WO2003102188A1 - Recepteur d'androgene mute, cellules cancereuses l'exprimant, procede de construction de ce recepteur et utilisations correspondantes - Google Patents
Recepteur d'androgene mute, cellules cancereuses l'exprimant, procede de construction de ce recepteur et utilisations correspondantes Download PDFInfo
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- WO2003102188A1 WO2003102188A1 PCT/JP2003/006942 JP0306942W WO03102188A1 WO 2003102188 A1 WO2003102188 A1 WO 2003102188A1 JP 0306942 W JP0306942 W JP 0306942W WO 03102188 A1 WO03102188 A1 WO 03102188A1
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Definitions
- the present invention relates to a mutant androgen receptor derived from a human prostate cancer cell, a human prostate cancer cell line expressing the same, a method for producing the same, and an anti-proliferative agent which uses the same to inhibit the growth of androgen-independent prostate cancer. It relates to the method of screening androgen agents. Background art
- Androgen is a generic term for steroid hormones that have androgenic action, and includes testosterone synthesized in the testes, dehydroepiandrosterone synthesized in the adrenal cortex, and androstenedione. Because about 95% of androgens are secreted from the testes, androgen-suppressing endocrine therapy often involves surgical (experimental) or drug (estrogen, LHRH agonist) castration to block testis-derived testosterone. I have.
- DHT dihydrotestosterone
- a cancer cell line that expresses the mutant AR is a very useful research tool in developing drugs that are useful for preventing and treating androgen-independent cancers based on AR mutations.
- all of the mutant AR-expressing cancer cells that have been reported so far have been obtained clinically from relapsed or untreated prostate cancer, and mutations have been induced in vitro in AR-expressing cancer cells.
- Most ARs clinically found in androgen-independent cancers have a single mutation at the amino acid level, and there are no reports of ARs that have mutations in two or more amino acids. Is very rare.
- an object of the present invention is to provide a clinically found androgen-independent cancer cause.
- a cancer cell line expressing an AR having the same mutation as that of the original mutant AR using an in vitro method and a mutant AR obtained by the method and a cancer cell line expressing the same.
- a method for screening effective drugs for prevention and treatment of androgen-independent relapse cancer against endocrine therapy is provided.
- the present inventors have made intensive studies to achieve the above object, and as a result, when human prostate cancer cells sensitive to a predetermined antiandrogen agent are cultured for a long period of time in the presence of the antiandrogen agent, anti-androgen Mutations that significantly reduce the efficacy of the drug or conversely show an agonist-like effect are induced in AR, resulting in the development of anti-androgen-resistant, i.e., androgen-independent cancer, which begins to grow I found that.
- the mutation site of AR was found to be from a relapsed cancer in a patient who had been treated with endocrine therapy with an antiandrogen. It was found to be in good agreement with that of clinically obtained mutant AR.
- the present inventors have further studied based on these findings, and as a result, have completed the present invention.
- Cancer cells susceptible to a predetermined anti-androgen agent are cultured in the presence of the anti-androgen agent to select a cancer cell line in which proliferation has been observed, and the androgen receptor in the cancer cell line is selected.
- a method for producing the anti-androgen-resistant cancer cell line expressing a mutant androgen receptor which comprises analyzing a nucleotide sequence of a gene and selecting a strain having a mutation in the sequence;
- a cancer cell line that expresses a mutant androgen receptor and is sensitive to a predetermined anti-androgen agent is cultured in the presence of the anti-androgen agent to select a cancer cell line in which proliferation is observed. Analyzing the nucleotide sequence of a mutant androgen receptor gene in a cancer cell line and selecting a strain having another mutation in the sequence. A method for producing the anti-androgen-resistant cancer cell line expressing the multiple mutant androgen receptor;
- Non-induced resistance cancer characterized in that cells of androgen-sensitive cancer are cultured in the presence of a test substance, and the presence or absence of a cancer cell line that can grow under the conditions is examined over time.
- a predetermined androgen receptor and a test substance are brought into contact with a mammalian cell containing a gene whose expression can be analyzed under the control of the PSA promoter, and A method for screening for the modulation of the androgen receptor, comprising analyzing gene expression;
- a polynucleotide comprising the protein of the above-mentioned [29] or the polynucleotide encoding the partial peptide of the above-mentioned [30];
- a diagnostic agent comprising the polynucleotide of the above-mentioned [31];
- administering an effective amount of two or more antiandrogen agents exhibiting antiandrogen activity to different mutant androgen receptors comprising: Prevention and treatment of hormone-sensitive cancer in the non-dependent stage;
- An antibody against a protein or a salt thereof which has the same or substantially the same amino acid sequence as the amino acid sequence in which threonine of amino acid number 882 is substituted with alanine in the amino acid sequence represented by SEQ ID NO: 2,
- tributophan of amino acid number 746 is An antibody that does not recognize a protein containing the same or substantially the same amino acid sequence as the amino acid sequence substituted with leucine or cysteine, or a salt thereof;
- the antibody of the above-mentioned [50] which is a neutralizing antibody that suppresses the transcription factor activity of the protein of the above-mentioned [2.9];
- a diagnostic agent comprising the antibody according to any of [50] to [52];
- a medicament comprising the compound of the above-mentioned [56] or a salt thereof; [58] The medicament of the above-mentioned [57], which is an agent for preventing and treating hormone-sensitive cancer in androgen-dependent and androgen-independent phases. ;
- a method for diagnosing the transition of a hormone-sensitive cancer into an androgen-independent phase comprising using the quantification method according to the above [59] or [60];
- FIG. 1 is a drawing showing the effect of bicalamide on the proliferation of LNCaP-cxDll, LNCaP-cxD2 and LNCaP-FGC cells.
- FIG. 2 is a drawing showing the effect of bicalutamide on PSA production of LNCaP-cxDll, LNCaP-cxD2 and LNCaP-FGC cells.
- LNCaP-cxDIK LNCaP-cxD2 and LNCaP-FGC cells suspended in a medium (RPMI1640 + 103 ⁇ 4 DCC-FBS) were seeded at 40,000 cells / plate in a 24-well plate, and the next day, bicalutamide was added.
- Fig. 3 is a drawing showing the activity of bicalutamide to promote the transcription of wild-type or mutant (W741C, W741 AR.
- Vector DNA in which various ARs are inserted and vector DNA in which a luciferase gene is linked downstream of an androgen-responsive promoter. After transfusion, 0.01 was measured for luciferase activity after addition of IM bicalutamide. Mean ⁇ standard error, n 4.
- FIG. 4 is a drawing showing the antagonistic effect of compound A on W741C type mutant AR.
- FIG. 5 is a drawing showing the activation of gene transcription under the control of the PSA promoter by wild-type AR activated by DHT.
- Cotransfection of COS-7 cells with the vector DNA with the wild-type AR inserted and the vector DNA with the luciferase gene linked downstream of one PSA promoter or two PSA promoters connected in tandem. Luciferase activity after addition of IM DHT was measured. Mean ⁇ standard error, n 6.
- the mutant AR of the present invention is obtained by replacing the tryptophan of amino acid number 746 with leucine in the amino acid sequence represented by SEQ ID NO: 2 (Proc. Natl. Acad. ScI. USA, 85: 7211-7215, 1988).
- the tributophan of amino acid number 746 is replaced with leucine or cysteine, and It is a protein containing the same or substantially the same amino acid sequence as the amino acid sequence in which threonine is replaced by alanine (sometimes abbreviated as “W746L (C) + T882A”).
- the mutant AR of the present invention can be used, for example, in any cell (eg, spleen cell, nerve cell, glial cell, england) of mammals (eg, human, guinea pig, rat, mouse, mouse, egret, pig, sheep, horse, monkey, etc.).
- mammals eg, human, guinea pig, rat, mouse, mouse, egret, pig, sheep, horse, monkey, etc.
- amino acid sequence substantially the same as W746L or W746L (C) + T882A examples include, for example, about 50% or more, preferably about 70% or more, more preferably about 80% or more, and each amino acid sequence.
- amino acid sequence having an identity of about 90% or more, most preferably about 95% or more may be mentioned.
- Examples of the protein having an amino acid sequence substantially the same as W746L or W746L (C) + T882A include, for example, an amino acid sequence substantially the same as W746L or W746UC T882A, and substantially the same as each amino acid sequence.
- a protein having a high activity is preferred.
- substantially equivalent activities include, for example, ligand binding activity (including endogenous ligand androgen, as well as other steroid hormones, agonists such as anti-androgens, and angiogonists), cell proliferation activity, And the effect of increasing the expression of genes under the control of an androgen-responsive promoter such as PSA (prostate-specific antigen). Substantially the same indicates that their activities are the same in nature. Therefore, activities such as ligand binding activity, cell growth activity, and gene expression increasing activity under the control of a promoter responsive to androgen are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 2 times).
- the quantitative factors such as the degree of activity and the molecular weight of the protein may be different. These activities can be measured according to a method known per se, for example, according to a screening method described later.
- the activity substantially equivalent to that of W746L is bicalutamide or Indicates that the analog exhibits agonist activity, that is, cell proliferation activity in the presence of bicalutamide and PSA expression induction activity.
- the activity substantially the same as that of W746L (C) + T882A is that in addition to the bicalucamide response similar to W746L, flutamide or an analog thereof exhibits agonist activity, that is, in the presence of fluramide. Cell proliferating activity and PSA expression inducing activity.
- the mutant AR of the present invention includes: (1) one or two or more of W746L or W746L (C) + T882A (preferably about 1 to 30, more preferably about 1 to 10, and more preferably Is an amino acid sequence in which several (1 to 5) amino acids have been deleted.
- the protein has an N-terminus (amino terminus) at the left end and a C-terminus (aminopropyl terminus) at the right end according to the convention of peptide labeling.
- the mutant AR of the present invention including W746L or W746L (CT88A), usually has a carboxyl group (—COOH), a carboxylate (—C ⁇ —), an amide (—CONH 2 ) It can be any of tell (—COOR).
- R in the ester e.g., methyl, Echiru, n_ propyl, alkyl groups such as isopropyl, n- butyl, Shikurobe pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl , 2 Ariru groups such as ⁇ one naphthyl, for example, benzyl, phenethyl, such as full Eniru C I 2 alkyl or flight one naphthylmethyl etc. ⁇ - naphthyl - C ⁇ etc. 2 alkyl group (: 7 _ 14 Ararukiru group
- a Viva yloxymethyl group commonly used as an ester for oral use is used.
- the amino group is amidated or esterified are also included in the mutant AR of the present invention.
- the ester for example, the above-mentioned C-terminal ester and the like are used.
- the mutant AR of the present invention is the protein mentioned above, Amino group protecting groups Mechio two emissions residues of N-terminal (e.g., formyl group, Ashiru group such as C 2 _ 6 Al force Noiru group such Asechiru ), N-terminally cleaved in vivo and the resulting dalminyl group undergoes pyroglutamine oxidation, substituents on the side chains of amino acids in the molecule (eg, _OH, _SH) , it is protected with an amino group, imidazo Ichiru group, i Ndoru group, etc. Guanijino group) appropriate protecting groups (e.g., formyl group, etc.
