours after the introduction of the antisense oligonucleotide compared to the control oligonucleotide (SEQ ID NO: 15).
  • the amount of expression of the NEPHA gene was reduced to 57% when the antisense (SEQ ID NO: 14) was used, compared to the control oligonucleotide (SEQ ID NO: 16).
  • apoptosis was calculated to be 224% when the control oligonucleotide (SEQ ID NO: 13) was introduced and the control oligonucleotide (SEQ ID NO: 13) was assumed to be 100%. Assuming that the oligonucleotide (SEQ ID NO: 16) was 100%, it was increased to 185% when the antisense oligonucleotide (SEQ ID NO: 14) was introduced.
  • the oligonucleotide of the present invention has low toxicity and is useful as an excellent agent for preventing and / or treating cancer. Further, it can be obtained by screening using a transformant transformed with a recombinant DNA in which a repo overnight gene is ligated downstream (under expression control) of the transcription regulatory region of the receptor protein gene of the present invention.
  • a compound, preferably a compound having an activity of inhibiting the expression of the receptor protein of the present invention or a salt thereof is also low toxicity and is useful as an excellent agent for preventing and / or treating cancer.

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Abstract

L'invention concerne des oligonucléotides possédant des séquences de base qui sont identiques ou sensiblement identiques à une séquence de base représentée par SEQ ID NO:13 ou 14, lesquels oligonucléotides sont d'excellents agents anticancéreux.
PCT/JP2003/004272 2002-04-05 2003-04-03 Produits de prevention et/ou remedes pour le cancer WO2003085106A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038503A2 (fr) * 1999-11-24 2001-05-31 Sugen, Inc. Nouvelles proteines kinases humaines et enzymes analogues
WO2001074851A2 (fr) * 2000-03-30 2001-10-11 Curagen Corporation Nouvelles proteines et acides nucleiques codant pour ces proteines
WO2002008253A2 (fr) * 2000-07-26 2002-01-31 Merck Patent Gmbh Nouveau membre de la famille du recepteur epha
WO2002008399A2 (fr) * 2000-07-21 2002-01-31 Incyte Genomics, Inc. Kinases humaines
US20020025570A1 (en) * 2000-06-09 2002-02-28 Pe Corporation (Ny) Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof
WO2002083735A1 (fr) * 2001-04-06 2002-10-24 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice et son adn

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038503A2 (fr) * 1999-11-24 2001-05-31 Sugen, Inc. Nouvelles proteines kinases humaines et enzymes analogues
WO2001074851A2 (fr) * 2000-03-30 2001-10-11 Curagen Corporation Nouvelles proteines et acides nucleiques codant pour ces proteines
US20020025570A1 (en) * 2000-06-09 2002-02-28 Pe Corporation (Ny) Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof
WO2002008399A2 (fr) * 2000-07-21 2002-01-31 Incyte Genomics, Inc. Kinases humaines
WO2002008253A2 (fr) * 2000-07-26 2002-01-31 Merck Patent Gmbh Nouveau membre de la famille du recepteur epha
WO2002083735A1 (fr) * 2001-04-06 2002-10-24 Takeda Chemical Industries, Ltd. Nouvelle proteine receptrice et son adn

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