- the mutant AR of the present invention includes those derived from humans including W746L or W746L (C) + T882A. More preferred
- the partial peptide of the mutant AR of the present invention includes, for example, a mutant AR molecule of the present invention.
- a normal AR amino acid sequence represented by SEQ ID NO: 2 and its polymorphism
- having a partial amino acid sequence corresponding to a region required for binding to an androgen is used.
- AR is arranged in the order of N-terminal transcription activation domain, DNA binding domain, hinge region, and ligand binding domain in the same manner as other nuclear receptors, and the ligand binding domain is represented by SEQ ID NO: 2.
- the amino acid sequence represented is the amino acid sequence represented by amino acid numbers 672-292.
- the number of amino acids in the partial peptide of the present invention is not particularly limited as long as it has a partial amino acid sequence corresponding to a region required for binding to normal AR androgen, but is at least 20 or more, preferably 50 or more. And more preferably a peptide having 100 or more amino acid sequences.
- amino acid sequence substantially identical to the partial peptide of the present invention refers to an amino acid sequence having about 50% or more, preferably about 70% or more, more preferably about 80% or more, and more preferably about 80% or more. 90% or more, most preferably about 95% or more homology 2 shows the amino acid sequence.
- substantially the same activity has the same meaning as described above. “Substantially the same activity” can be measured in the same manner as described above.
- the partial peptide of the present invention has one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence deleted. Or one or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are added to the amino acid sequence. Or 1 or 2 or more (preferably about 1 to 10, more preferably several, and more preferably about 1 to 5) amino acids in the amino acid sequence are replaced with other amino acids. (However, the 746th (and 882nd) amino acids are conserved).
- the partial peptide of the present invention may have a carboxyl group (one CO OH), a carboxylate (_C—0—), an amide (—C ⁇ NH 2 ) or an ester (—COOR) at the C-terminus.
- R in the ester is as defined above.
- 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 an N-terminal methionine residue in which the amino group of the methionine residue is protected by a protecting group, and is formed by cleavage of the N-terminal side in vivo.
- a protecting group examples include those in which Gin is pyroglutamine-oxidized, those in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, and those in which a sugar chain is bonded, such as a so-called glycopeptide.
- Examples of the salt of the mutant AR of the present invention or a partial peptide thereof include a physiologically acceptable salt with an acid or a base, 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) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,
- a salt with methanesulfonic acid or benzenesulfonic acid is used.
- the mutant AR or a salt thereof of the present invention can be produced by isolating and purifying the mutant AR from animal cells having the ability to produce the mutant AR, using a method for purifying a receptor protein known per se. For example, after homogenizing human or mammalian tissues or cells, extraction with an acid or the like is performed, and the extract is purified and isolated by a combination of chromatography such as reverse phase chromatography and ion exchange chromatography. Can be.
- the animal cells having the ability to produce the mutant AR of the present invention include the above-mentioned human or mammalian cells or tissues (particularly, anti-androgens (eg, bicalutamide or bicalutamide and flutamide) prior to administration.
- an anti-androgen eg, bicalutamide or its analog
- a cancer cell line expressing a normal AR or AR with T882A mutation eg, LNCaP cell line
- a cell line derived from an adenocarcinoma relapsed cancer examples include an anti-androgen drug-resistant cancer cell line that has been cultured and selected using cell proliferation as an index.
- mutant AR of the present invention or a salt thereof can also be produced by culturing a transformant transformed with a recombinant vector containing DNA encoding the mutant AR of the present invention described below.
- W746L or W746L (based on the amino acid sequence of CT882A) can be produced by the protein synthesis method described below or a method analogous thereto.
- a commercially available resin for protein synthesis can be used.
- a resin for protein synthesis examples include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, Hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyihydroxymethyl) phenoxy resin, 4_ (2', 4'-dimethoxyphenyl-Fmocamino Ethyl) phenoxy resin and the like.
- an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods.
- the protein is cleaved from the resin and at the same time various protecting groups are removed.
- an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain a target protein or an amide thereof.
- various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
- the carbopimides include DC ⁇ , ⁇ ′-diisopropyl carbopimide, ⁇ -ethyl- ⁇ ′-(3-dimethylaminoprolyl) carbopimide, and the like.
- the protected amino acid is directly added to the resin together with a racemization inhibitor (for example, HOBt, HOOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride or HOBt ester or HOOBt ester. After performing, it can be added to the resin.
- 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-dimethylacetoamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, chloroform, and trifluoroethanol.
- Alcohols, sulfoxides such as dimethyl sulfoxide, 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: 50.
- 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, tertiary pentyl oxycarbonyl, isopolnyloxycarbonyl, 4-methoxybenzyloxycarponyl, CutZ, Br-Z, and adamantyl.
- Oxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylpho Suhuinochi oil and Fmoc are used.
- the carboxyl group may be, for example, alkyl-esterified (eg, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.).
- alkyl-esterified eg, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.
- aralkyl esterification eg, benzyl ester, 412 trobenzylyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhydryl esterification
- phenacyl esterification It can be protected by benzyloxycarbonyl hydrazide, evening butyroxycarbonyl hydrazide, trityl hydrazide and the like.
- the hydroxyl group of serine can be protected, for example, by esterification or etherification.
- a group suitable for this 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 ethoxycarponyl group, and the like are used.
- Examples of a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t-butyl group.
- the protecting group of the phenolic hydroxyl group of tyrosine for example, Bz and Cl 2 -Bz l, 2-nitrobenzyl, Br-Z, t-butyl are used.
- a protecting group for imidazole of histidine for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, labile, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
- activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, phenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, H0NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt).
- active esters eg, phenol, 2,4,5-trichlorophenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, H0NB, N-hydroxysuccinimide, N-hydroxyphthalimide, and an ester with HOBt.
- a corresponding phosphoric amide is used as the activated amino group of the raw material.
- 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, methanesulfonic acid, or the like. , Trifluoromethanesulfonic acid, trifluoroacetic acid or these Acid treatment with a mixed solution of the above, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used.
- the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about ⁇ 20 ° C. to 40 ° C.
- anisol for example, anisol, phenol, thioanisole, methacresol, paracresol, dimethylsulfur
- a cation scavenger such as amide, 1,4-butanedithiol, 1,2-ethanedithiol and the like.
- the 2,4-dinitro phenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributophane is 1,2-ethanedithiol, as described above.
- an amide form of a protein for example, first, after amidating and protecting the ⁇ -carboxyl group of the carboxy-terminal amino acid, a peptide (protein) chain is extended to a desired chain length on the amino group side. After that, a protein in which only the protecting group for the amino group at the ⁇ -terminal of the peptide chain was removed and a protein in which only the protecting group for the carboxyl group at the C-terminus was removed were prepared. Condensate in 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.
- an ester of a protein for example, after condensing the ⁇ -carboxyl group of the amino acid at the carboxy terminal 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.
- mutant AR of the present invention or the partial peptide of the present invention (hereinafter, the “partial peptide of the present invention or its amide or its ester or its salt” is simply referred to as the “partial peptide of the present invention” and the “mutant AR of the present invention. Or its salt) is simply referred to as “mutant AR of the present invention. ) May be produced according to a peptide synthesis method known per se, or by cleaving the mutant AR of the present invention or a protein containing the mutant AR of the present invention with an appropriate peptidase. it can. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
- the mutant AR of the present invention or the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. .
- the mutant AR of the present invention or the partial peptide of the present invention obtained by the above method is in a free form, it can be converted to an appropriate salt by a known method. It can be converted to a free form by a known method.
- the polynucleotide encoding the mutant AR of the present invention may be any polynucleotide as long as it contains the aforementioned base sequence (DNA or RNA, preferably DNA) encoding the mutant AR of the present invention.
- the polynucleotide is RNA such as DNA or mRNA encoding the mutant AR 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, whether it is a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand) Good.
- the method of the present invention can be carried out, for example, by the method described in the well-known extraordinary experimental medicine “New PCR and its Applications” 15 (7), 1997, or a method analogous thereto
- the mRNA of the mutant AR can be quantified.
- the DNA encoding the mutant AR of the present invention may be 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.
- the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
- amplification can be performed directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of total RNA or mRNA fraction from the cells and tissues described above.
- the DNA encoding the mutant AR of the present invention includes, for example, a nucleotide sequence (G2237T) in which the nucleotide of nucleotide number 2237 in the nucleotide sequence represented by SEQ ID NO: 1 has been substituted with thymine, or SEQ ID NO: DNA containing the base sequence (G2237 (2238) T + A2644G) in which the base number 2237 or 2238 in the base sequence represented by 1 is substituted with thymine and the base number 2644 is substituted with guanine, or It has a base sequence that hybridizes with the base sequence under high stringent conditions, and has substantially the same activity as the mutant AR of the present invention (eg, ligand binding activity, cell growth activity, PSA (prostate specific antigen) ) Any DNA can be used as long as it encodes a variant AR having an increasing effect. -
- Examples of the DNA capable of hybridizing with G2237T or G2237 (2238) T + A2644G include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more of each base sequence.
- DNA having a base sequence having at least% identity is used.
- Hybridization can be performed by a method known per se or a method analogous thereto, for example, described in Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be performed according to the method described above. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, Can be carried out in accordance with an intelligent condition.
- the high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 20 mM, and a temperature of about 50 to 70, preferably about 60 to 6 mM.
- the condition at 5 is shown.
- the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.
- a part of the nucleotide sequence of the DNA encoding the mutant AR of the present invention or a nucleotide containing a part of the nucleotide sequence complementary to the DNA is a DNA encoding the following partial peptide of the present invention. Is used to mean not only RNA but also RNA.
- the antisense nucleotide (nucleic acid) capable of inhibiting the replication or expression of the mutant AR gene of the present invention has been cloned or determined. Design and synthesize based on information.
- Such nucleotides (nucleic acids) can specifically hybridize with the mutant AR gene RNA of the present invention if temperature conditions and the like are precisely controlled, and can inhibit the synthesis or function of the RNA.
- the expression of the mutant AR gene of the present invention can be regulated and controlled through the interaction with the mutant AR-related RNA of the present invention.
- Polynucleotides that are complementary to the selected sequence of the AR-related RNA of the present invention, and nucleotides that can specifically hybridize with the mutant AR-related RNA of the present invention, are those of the present invention in vivo and in vitro. It is useful for regulating and controlling the expression of the AR gene, and is also useful in treating diseases, especially hormone-sensitive cancers (eg, prostate cancer) in the androgen-dependent and androgen-independent phases or transitioning to androgen-independent phases ( Useful for diagnosis of relapse).
- hormone-sensitive cancers eg, prostate cancer
- corresponding means having homology or being complementary to a nucleotide, base sequence or a specific sequence of a nucleic acid including a gene.
- “Corresponding” between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to an amino acid of a peptide (protein) specified by a sequence derived from a nucleotide (nucleic acid) sequence or its complement. I have. 5'-end hairpin loop, 5'-end 6—base pair of repeat AR gene of the present invention.
- terminal palindrome region and the 3 ′ terminal hairpin loop can be selected as preferred target regions, but any region within the mutant AR gene can be selected as the target.
- Antisense nucleotides include 2-oxy-D-ribose-containing deoxynucleotides, D-lipose-containing nucleotides, N-glycosides of purine or pyrimidine bases, and other types of poly-nucleotides.
- Nucleotide or other polymer having a non-nucleotide backbone eg, commercially available protein nucleic acid and synthetic sequence-specific nucleic acid polymer
- other polymer containing a special bond provided that the polymer is contained in DNA or RNA
- RNA including nucleotides having a configuration that allows base pairing and base attachment as found
- They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified oligonucleotides).
- nucleotide such as one having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, , Phosphorothioate, phosphorodithioate, etc., such as proteins (nucleases, nuclease inhibitors, toxins, Those having side-chain groups such as antibodies, signal peptides, poly-L-lysine, etc., and sugars (eg, monosaccharides), and those having an interactive compound (eg, acridine, psoralen, etc.) Containing chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.
- an intramolecular nucleotide such as one having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.),
- nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. These modifications include methylated purines and pirimi It may contain gin, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., one or more hydroxyl groups are replaced with halogens, aliphatic groups, etc., or converted to functional groups such as ethers, amines, etc. It may be done.
- the antisense nucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
- modified nucleic acid include, but are not limited to, a sulfur derivative of a nucleic acid, a thiophosphate derivative, and a polynucleoside amide that is resistant to degradation of an oligonucleoside amide.
- the antisense nucleic acid of the present invention can be preferably designed according to the following policy.
- the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form.
- additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase the uptake of nucleic acids ( For example, phospholipids, cholesterol, etc.) can be used.
- Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
- Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond.
- Other groups include those at the 3 'or 5' end of the nucleic acid.
- Differently placed capping groups that prevent degradation by nucleases such as exonucleases and RNases. Examples of such a capping group 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 can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the mutant AR of the present invention. .
- 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.
- 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. Alternatively, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using an mRNA fraction prepared from the cells and tissues described above.
- RT-PCR method reverse transcriptase polymerase chain reaction
- the DNA encoding the partial peptide of the present invention includes, for example, (1) DNA having a partial nucleotide sequence of DNA having G2237T or G2237 (2238) T + A2644G, or (2) the respective nucleotide sequence Has a nucleotide sequence that hybridizes under high stringent conditions, and has substantially the same activity as the partial peptide of the present invention (eg, increased ligand binding activity, cell growth activity, PSA (prostate cancer specific antigen)) DNA having a partial base sequence of the DNA encoding the mutant AR having the above-mentioned action is used.
- DNA having a partial nucleotide sequence of DNA having G2237T or G2237 (2238) T + A2644G or
- the respective nucleotide sequence Has a nucleotide sequence that hybridizes under high stringent conditions, and has substantially the same activity as the partial peptide of the present invention eg, increased ligand binding activity, cell growth activity, PSA (prostate cancer specific antigen
- Examples of the DNA capable of hybridizing with G2237T or G2237 (2238) T + A2644G include, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about A DNA having a nucleotide sequence having a homology of 95% or more is used.
- Mutation of the present invention AR or its partial peptide (hereinafter referred to simply as the mutation of the present invention (Abbreviated as AR in some cases.) Can be used as a means for cloning DNA that completely encodes the DNA by PCR using a synthetic DNA primer having a partial base sequence of the mutant AR of the present invention, or The DNA incorporated in an appropriate vector can be selected by hybridization with a DNA fragment encoding a part or the entire region of the mutant AR of the present invention or labeled with a synthetic DNA. Hybridization can be performed according to, for example, the method described in Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). In addition, when using a commercially available library, it can be performed according to the method described in the attached instruction manual.
- Conversion of DNA base sequence can be performed using known kits such as Mutan TM -Super Express Km (Takara Shuzo Co., Ltd.), Mutan TM- K (Takara Shuzo Co., Ltd.), and the like.
- the method can be carried out according to a method known per se such as the du 1 ex method or the Kunke 1 method, or a method analogous thereto.
- the DNA encoding the cloned mutant AR can be used as it is or, if desired, 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 be added using an appropriate synthetic DNA adapter.
- the expression vector for the mutant AR of the present invention may be prepared, for example, by (i) cutting out a DNA fragment of interest from DNA encoding the mutant AR of the present invention, and (mouth) converting the DNA fragment into a promoter in an appropriate expression vector. It can be manufactured by connecting to the downstream of.
- Escherichia coli-derived plasmids eg, pBR322, pBR325, pUC12, pUC13
- Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC194
- yeast-derived plasmids eg, , PSH19, pSH15
- bacteriophages such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as pAl-11, ⁇ 1, ⁇ Rc / CMV, pRcZRSV, pcDNAI / Neo and the like are 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.
- SR promoter overnight, SV40 promoter, LTR motor, CMV promoter, HSV-TK promoter, etc.
- CMV promoter, SRa It is preferable to use a promoter or the like.
- the host is a Eshierihia bacteria of the genus, trp promoter, lac-flops Romo - evening -, re cA promoter, AP L promoter, lpp promoter, etc.
- the host is a bacterium of the genus Bacillus, SPO l promoter, SP_rei_2 flop port motor - and p en p promoter, if the host is a yeast, PH_rei_5 flop port motor, PGK promoter, GAP promoter, etc.
- ADH promoter one is preferable.
- polyhedrin promoter, P10 promoter and the like are preferable.
- the expression vector may contain, in addition to the above, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), if desired. Anything can be used.
- the selection marker include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], ampicillin phosphorus resistant gene (hereinafter sometimes abbreviated as Am p r), neomycin resistance gene (hereinafter sometimes referred to as Ne o r, G418 resistance), and the like.
- the target gene when used as a selection marker using CHO (dh fr-) cells, the target gene can 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 AR of the present invention.
- the host is a bacterium belonging to the genus Escherichia, a PhoA signal sequence, a 0-A signal sequence, etc.
- the host is a Bacillus genus, a human amylase-signal sequence, a subtilisin signal sequence, etc.
- yeast MF-signal sequence, SUC2 signal sequence, etc.
- insulin signal sequence ⁇ -interferon signal sequence, Antibody molecules and signal sequences can be used.
- Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
- Escherichia examples include Escherichia coli K12 ⁇ DH1 [Processings of the National Academy of Sciences of the United States] (Proc. Natl. Acad. Sci. US A), 60, 160 (1968)], JM103 [Nucleic Acids * Research, (Nucleic Acids Research), 9, 309 (1981)], JA221 [Journal of Ob. ⁇ Molecular ⁇ Biology (Journal of Molecular Biology)], Volume 120, 517 (1978)], HB 101 [Journal 'ob' Molecular Biology ⁇ Biology, Volume 41, 459 (1969)], C 600 [ Genetics, 39, 440 (1954)].
- Bacillus bacteria examples include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95 vol. , 87 (1 984)].
- yeast examples include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC 1913, NCYC2036, Pichia Pastoris (Pichia pastoris) is used.
- Insect cells include, for example, when the virus is Ac NPV, a cell line derived from the larvae of the night larva (Spodopte-ra frugiperda cell; S f cell), MG1 cells derived from the midgut of Trichoplusia ni, and eggs of Trichoplusia ni High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
- Sf cells include Sf9 cells (ATCC CRL1711) And Sf21 cells (above, Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
- insects for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
- animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient chick cell hamster cell CHO (hereinafter CHO (dh fr)) Cells), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells.
- CHO cell Chinese hamster cell CHO
- dh fr gene-deficient chick cell hamster cell CHO
- mouse L cells mouse AtT-20
- mouse myeloma cells rat GH3, and human FL cells.
- transformation is performed with an expression vector containing the DNA encoding the AR.
- a transformed transformant is obtained.
- a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
- the carbon source include glucose, dextrin, soluble starch, and sucrose.
- the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like.
- the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
- yeast extract, vitamins, growth promoting factors and the like may be added.
- the pH of the medium is preferably about 5-8.
- a medium for cultivating a bacterium belonging to the genus Escherichia for example, an M9 medium containing glucose and casamino acid [Miller, Journal of 'perimentin in more than 10 liters, Journal of Science Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
- a drug such as 3i3-indolylacrylic acid can be added to make the promoter work efficiently if necessary.
- culturing is usually performed at about 15 to 43: for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
- the cultivation is usually performed at about 30 to 40 for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
- ⁇ of the medium is adjusted to about 5 to 8.
- Culture is usually performed at about 20 to 35 and for about 24 to 72 hours, and aeration and agitation are added as necessary.
- the culture medium is 10% serum which is immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)). And the like, to which the additives described above are appropriately added.
- the ⁇ of the culture medium is adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 for about 3 to 5 days, and aeration and agitation are added as necessary.
- examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], a DMEM medium [Virology, 8, 396 (1959)), RPM I 1640 medium [Journal of the American Medical Association at Association (The Journal of the American Medical Association) 199, 519 (1967) And 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)].
- the pH is about 6-8. Culture is usually performed at about 30 to 40 to about 15 to 60 hours, and aeration and agitation are added as necessary.
- the mutant AR of the present invention can be produced inside or outside the cells of the transformant.
- the mutant AR of the present invention can be separated and purified from the above culture by, for example, the following method.
- the cells or cells are collected by a known method, suspended in an appropriate buffer, and then subjected to ultrasonication, lysozyme and z or freeze-thawing. After the cells or cells are disrupted by, for example, the method of obtaining a crude extract of the mutant AR of the present invention 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 AR of the present invention contained in the culture supernatant or extract obtained in this manner can be performed by appropriately combining known separation and purification methods.
- known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
- Methods that use differences in molecular weight, methods that use differences in charge, such as ion-exchange chromatography, methods that use specific affinity, such as affinity chromatography, and methods that use hydrophobicity, such as reversed-phase high-performance liquid chromatography A method using a difference, a method using an isoelectric point difference such as an isoelectric focusing method, and the like are used.
- mutant AR of the present invention When the mutant AR of the present invention thus obtained is obtained in a free form, it can be converted to a salt by a method known per se or a method analogous thereto, and conversely, when it is obtained in a salt, it is known per se It can be converted to a free form or another salt by a method or a method analogous thereto.
- the mutant AR of the present invention produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
- the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
- the activity of the thus-produced mutant AR of the present invention or a salt thereof can be measured by a binding experiment with a labeled ligand and an enzyme immunoassay using the antibody of the present invention (described in detail below). .
- the antibody against the mutant AR of the present invention may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the AR of the present invention.
- the antibody against the mutant AR of the present invention can be produced by using the mutant AR of the present invention as an antigen according to a known method for producing an antibody or antiserum.
- the antibody of the present invention is characterized in that it specifically recognizes the mutant AR of the present invention but does not recognize normal AR. Therefore, as the antigen to be used, a peptide containing amino acid number 746 of SEQ ID NO: 2 and a partial amino acid sequence in the vicinity thereof and / or a peptide containing amino acid number 882 and a partial amino acid amino acid sequence in the vicinity thereof is preferable.
- Another antibody of the present invention is an amino acid having the amino acid sequence represented by SEQ ID NO: 2 in which tributophan of amino acid No. 7466 is substituted with leucine or cysteine. It specifically recognizes a protein having the same or substantially the same amino acid sequence as the acid sequence (W746L (C)) or a salt thereof.
- Yet another antibody of the present invention specifically recognizes a protein having the same or substantially the same amino acid sequence as T882A or a salt thereof, but has the same or substantially the same as W746L (C).
- the mutant AR 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 or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability 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 the mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
- a monoclonal antibody-producing hybridoma can be prepared.
- the antibody titer in the antiserum can be measured, for example, by reacting the labeled mutant AR described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody.
- the fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, Vol. 256, p. 495 (1975)].
- the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
- PEG polyethylene glycol
- myeloma cells include NS-1, P3U1, SP20 and the like, and P3U1 is preferably used.
- the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG1000 to PEG6000) is about 10 to 80%. Incubation at about 20 to 40, preferably about 30 to 37 "C for about 1 to 10 minutes allows efficient cell fusion.
- a high-pridoma culture supernatant is applied to a solid phase (eg, a microplate) on which the antigen of the mutant AR of the present invention is adsorbed directly or together with a carrier. Then, add an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the cells used for cell fusion are mice, use an anti-mouse immunoglobulin antibody) or protein A, and bind to the solid phase.
- a radioactive substance or enzyme if the cells used for cell fusion are mice, use an anti-mouse immunoglobulin antibody
- protein A protein A
- a method for detecting a monoclonal antibody that has been bound adding a hybridoma culture supernatant to a solid phase to which anti-immunoglobulin antibody or protein A has been adsorbed, adding a mutant AR labeled with a radioactive substance, an enzyme, etc., and binding to the solid phase And a method for detecting the obtained monoclonal antibody.
- the selection of the monoclonal antibody can be carried out according to a method known per se 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 the hybridoma can grow.
- RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or for hybridoma culture
- a serum-free medium SFM-101, Nissui Pharmaceutical Co., Ltd.
- the culturing temperature is usually 20 to 40, preferably about 37.
- the culturing time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks.
- the culture can be usually performed under 5% carbon dioxide.
- the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
- Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies.Immunoglobulin separation and purification methods (e.g., salting out, alcohol precipitation, isoelectric focusing) Antibodies can be collected by the method, electrophoresis, adsorption / desorption using an ion exchanger (e.g., DEAE), ultracentrifugation, gel filtration, antigen-bound solid phase, or an active adsorbent such as protein A or protein G. Specific purification method for obtaining an antibody by dissociating a bond].
- an ion exchanger e.g., DEAE
- the polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, a complex of an immunizing antigen (the antigen of the mutant AR 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 the antibody-containing substance and separating and purifying the antibody.
- the type of carrier protein and the mixing ratio of the carrier and the hapten are determined by the antibody against the hapten immunized by cross-linking the carrier.
- any kind may be cross-linked at any ratio.
- serum albumin, thyroglobulin, keyhole's lindet hemocyanin, etc. may be used in a weight ratio of hapten.
- a method of coupling at a rate of about 20 to 20 and preferably about 1 to 5 is used.
- various condensing agents can be used for force coupling between the hapten and the carrier.
- an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.
- the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
- Complete Freund's adjuvant or 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 the blood, ascites, etc., preferably from the 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 polyclonal antibodies can be performed according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of monoclonal antibodies. You.
- the DNA encoding the mutant AR of the present invention or a salt thereof, a partial peptide thereof or an amide thereof or an ester thereof or a salt thereof comprises: (1) prevention of a disease which can be improved by overexpression of the mutant AR of the present invention and or A therapeutic agent, (2) a gene diagnostic agent, (3) a method of screening the compound of the present invention that changes the expression level of the mutant AR or a salt thereof, (4) a compound that changes the expression level of the mutant AR of the present invention.
- a method for screening compounds eg, agonists, antagonists, etc.
- a preventive and / or therapeutic agent for various diseases containing a compound (agonist, antagonist) that changes the binding property between the mutant AR and the ligand of the present invention
- quantification of the mutant AR of the present invention (8) the present invention It can be used for a drug containing the antibody of the present invention, (9) a drug containing an antisense DNA, (10) a DNA-transferred animal, and the like.
- the binding of the ligand to human-to-mammal specific receptors can be improved.
- Eg, agonist, antagonist, etc. can be screened, and the agonist or antagonist can be used as an agent for preventing or treating various diseases.
- the mutant AR of the present invention, the DNA encoding the mutant AR of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention) and the antibody against the mutant AR of the present invention (hereinafter referred to as the antibody of the present invention) ) Will be specifically described below.
- the mutant AR of the present invention is characterized in that bicalidamide or an analog thereof, or furufumid or an analog thereof acts as an agonist.
- bicalidamide or an analog thereof, or furufumid or an analog thereof acts as an agonist.
- growth is conversely suppressed when AR stimulation is excessive, so that (1) mutant AR of the present invention or (2) DNA of the present invention is locally delivered to cancer cells and excessively When present, it is possible to suppress the growth of anti-androgen drug-resistant cancer cells.
- the mutant AR of the present invention is used as the above-described prophylactic / therapeutic agent, it can be formulated according to a conventional method.
- the DNA of the present invention when used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention is used alone or inserted into an appropriate vector such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. Thereafter, it can be administered according to conventional means.
- the DNA of the present invention can be administered as it is or together with adjuvants for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.
- the mutant AR of the present invention or (2) the DNA of the present invention can be orally administered as tablets, capsules, elixirs, microcapsules and the like, or coated with water or other pharmaceuticals, if necessary. It can be used parenterally in the form of an injectable preparation such as a sterile solution with an acceptable solution, or a suspension.
- an injectable preparation such as a sterile solution with an acceptable solution, or a suspension.
- Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- binders such as gelatin, corn starch, tragacanth, gum arabic
- excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- sweeteners such as sucrose, lactose or saccharin
- flavoring agents such as peppermint, cocoa oil or cherry.
- the unit dosage form is a capsule
- the above type of material
- aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
- Agent for example, alcohol (eg, It may be used in combination with an alcohol, a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant (eg, Polysorbate 80 TM , HCO-50), and the like.
- the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
- prophylactic and therapeutic agents examples include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
- buffers for example, phosphate buffer and sodium acetate buffer
- soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
- stabilizers for example, human serum Albumin, polyethylene glycol, etc.
- preservatives eg, benzyl alcohol, phenol, etc.
- antioxidants antioxidants and the like.
- the prepared injection solution is usually filled in a suitable ampoule.
- the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg, humans, rats, puppies, higgs, bush, puppies, cats,
- the dosage of the mutant AR of the present invention varies depending on the administration target, target organ, symptoms, administration method, and the like.
- oral administration in general, for example, in a cancer patient (60 kg), one dose is used.
- parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (60 kg). It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg by intravenous injection.
- the dose can be administered in terms of 6 O kg.
- the dosage of the DNA of the present invention varies depending on the administration subject, the target organ, the condition, the administration method, and the like. However, in the case of oral administration, for example, in a cancer patient (as 6 O kg), one dose is generally used. About 0.1 mg / day: I 0 Omg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
- the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
- the dose in the case of injection, it is usually, for example, a cancer patient (as 6 O kg) About 0.01 to 30 mg per day, preferably about 0.1 to 20 mg per day, It is convenient to administer preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
- the DNA of the present invention can be used as a probe to produce a DNA encoding the mutant AR of the present invention in mammals (eg, humans, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Or mRNA (that is, normal AR gene or mRNA abnormality), thus obtaining resistance to cancer gene diagnostics, especially antiandrogens (eg, Bicalumide, Fluidumide, etc.) It is useful as a genetic diagnostic agent for the appearance and increase of cancer cells.
- mammals eg, humans, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.
- antiandrogens eg, Bicalumide, Fluidumide, etc.
- the above-described genetic diagnosis using the DNA of the present invention includes, for example, the known Northern hybridization and the PCR_SS CP method (Genomics, Vol. 5, pp. 874-879 (1989), Processings).
- ⁇ Ob ⁇ The ⁇ National Academy ⁇ Ob ⁇ Sciences ⁇ Ob ⁇ U.S.A. (Proceedings of the National Academy of Sciences of the United States of America), Vol. 86, pp. 2766-2770 (1989) ) Etc.
- the present invention provides, for example, (i) non-human mammals (eg, rats, egrets, higgs, bushus, dogs, cats, dogs, monkeys, etc.), from blood, from specific organs, and from organs. (Ii) A method for screening a compound that alters the expression level of the mutant AR of the present invention by measuring the mRNA amount of the mutant AR of the present invention contained in the isolated tissue or cell, or (ii) the transformant or the like. provide.
- non-human mammals eg, rats, egrets, higgs, bushus, dogs, cats, dogs, monkeys, etc.
- a method for screening a compound that alters the expression level of the mutant AR of the present invention by measuring the mRNA amount of the mutant AR of the present invention contained in the isolated tissue or cell, or (ii) the transformant or the like. provide.
- the measurement of the mRNA amount of the mutant AR of the present invention is specifically performed as follows. (i) For non-human mammals having cells expressing the normal AR or the mutant AR of the present invention (for example, mouse, rat, rabbit, sheep, sheep, pig, pig, cat, dog, monkey, etc.) After a certain period of time after applying a drug or physical stress, blood, or a specific organ, or a tissue or cell isolated from an organ is removed. obtain.
- the mRNA of the mutant AR of the present invention contained in the obtained cells can be quantified by, for example, extracting mRNA from cells or the like by an ordinary method and using a technique such as TaqMan PCR, for example.
- the analysis can also be performed by performing a Northern plot by the above method.
- a transformant expressing the mutant AR of the present invention or an animal cell capable of expressing the mutant AR of the present invention is prepared according to the above-described method, and the transformant or the AR of the present invention contained in the above animal cell is prepared.
- a predetermined time (30 minutes to 24 hours, preferably 30 minutes or 24 hours before administration of a drug or physical stress to a non-human mammal having cells expressing a normal AR or a mutant AR of the present invention) Is from 30 minutes to 12 hours ago, more preferably from 1 hour to 6 hours ago, or after a certain time (from 30 minutes to 3 days, preferably from 1 hour to 2 days, more preferably after 1 hour To 24 hours), or the test compound is administered simultaneously with the drug or physical stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 2 days) Time to 24 hours), by quantifying and analyzing the mRNA amount of the mutant AR of the present invention contained in the cells,
- test compound When culturing a transformant or an animal cell having the ability to express the mutant AR of the present invention according to a conventional method, the test compound is mixed in a medium, and after culturing for a certain period of time (1 to 7 days, preferably Is 1 day to 3 days, more preferably 2 days to 3 days after), the mRNA amount of the mutant AR of the present invention contained in the transformant or an animal cell having the ability to express the mutant AR of the present invention is determined. This can be done by analysis.
- the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the expression level of the mutant AR of the present invention.
- the expression level of the mutant AR of the present invention A compound that excessively enhances the cell stimulating activity (eg, cell growth stimulating activity, PSA expression inducing activity, etc.) via the mutant AR of the present invention by increasing the expression of the mutant AR of the present invention.
- the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation products. These compounds may be novel compounds or known compounds.
- the compound that excessively enhances the cell stimulating activity can suppress the growth of anti-androgen-resistant cancer, for example, when used in combination with an anti-androgen agent, and can inhibit hormone-sensitive cancer in the androgen-independent phase (for example, prostate). It is useful as a prophylactic and therapeutic agent for cancer.
- the compound that attenuates the cell stimulating activity reduces the physiological activity of the mutant AR of the present invention, it is useful as a prophylactic / therapeutic agent for hormone-sensitive cancer in androgen-independent stage (eg, prostate cancer).
- hormone-sensitive cancer in androgen-independent stage eg, prostate cancer
- a compound or a salt thereof obtained by using the screening method of the present invention when used as a pharmaceutical composition, it can be used in a conventional manner.
- tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the mutant AR of the present invention.
- the preparations obtained in this way are safe and have low toxicity, so they are administered to mammals (eg, humans, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.). be able to.
- the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
- oral administration for example, in a cancer patient (as 6 O kg)
- one dose is generally used.
- the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
- injection it is usually used, for example, in cancer patients (as 6 O kg).
- the dose can be administered in terms of 60 kg.
- a preventive and / or therapeutic agent for various diseases containing a compound that alters the expression level of mutant AR of the present invention
- Compounds that alter the expression level of the mutant AR of the present invention are hormone-dependent and hormone-dependent. It can be used as a preventive and therapeutic agent for mon-independent cancers (eg, prostate cancer).
- the compounds 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.
- Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- binders such as gelatin, corn starch, tragacanth, gum arabic
- excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- sweeteners such as sucrose, lactose or saccharin
- flavoring agents such as peppermint, cocoa oil or cherry.
- the unit dosage form is a capsule
- the above type of material
- aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D_mannitol, sodium chloride, etc.) and the like are used.
- auxiliary agents eg, D-sorbitol, D_mannitol, sodium chloride, etc.
- an alcohol e.g., E evening Nord
- polyalcohol e.g., propylene glycol, polyethylene glycol
- nonionic surfactant eg, polysorbate WINCH 8 0 TM, HCO - 5 0
- oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
- prophylactic / therapeutic agent examples include a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride). ), Stabilizers (eg, human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
- a buffer eg, phosphate buffer, sodium acetate buffer
- a soothing agent eg, benzalkonium chloride, procaine hydrochloride
- Stabilizers eg, human serum albumin, polyethylene glycol, etc.
- preservatives eg, benzyl alcohol, phenol, etc.
- antioxidants antioxidants and the like.
- the prepared injection solution is usually filled into a suitable ampoule.
- the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg, humans, rats, puppies, higgs, bush,
- 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 patient with cancer (as 60 kg)
- parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
- injection it is usually used, for example, in cancer patients (as 6 O kg).
- the dose can be administered in terms of 60 kg.
- a method for screening a compound eg, an agonist, an angelist, etc.
- the mutant AR of the present invention By using the mutant AR of the present invention or constructing an expression system of the recombinant mutant AR and using the receptor binding system using the expression system, the binding between the ligand and the mutant AR of the present invention can be achieved.
- the binding between the ligand and the mutant AR of the present invention can be achieved.
- peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. or salts thereof can be efficiently screened.
- Such compounds include (a) compounds having an activity of promoting cell stimulating activity (eg, cell proliferation activity, PSA expression inducing activity, etc.) via the mutant AR of the present invention (so-called mutations of the present invention). (Agonists against AR), (mouth) compounds having no cell-stimulating activity (so-called antagonists to mutant AR of the present invention), (8) compounds that enhance the binding force between ligand and mutant AR of the present invention, or (2) ) Includes compounds that reduce the binding strength between the ligand and the mutant AR of the present invention.
- the present invention provides: (i) a case where the mutant AR of the present invention is brought into contact with a ligand. And (ii) comparing the mutant AR of the present invention with a ligand and a test compound when contacting the ligand and a test compound.
- a method for screening a salt is provided.
- the binding amount of a ligand (androgen) to AR of the present invention cell stimulating activity (eg, cell proliferation activity, PSA expression induction activity)
- cell stimulating activity eg, cell proliferation activity, PSA expression induction activity
- the present invention provides
- the labeled ligand and a test compound are contacted with the mutant AR of the present invention.
- a method for screening a compound or a salt thereof, which alters the binding property between the compound and the mutant AR of the present invention is characterized by measuring the amount of a labeled ligand bound to a cell containing the same (eg, an animal cell capable of expressing a mutant AR), and comparing the amount of the labeled ligand to the cell.
- a cell containing the mutant AR of the present invention eg, an animal cell capable of expressing the mutant AR
- a compound that activates the mutant AR of the present invention for example, an androgen or an anti-androgen such as bicalidamide or flutamide.
- a compound that activates the mutant AR of the present invention and a test compound containing the mutant AR of the present invention are treated with a compound that activates the mutant AR of the present invention.
- a compound that activates the mutant AR of the present invention for example, an androgen or an anti-androgen such as bicalidamide or flutamide.
- a test compound containing the mutant AR of the present invention for example, cell proliferation activity, PSA expression inducing activity, etc.
- Cell stimulating activity eg, cell proliferation activity, PSA expression inducing activity, etc.
- Provided is a method for screening a compound or a salt thereof, which changes the binding property between a ligand and a variant AR of the present invention, which is characterized by comparison.
- the mutant AR of the present invention used in the screening method of the present invention may be any as long as it contains the above-described mutant AR of the present invention.
- Animal cells and the like having the ability to express are suitable.
- the method described above is used to produce the mutant AR of the present invention, but it is preferable to express the DNA of the present invention in mammalian cells or insect cells.
- a complementary DNA is used as the DNA fragment encoding the target protein portion, but is not necessarily limited to this.
- gene fragments or synthetic DNA may be used.
- the DNA fragment In order to introduce the DNA fragment encoding the AR of the present invention into host animal cells and express them efficiently, the DNA fragment must be expressed by nuclear polyhedrosis virus belonging to baculovirus using insects as a host.
- NP V polyhedrin promoter
- SV40-derived promoter retrovirus promoter
- metamouth thionein promoter human heat shock promoter
- cytomegalovirus promoter SR ⁇ promoter
- the quantity and quality of the expressed receptor can be examined by a method known per se. For example, the method can be carried out according to the method described in the literature [Nambi, P. et al., The Journal of Biological 'Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. .
- the mutant AR of the present invention may be a mutant AR purified according to a method known per se, or a cell containing the mutant AR may be used. .
- the cell when a cell containing the mutant AR of the present invention is used, the cell may be immobilized with daltaraldehyde, formalin, or the like.
- the immobilization method can be performed according to a method known per se.
- the cell containing the mutant AR of the present invention refers to a host cell that has expressed the mutant AR, and the host cell is preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
- the amount of mutant AR in a cell containing the mutant AR is preferably 10 3 to 10 8 molecules, and more preferably 10 5 to 10 7 molecules per cell.
- the mutant AR fraction an AR fraction derived from a mutant AR-expressing cell obtained by natural or artificial mutation or a recombinant mutant AR fraction having an activity equivalent thereto is desirable.
- the equivalent activity means equivalent ligand binding activity, cell stimulating activity, and the like.
- a labeled ligand As the labeled ligand, a labeled ligand (androgen), a labeled ligand analog compound and the like are used. For example, testosterone, DHT, etc. labeled with [ 3 H], [ 125 I], [ 14 C], [ 35 S] and the like are used.
- a buffer suitable for screening To prepare a mutant AR sample.
- the buffer may be any buffer such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer that does not inhibit the binding between the ligand and the mutant AR.
- a surfactant such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, and dexcholate can be added to the buffer.
- protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide suptin can be added for the purpose of suppressing receptor degradation and ligand degradation by proteases.
- reaction tube containing a large excess of unlabeled ligand to determine the amount of non-specific binding (NSB).
- the reaction is carried out at about 0 to 50 ⁇ , preferably at about 4 ° C. to 37, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
- the reaction solution is filtered through a glass fiber filter paper, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured using a liquid scintillation counter or an oven.
- the specific binding amount (B-NSB) is For example, a test compound having 50% or less can be selected as a candidate substance having a competitive inhibitory ability.
- cells containing the mutant AR of the present invention are cultured on a multiwell plate or the like.
- replace the cells with fresh medium or an appropriate buffer that is not toxic to the cells add the test compound, etc., incubate for a certain period of time, and then evaluate the proliferation of the cells. Extract or collect the supernatant, and quantitate the product produced according to each method.
- an inhibitor for the degrading enzyme may be added to perform the assay.
- cells expressing the appropriate mutant AR are required.
- the cells expressing the mutant AR of the present invention are preferably cells expressing the mutant AR obtained by natural or artificial mutation, and cell lines expressing the above-mentioned recombinant mutant AR.
- test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
- the cleaning kit includes a mutant AR of the present invention or a cell containing the mutant AR of the present invention (such as an animal cell capable of expressing the mutant AR).
- screening kit of the present invention examples include the following. 1. Screening reagent
- CHO cells expressing the mutant AR of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 wells, and cultured for 2 days in 37 :, 5% CO 2 , 95% air.
- the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an effect of changing the binding property between the ligand and the mutant AR of the present invention.
- Compounds having cell stimulating activity eg, cell proliferation activity, PSA production, etc.
- AR of the present invention e.g., cell proliferation activity, PSA production, etc.
- agonists against mutant AR of the present invention e.g., agonists against mutant AR of the present invention
- mouth Compounds not having the cell stimulating activity
- a compound that reduces force e.g, cell proliferation activity, PSA production, etc.
- Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, and fermentation products. These compounds may be novel compounds or known compounds.
- the antagonist of the present invention against the mutant AR of the present invention can suppress the physiological activities of ligand (androgen) and agonist (some antiandrogen agents such as bicalutamide and flutamide) against the mutant AR of the present invention, It is useful as a prophylactic and therapeutic agent for androgen-independent (antiandrogen-resistant) cancer, especially prostate cancer.
- Compounds that decrease the binding force between the ligand and the mutant AR of the present invention include safe and low-toxic drugs (eg, androgen-free drugs) for reducing the biological activity of the ligand (agonist) for the mutant AR of the present invention.
- Dependence is useful as a cancer, especially a prophylactic / preventive agent for prostate cancer.
- 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 pharmaceutical composition, it can be produced and used according to conventional means. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the mutant AR of the present invention.
- the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg, humans, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.) be able to.
- mammals eg, humans, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, 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 (as 60 kg); About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
- parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (60 kg). It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg, by intravenous injection. In the case of other animals, the dose can be administered in terms of 6 O kg.
- the antagonist of the present invention against the mutant AR of the present invention can suppress the physiological activities of ligand (androgen) and agonist (some antiandrogen agents such as bicalutamide and flutamide) against the mutant AR of the present invention, It can be used as an agent for preventing and treating androgen-independent (antiandrogen-resistant) cancer, especially prostate cancer.
- the compound can be formulated according to a conventional method. ''
- the compound can be sterilized with orally or as water or other pharmaceutically acceptable liquids, such as tablets, capsules, elixirs, microcapsules, and the like, optionally coated with sugar.
- known carriers or flavors that can physiologically recognize the compound It can be manufactured by mixing the compound with excipients, excipients, vehicles, preservatives, stabilizers, binders and the like in the unit dosage form generally required for the practice of the formulation.
- the amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
- Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- binders such as gelatin, corn starch, tragacanth, gum arabic
- excipients such as crystalline cellulose, corn starch, gelatin, alginic acid And swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
- sweeteners such as sucrose, lactose or saccharin
- flavoring agents such as peppermint, cocoa oil or cherry.
- the unit dosage form is a capsule
- the above type of material
- aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (for example, D_sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
- an alcohol e.g., E evening Nord
- polyalcohol e.g., propylene glycol, polyethylene glycol
- nonionic surfactant eg, polysorbate one preparative 8 0 TM, HCO - 5 0
- oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
- prophylactic and therapeutic agents examples include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
- buffers for example, phosphate buffer and sodium acetate buffer
- soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
- stabilizers for example, human serum Albumin, polyethylene glycol, etc.
- preservatives eg, benzyl alcohol, phenol, etc.
- antioxidants antioxidants and the like.
- the prepared injection solution is usually filled in a suitable ampoule.
- the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg, humans, rats, puppies, higgs, bush, puppies, cats,
- the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
- the daily dose is generally one day.
- the single dose varies depending on the administration target, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (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 1 Omg per day by intravenous injection.
- the dose can be administered in terms of 6 O kg. (7) Quantification of mutant AR of the present invention
- the antibody of the present invention can specifically recognize the AR of the present invention, it can be used for quantification of the mutant AR of the present invention in a test wave, particularly for quantification by sandwich immunoassay. That is, the present invention provides, for example, U) reacting the antibody of the present invention competitively with the detected and labeled mutant AR of the present invention, and binding the labeled mutant AR of the present invention to the antibody. Quantifying the mutant AR of the present invention in a test wave,
- the present invention provides a method for quantifying the mutant AR of the present invention in a test solution, which is characterized by
- the mutant AR of the present invention can be measured using a monoclonal antibody against the mutant AR of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and can also be detected by tissue staining or the like.
- the antibody molecule itself may be used, or the F (ab ') 2 , Fab ⁇ or Fab fraction of the antibody molecule may be used.
- the assay method using an antibody against the mutant AR 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 the antigen (eg, the amount of the mutant AR) in the test solution.
- Any measurement method may be used as long as the amount 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, competition method, immunometric method and sandwich method are preferably used. From the viewpoint of specificity, it is particularly preferable to use the sandwich method described below.
- 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, [1 2 5 I], [1 3 1 I], [3 H], and [1 4 C] used.
- the above-mentioned enzyme those which are stable and have high specific activity are preferable.
- 3-galactosidase, 3-dalcosidase, alkaline phosphatase, peroxidase, and lignoic acid dehydrogenase are used.
- the fluorescent substance for example, fluorescein, fluorescein isothiosinate and the like are used.
- the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
- a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
- 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.
- 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 wave 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, or may be performed simultaneously or at staggered times.
- the labeling agent and the method of insolubilization may be the same as 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 for the primary reaction and the secondary reaction is preferably an antibody having a different site to which the mutant AR of the present invention binds. Used.
- Measuring system other than the sandwich method using the monoclonal antibody of the present invention can be used for the competition method, the immunometric method, the nephrometry method, and the like.
- the competition method after the antigen in the test wave and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody ( B / F separation), measure the amount of B or F label, and quantify the amount of antigen in the test wave.
- a soluble antibody is used as the antibody
- BZF separation is performed using polyethylene glycol
- a liquid phase method using a second antibody against the above antibody or an immobilized antibody is used as the first antibody.
- An immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
- the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. 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 wave.
- 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 sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.
- the AR of the present invention or a salt thereof can be quantified with high sensitivity.
- the antibody of the present invention can be used for specifically detecting the mutant AR of the present invention present in a subject such as a body fluid or a tissue.
- preparation of an antibody column used for purifying the mutant AR of the present invention, detection of the mutant AR of the present invention in each fraction during purification, and analysis of the behavior of the mutant AR of the present invention in test cells It can be used for such purposes.
- the neutralizing activity of the antibody against the mutant AR of the present invention against the mutant AR of the present invention means an activity of inactivating a cell stimulating activity involving the mutant AR of the present invention. Therefore, when the antibody has neutralizing activity, it can inactivate the cell stimulating activity (eg, cell proliferation activity, PSA production, etc.) involving the mutant AR. Therefore, it can be used for the prevention and / or treatment of diseases caused by the cell proliferation stimulation of the mutant AR, for example, androgen-independent (anti-androgen drug-resistant) cancer, particularly prostate cancer.
- the therapeutic or prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used as it is as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form, in humans or mammals (eg, rat, porch egret, hidge, bush, Can be administered orally or parenterally to mice, cats, dogs, monkeys, etc.).
- the dose varies depending on the administration subject, target disease, symptoms, administration route, etc.For example, when used for treatment and prevention of adult cancer patients, the antibody of the present invention is used as a single dose.
- 0.0 1-2 OmgZk g body weight Preferably about 0.1 to 1 Omg Z kg body weight, more preferably about 0.1 to 5 mg Z kg body weight, about 1 to 5 times a day, preferably about 1 to 3 times a day, intravenous injection Conveniently for administration. In the case of other parenteral administration and oral administration, an equivalent dose can be administered. If the symptoms are particularly severe, the dose may be increased accordingly.
- the antibodies of the present invention can be administered by themselves or as a suitable pharmaceutical composition.
- the pharmaceutical composition used for the above administration contains the above 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.
- compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules and the like). ), Syrups, emulsions, suspensions and the like.
- Such a composition is produced by a method known per se and contains a carrier, diluent or excipient commonly used in the field of pharmaceuticals. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
- compositions for parenteral administration for example, injections, suppositories, etc. are used.
- Injections are in the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, etc. Is included.
- Such injections are prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injections.
- aqueous liquid for injection for example, physiological saline, isotonic solution containing glucose and other auxiliary agents and the like are used, and suitable solubilizing agents, for example, alcohol (eg, ethanol), polyalcohol (eg, , Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (.polyoxyethylene (5 O mol) adduct of hydrogenat ed castor oil)), etc. You may.
- alcohol eg, ethanol
- polyalcohol eg, Propylene glycol, polyethylene glycol
- nonionic surfactants eg, polysorbate 80, HCO-50 (.polyoxyethylene (5 O mol) adduct of hydrogenat ed castor oil)
- oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
- each dosage unit dosage form is 5 to 500 mg, especially for injections.
- the antibody contains 5 to 100 mg, and 10 to 25 Omg of the above antibody in other dosage forms.
- compositions may contain another active ingredient as long as the composition does not cause an undesirable interaction with the above-mentioned antibody.
- the antisense DNA capable of binding to the DNA of the present invention complementarily and suppressing the expression of the DNA has a cell stimulating activity (eg, cell proliferation activity, PSA production, etc.) involving the mutant AR. Can be blocked at the translation level. Therefore, it can be used for the prevention and Z or treatment of diseases caused by the cell proliferation stimulation of the mutant AR, for example, androgen-independent (antiandrogen drug-resistant) cancer, particularly prostate cancer.
- a cell stimulating activity eg, cell proliferation activity, PSA production, etc.
- diseases caused by the cell proliferation stimulation of the mutant AR for example, androgen-independent (antiandrogen drug-resistant) cancer, particularly prostate cancer.
- the antisense DNA when used, the antisense DNA is inserted alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, and the like. Therefore, it can be implemented.
- the antisense DNA can be administered as it is or in the form of a formulation together with a physiologically acceptable carrier such as an auxiliary agent for promoting uptake, and then administered using a gene gun or a catheter such as a hydrogel catheter.
- the antisense DNA 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 expression thereof.
- the present invention provides a non-human mammal having a DNA encoding an exogenous mutant AR of the present invention (hereinafter, abbreviated as the exogenous DNA of the present invention).
- the present invention (1) a non-human mammal having the exogenous DNA of the present invention
- the non-human mammal having the exogenous DNA of the present invention (hereinafter abbreviated as the DNA transfer product of the present invention) is preferably used for unfertilized eggs, fertilized eggs, germ cells including spermatozoa and their progenitor cells, and the like.
- the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, and the microinjection method are used during the embryonic development stage in non-human mammal development (more preferably, at the single cell or fertilized egg cell stages and generally before the 8-cell stage). It can be produced by transferring the target DNA by the injection method, particle gun method, DEAE-dextran method, etc.
- exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, and the like by the DNA transfer method, and can be used for cell culture, tissue culture, and the like.
- the DNA-transferred animal of the present invention can also be produced by fusing the cells with the above-mentioned germ cells by a cell fusion method known per se.
- mice for example, Japanese lions, bushes, higgies, goats, egrets, dogs, cats, guinea pigs, hamsters, mice, rats and the like are used.
- rodents and biological cycles are relatively short in terms of the creation of disease animal model systems, and rodents that are easy to breed, especially mice (for example, pure strains such as C57 BLZ6 strain, DBA2 strain, and hybrid strains) Preferred are BGC SFi system, BDFi system, B6D2Fi system, BALBZc system, ICR system, etc.) or rat (for example, Wistar, SD, etc.).
- mammal in the recombinant vector that can be expressed in mammals, human and the like can be mentioned in addition to the above-mentioned non-human mammals.
- the exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from a mammal, not the mutant AR gene originally possessed by a non-human mammal.
- the exogenous DNA of the present invention may be derived from a mammal of the same species or a different species as the target animal.
- a mammal of the same species or a different species as the target animal In transferring the DNA of the present invention to a subject animal, thus, it is generally advantageous to use the DNA as a DNA construct downstream of a promoter capable of being expressed in animal cells.
- the human DNA of the present invention when transferred, it is derived from various mammals having the DNA of the present invention, which are highly homologous thereto (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.).
- the present invention is achieved by microinjecting the DNA construct (eg, a vector, etc.) to which the human DNA of the present invention is bound downstream of various promoters capable of expressing the DNA of the present invention into a fertilized egg of a target mammal, for example, a mouse fertilized egg.
- a DNA transgenic mammal that highly expresses DNA can be created.
- the expression vector of the AR of the present invention includes a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, a plasmid derived from yeast, a bacteriophage such as ⁇ phage, a retrovirus such as Moroni leukemia virus, a vaccinia virus or a baculovirus. Animal viruses and the like 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.
- promoters that regulate the above DNA expression include: (1) Promoter of DNA derived from viruses (eg, Simian virus, cytomegalovirus, Moroni leukemia virus, JC virus, breast cancer virus, polio virus, etc.), 2 Promoters from various mammals (humans, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.), such as albumin, insulin II, perobrakin II, Eras Yuichi, erythropoietin, endothelin, muscle creatine Kinase, glial fibrillary acidic protein, daltathione S-transferase, platelet-derived growth factor / 3, keratin Kl, ⁇ 10 and ⁇ 14, collagen type I and type II, cyclic AMP-dependent protein kinase / 3 I subunit G, dystrophy , Tartrate-resistant alkaline phosphatase, atrial sodium diuretic factor, endothelial receptor
- the vector preferably has a sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal (generally referred to as terminator).
- terminator a sequence that terminates transcription of the target messenger RNA in a DNA-transferred mammal.
- the sequence of DNA can be used, and preferably, SV40 of the simian virus and Mineiichi are used.
- the splicing signal of each DNA, the enhancer region, a part of the intron of eukaryotic DNA, etc. are used to further express the target exogenous DNA at 5 'upstream of the promoter region, between the promoter region and the translation region, or translation. Connection to the 3 'downstream of the region is also possible depending on the purpose.
- the translated region of the mutant AR of the present invention may be a cell derived from androgen-independent cancer, particularly prostate cancer, of humans or various mammals (eg, egrets, dogs, cats, guinea pigs, hamsters, rats, mice, etc.)
- genomic DNA from DNA isolated from the anti-androgen drug-resistant cancer cell line obtained by the method of the present invention, or by a method known from RNA from the above cells (or cell lines).
- the prepared complementary DNA can be obtained as a raw material.
- the translation region of the normal AR can be isolated by a conventional method, and the mutated translation region can be prepared by the point mutagenesis method.
- the non-human mammal having the exogenous DNA of the present invention can be subcultured in a normal breeding environment as an animal having the DNA after confirming that the exogenous DNA is stably maintained by the cross. Furthermore, the desired foreign DNA can be incorporated into the above-mentioned plasmid and used as a raw material.
- DNA construct with promoter Can be prepared by ordinary DNA engineering techniques. 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 germ cells of the produced animal after DNA transfer means that all the offspring of the produced animal have the exogenous DNA of the present invention in all of the germ cells and somatic cells. .
- the offspring of this type of animal inherited from the exogenous DNA of the present invention have the exogenous DNA of the present invention in all of its germ cells and somatic cells.
- a homozygous animal having the introduced DNA on both homologous chromosomes is obtained, and by crossing the male and female animals, it is possible to breed so that all offspring have the DNA.
- the exogenous DNA of the present invention is highly expressed, and the function of endogenous normal DNA is impaired by inhibiting the function of endogenous normal DNA. It may be active refractory and can be used as a model animal for the disease. For example, using the exogenous DNA-transferred animal of the present invention to elucidate the pathological mechanism of androgen-independent (anti-androgen drug-resistant) cancer, particularly prostate cancer, and to study methods for preventing and treating this disease. Is possible.
- DNA-transferred animal of the present invention include, for example,
- the DNA-transferred animal of the present invention it is possible to examine clinical symptoms of a disease associated with AR, including a function inactive refractory disease of normal AR, etc. More detailed pathological findings in each organ of the disease model were obtained, It can contribute to the development of new treatment methods and the research and treatment of secondary diseases caused by the disease.
- 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. Furthermore, it is possible to investigate the specificity of the protein-producing cells of the present invention, apoptosis, their relationship with differentiation or proliferation, or the signal transduction mechanism in them, and to investigate their abnormalities. It is an effective research material for elucidation. '
- the DNA-transferred animal of the present invention in order to use the DNA-transferred animal of the present invention to develop a therapeutic agent for an AR-related disease of the present invention, including a function inactive refractory disease of a normal AR, the above-described test method and quantification method By using such a method, it is possible to provide an effective and rapid method for screening for a therapeutic agent for the disease.
- using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention it is possible to examine and develop a method for treating DNA associated with the AR of the present invention.
- the present invention also provides a method for culturing cancer cells sensitive to a predetermined antiandrogen agent in the presence of the antiandrogen agent, selecting a cancer cell line in which proliferation has been observed, and selecting the AR gene in the cancer cell line.
- a method for producing the anti-androgen drug-resistant cancer cell line expressing the mutant AR comprising analyzing the nucleotide sequence of the anti-androgen drug-resistant cancer cell line and selecting a strain in which the sequence has a mutation.
- the starting cancer cell is not particularly limited as long as it is sensitive to a predetermined antiandrogen, and is preferably a cancer cell expressing normal AR (for example, having the amino acid sequence represented by SEQ ID NO: 2), preferably Examples include prostate cancer cells or cancer cells that express a mutant AR that is resistant to other antiandrogens.
- “sensitive (resistant) to a predetermined antiandrogen agent” means that it cannot grow (can grow) in the presence of the antiandrogen agent.
- Cancer cells that express normal AR include biopsy samples obtained from androgen-dependent cancer patients and cancer cells derived from removed prostate.Cells that express mutant AR include other antiandrogen agents. Examples of cancer cells or LNCaP cell lines (expressing the T882A mutant AR) from patients with relapse of androgen-independent cancer after endocrine therapy It is.
- the culturing of cancer cells can be carried out using a method usually used for culturing animal cells or with appropriate modifications.
- 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 [The Journal of the American Medical Association (1992), 519 (1967)], 199 medium [Processing of the American Medical Association] Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)] is used. Since androgen or other steroids contained in serum may induce cell proliferation, it is preferable to use serum pretreated with dextran-coated charcoal.
- the pH of the medium is about 6-8.
- Culture is usually performed at about 30:40, and aeration and agitation are added as necessary.
- a cancer cell is added to any one of the above-mentioned media containing 0.0001 to 100 antiandrogen agents (for example, furumin mid, bicalum mid, and nitrum midid, etc.) in an amount of 1 to 1,000,000 cells / m 1.
- antiandrogen agents for example, furumin mid, bicalum mid, and nitrum midid, etc.
- culture in a culture vessel such as a multiwell plate under the above culture conditions. Observe the presence or absence of cell proliferation over time while changing the medium at appropriate intervals according to the usual method.
- RNA was isolated from clones in which cell proliferation was observed.
- the cDNA of the AR was amplified and isolated using the RT-PCR method with an oligonucleotide prepared based on a known normal ARc DNA sequence. By analyzing the base sequence after subcloning it into a direct sequence or an appropriate vector, a clone that actually has a mutation in AR is selected.
- the mutation site of the mutant AR obtained as described above is characteristic depending on the type of the antiandrogen used as the selection pressure.
- mutations are concentrated in the tributophan of amino acid No. 746 (preferably substituted by leucine or cysteine), and the amino acid represented by SEQ ID NO: 2 when using fluorinamide is used.
- Mutation concentrates on threonine at amino acid number 882 in the sequence (preferably replaced by alanine).
- a multiple mutant AR-expressing cancer cell line having a mutation at another site can be created. Therefore, the present invention selects cancer cell lines in which proliferation is observed by culturing cancer cells that express the mutant AR and are sensitive to a predetermined antiandrogen agent in the presence of the antiandrogen agent. Analyzing the nucleotide sequence of the mutated AR gene in the cancer cell line and selecting a strain having another mutation in the sequence, wherein the anti-androgen drug-resistant cancer cell line expressing the multiple mutated AR is characterized by: Provides a method for creating a mutant AR (eg, LNCaP cell or the like)
- AR is a nuclear receptor that has a transcription factor activity that activates the transcription of a specific target gene
- agonist or angelic agonist activity against mutant AR can be easily assayed by analyzing the expression of the target gene. can do.
- a R target gene include a group of genes having a consensus sequence called an androgen response element (A RE) further upstream of the promoter, and P S A and force reclein are typical examples.
- the mutated AR-expressing cancer cell line produced by the method of the present invention has an endogenous PSA gene. Can be measured and evaluated using an anti-PSA antibody or DNA encoding PSA or a part thereof.
- a promoter that responds to androgen in the obtained cancer cell line eg, a promoter having a native ARE equivalent sequence such as PSA promoter, or an ARE sequence and an SR promoter, an SV40 promoter, an LTR promoter
- a CMV promoter a chimeric promoter in combination with an animal cell promoter such as the HSVTK promoter, etc.
- the PSA promoter is useful in that it reflects well the behavior of the mutant AR in actual prostate cancer cells.
- a plurality of (preferably 2 to 5, more preferably 2 or 3) motors in tandem.
- luciferase, i3-galactosidase, chloramphenicol acetyltransferase, peroxidase and the like can be used as the repo overnight protein.
- DNA encoding the repo overnight protein can be inserted into the animal cell expression vector described above and introduced using the gene transfer method described above.
- Mutant AR-expressing cancer cells that are resistant to certain anti-androgens are still sensitive to certain other antiandrogens (eg, Bicalmin). Therefore, by culturing the mutant AR-expressing cancer cell line obtained as described above in the presence of a test substance, and examining the action on the mutant AR, it became resistant to the existing antiandrogen agent Other types of antiandrogens that can inhibit the growth of relapsed cancers can be screened. Therefore, the present invention also comprises culturing the mutant AR-expressing cancer cell line obtained as described above in the presence of a test substance, and has an antagonistic effect on the mutant AR expressed in the cancer cell line. The present invention also provides a method for screening an anti-androgen agent.
- certain anti-androgens eg, Flucanamide
- Bicalmin certain other antiandrogens
- Antagonism to mutant AR can be detected using cell proliferation as an indicator, but analysis and analysis of the expression of a gene that is transcriptionally activated by the mutant AR should be detected and evaluated in a shorter time.
- Examples of the gene to be used include a promoter that responds to the PSA gene and an androgen as described above (eg, a promoter having a native ARE equivalent sequence such as the PSA promoter, or an ARE sequence and an SR ⁇ promoter overnight, an SV40 promoter). , LTR promoters, CMV promoters, HSVTK promoters and other chimeric open-loop motors in combination with animal cell promoters, etc.).
- the present invention also provides an anti-androgen agent that exhibits an antagonistic effect on the mutant AR selected by the above method.
- the multiple mutant AR of the present invention W746L (C) + T882A, is resistant to both bicalutamide and flutamide, Compound A shown in the Examples is a mutant A in which such an existing antiandrogen is ineffective.
- the anti-androgen resistance that expresses the mutant AR in about 3 months at the latest Sex strains can be established.
- a known anti-androgen agent for example, Bicalumid ⁇ Fluidumid, etc.
- the present invention also provides a method for culturing cells of androgen-sensitive cancer in the presence of a test substance, and examining with time the presence or absence of a cancer cell line capable of growing under the conditions. It is intended to provide a method for screening a non-cancer- or hard-to-produce anti-androgen agent, and an anti-androgen agent to be resistant or non-causing to cancer selected by the method.
- the present invention provides a prophylactic / therapeutic agent for a hormone-sensitive cancer, preferably a prostate cancer, comprising an antiandrogen agent having an antagonistic effect on the mutant AR obtained by the above screening method.
- a prophylactic / therapeutic agent for a hormone-sensitive cancer preferably a prostate cancer
- an antiandrogen agent having an antagonistic effect on the mutant AR obtained by the above screening method can antagonize androgen against both normal and mutated AR, and thus can suppress the growth of cancer cells in both the androgen-dependent and androgen-independent phases.
- the present invention also provides a prophylactic / therapeutic agent for a hormone-sensitive cancer, preferably a prostate cancer, comprising an anti-androgen agent which is not resistant to cancer or hardly induced, obtained by the above screening method.
- a prophylactic / therapeutic agent for a hormone-sensitive cancer preferably a prostate cancer
- an anti-androgen agent which is not resistant to cancer or hardly induced, obtained by the above screening method.
- Such anti-androgens do not cause mutations in the AR that confer resistance to the drug, or are significantly less likely to occur than those of other anti-androgens, and therefore delay the transition to androgen-independent cancer. Useful for extending.
- the prophylactic / therapeutic agent can be manufactured and used according to conventional methods.
- tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the mutant AR of the present invention.
- the preparations obtained in this way are safe and have low toxicity, for example, mammals (eg humans, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.) Can be administered.
- mammals eg humans, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, 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 patient with cancer (60 kg)
- parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
- an injection it is usually used, for example, in a cancer patient (as 6 O kg). It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg, by intravenous injection.
- the dose can be administered in terms of 60 kg.
- the present invention also provides a drug therapy which is a combination of two or more anti-androgens that exhibit anti-androgenic activity against different mutant ARs. There have been many reports on AR mutations so far, but there are very few reports of mutant ARs that have two or more amino acid mutations.
- antiandrogens eg, bicalutamide and flucanamide
- antiandrogenic activity against different mutant ARs eg, W746L (C) and T882A
- each anti-androgen agent may be formulated according to the method described above, and these may be administered simultaneously, or two or more anti-androgens may be combined in one formulation.
- the dosage of the antiandrogen varies depending on the administration target, target organ, symptoms, administration method, and the like.
- oral administration in general, for example, in a patient with cancer (as 6 O kg), About 0.1 to 10 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg, respectively.
- parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
- a cancer patient 6 O kg
- the dose can be administered in terms of 60 kg.
- the method for producing a mutant AR-expressing cancer cell line of the present invention is useful for typing such an anti-androgen agent. That is, a mutant AR obtained by performing the method for various anti-androgen agents is analyzed to identify a mutation site that is easily induced by the anti-androgen agent, and a resistant cancer cell line expressing the same mutant AR is generated.
- Anti-androgens eg, flutamide, nilyumid, etc.
- anti-androgens are grouped into a group consisting of anti-androgens (eg, bicalutamide, etc.) that generate resistant cancer cell lines expressing other mutant ARs .
- An effective cocktail therapy can be performed by combining two or more of any antiandrogen agents classified into different groups by such a classification method.
- the information obtained by the above-mentioned method of classifying anti-androgen drugs is based on the fact that one anti-androgen drug is continuously administered until resistant cancer appears, and then other anti-androgen drugs classified into a different group from the anti-androgen drug are used. It is also useful in performing sequential endocrine therapies to block the growth of resistant cancers that have developed by administering antiandrogens.
- the present invention also comprises contacting a mammalian cell containing a gene under the control of the PSA promoter and capable of analyzing expression with an AR and a predetermined antiandrogen, and analyzing the expression of the gene. And a method for evaluating the responsiveness of the transcription factor activity to the anti-androgen agent.
- PSA Promoter means an ARE equivalent sequence at least upstream of an essential promoter region such as a TATA box in the 5 ′ upstream sequence of the prostate-specific antigen gene (AGAACA GCAAGTGCT; SEQ ID NO: 3), preferably a 35 base pair portion known as an upstream androgen response region (ARR) [in the case of a human PSA gene, GTGGTGCAGGGATCAGGGAGTCTCACAATCTCCTG; SEQ ID NO: 4 (J. Biol. Chem.
- Examples of the “genes whose expression can be analyzed” include PSA, luciferase, / 3-galactosidase, chloramphenicolase, and PSA, as described above in the screening of anti-androgens using the mutant AR-expressing cancer cell line of the present invention.
- Examples include DNA encoding repo overnight proteins such as cetyltransferase and peroxidase. These DNAs are inserted into the animal cell expression vector described above (but lacking the promoter) together with the PSA promoter, and the mammalian cells (eg, COS-7, Vero, CHO, CHO (dhfr), mouse L cell, mouse AtT-20, mouse myeloma cell, rat GH3, human FL cell, etc.).
- the mammalian cells eg, COS-7, Vero, CHO, CHO (dhfr), mouse L cell, mouse AtT-20, mouse myeloma cell, rat GH3, human FL cell, etc.
- the PSA gene When the gene whose expression can be analyzed is the PSA gene, the PSA gene is isolated from the genomic DNA of prostate cells using a conventional method in a form containing the PSA promoter, and then isolated using a vector for animal cell expression (however, (Lacking the promoter).
- a prostate cell having an endogenous PSA gene can be used as it is as a “mammalian cell containing a gene under the control of the PSA promoter that can be analyzed for expression”.
- the AR applied to the evaluation method of the present invention may be any AR, including normal AR derived from various mammals, mutant AR derived from androgen-independent relapsed cancer, and cancer cell lines obtained by the method of the present invention. Mutated AR derived from the gene, recombinant AR obtained by genetic engineering techniques, and the like. These ARs can be produced by isolation and purification using a method for purifying a receptor protein known per se. For example, after homogenizing mammalian cells, excised cancer lesions, the cancer cell strain of the present invention, AR-producing host cells, etc., extract them with an acid or the like, and then subject the extract to reverse phase chromatography, ion exchange chromatography, etc. Purification and isolation can be performed by combining the above chromatography.
- AR can be rapidly isolated and purified by affinity chromatography by adding a sequence encoding a tag such as His tag or GST tag to the AR coding sequence.
- the AR isolated as described above may be added to a mammalian cell containing a gene that can be analyzed for expression under the control of the PSA promoter, but the mammalian cell itself converts the AR. Preferably, it is expressed.
- a gene capable of expression analysis under the control of a PSA promoter and a gene encoding an AR are used in a host mammalian cell (eg, COS-7, Vero, CH ⁇ , CHO (dhf r-), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc.), normal mammalian prostate cells, and androgen-independent relapsed cancer
- a mutant AR-expressing cancer cell line eg, LNCaP cell line
- a mutant AR-expressing cancer cell line obtained by the method of the present invention is obtained by introducing a gene capable of analyzing the expression under the control of the PSA promoter. Transformed cells and the like are exemplified.
- the gene encoding AR may be inserted into the animal cell expression vector described above, and then introduced into mammalian cells using the gene transfer method described above.
- any known substance exhibiting an antagonistic action against normal or mutant AR eg, bicalutamide, fluramide, etc.
- Anti-androgen responsiveness means how a given anti-androgen affects the transcription factor activity of the test AR.
- a mammalian cell containing a gene whose expression can be analyzed under the control of the PSA motor is added to an AR (T882A) derived from the LN CaP-FGC cell line (ATCC No .: CRL-1740) or a mutant AR of the present invention.
- T882A When (B746L or W746LCC T882A) is contacted with bicalixamide, the former suppresses the expression of the gene, whereas the latter enhances the expression. That is, T882A is determined to be bicalutamide-sensitive, whereas W746L and W746 O + T882A are determined to be bicalutamide-resistant.
- an “AR modulator” refers to a substance that can positively or negatively regulate AR transcription factor activity, and includes both substances that have agonist activity against AR and substances that have angiogonist activity (antiandrogens). Including. Test substances include, for example, known or novel peptides, proteins, non-peptides. Tide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like.
- test substance When a predetermined AR and a test substance are brought into contact with mammalian cells containing a gene whose expression can be analyzed under the control of the PSA promoter and the expression of the gene is analyzed, if the expression is enhanced, the test substance is identified as It is a positive modulator of AR, and if its expression is suppressed, the test substance can be determined to be a negative modulator of AR (in other words, an antiandrogen).
- the present invention also provides a kit suitable for use in the above-described method for evaluating the anti-androgen responsiveness of AR transcription factor activity or the screening method for AR modules.
- the kit comprises, as a component, a mammalian cell containing a gene under the control of the PSA promoter and capable of expression analysis.
- a mammalian cell containing a gene under the control of the PSA promoter and capable of expression analysis.
- a DNA encoding a reporter protein such as PSA, luciferase,] 3-galactosidase, chloramphenico-l-acetyltransferase, or peroxidase is operably linked downstream of the PSA promoter.
- the PSA gene is isolated from the genomic DNA of prostate cells in a form containing a PSA promoter using a conventional method, and the isolated gene is used as an animal cell expression vector (provided that the promoter is May be similarly introduced into mammalian cells.
- the mammalian cells may further contain An expression vector containing DNA encoding AR may be included.
- the AR-encoding DNA may be inserted into the above-described animal cell expression vector, and introduced into mammalian cells using the above-described gene transfer method.
- bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-I UB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When there is an optical isomer of amino acid, it indicates L-form unless otherwise specified. And
- Ar g Arginine H is: histidine
- HOB t 1-hydroxybenztriazole
- HOOB t 3,4-dihydro-3-hydroxy-1-4-oxo-1
- sequence numbers in the sequence listing in the present specification indicate the following sequences.
- the ARE sequence in the human PSA promoter is shown.
- the ARR sequence in the human PSA promoter is shown.
- FIG. 2 shows oligonucleotides designed to function as primers for amplifying human PSA promoter.
- SEQ ID NO: 3 ARE sequence in human PSA promoter.
- SEQ ID NO: 4 A RR sequence in human PS A promoter.
- SEQ ID NO: 5 Oligonucleotide designed to function as a primer for amplifying human PSA promoter.
- SEQ ID NO: 6 Oligonucleotide designed to function as a primer for amplifying human PSA promoter.
- Example 1 Method for establishing LNCaP-cxD cell line
- LNCaP-FGC ATCC No .: CRL-1740
- a culture solution RPMI1640I103 ⁇ 4 Dextran Charcoal (DCC)-Fetal Bovine Serum (FBS)
- AtM bicalutamide trade name: Power Sodex
- LNCaP-cxDIK LNCaP-cxD2 and LNCaP-FGC were seeded on a 24-well plate at 40,000 cells / mL / well, and the next day, 0.2 M of bicalutamide was added, and the number of cells was counted 3 days after the addition. At this time, the concentration of PSA (Prostatic Specific Antigen) produced in an androgen-dependent manner in the culture supernatant was measured.
- PSA Prostatic Specific Antigen
- LNCaP-cxDIK Total RNA was extracted from LNCaP-cxD2 and LNCaP-FGC, converted to cDNA, and the nucleotide sequence of the AR gene was analyzed by PCR direct sequencing. For LNCaP-cxDll, only the androgen-binding region of the AR gene was sequenced.
- both ARs of LNCaP-cxDll and LNCaP-cxD2 contained a mutation in the gene sequence accompanied by an amino acid mutation in the androgen binding region.
- Codon 746 Normally, the notation that the total number of codons in AR is 919 is used.
- TGG tryptophan
- TGT cystine
- Cos- 7 5 to 150 cm 2 flask, 000 were plated 000 eel Is, 24 hours after culture in the culture medium (DMEM + 10% Dextran Ch arcoal (DCC) -Fetal Bovine Serum (FBS) + 2mM glutamine), wild-type
- the vector DNA into which AR or mutant AR (W741C and W741L type mutations) were inserted and the vector DNA having luciferase gene ligated downstream of androgen responsive promoter were cotransfected by ribosome method. After 2 hours, the medium was changed. After culturing for 3 hours, 0.01 or M of bicalutamide was added. After culturing for 24 hours, luciferase activity was measured, and the transcriptional activity of AR by bicalutamide was examined.
- genomic DNA was converted into type III, and 5'-GGAGCTCGAATTCCACATTGTTTGCTGCACGTTGG-3 '(SEQ ID NO: 5) and 5'-CAAGCTTTGGGGCTGGGGAGCCTCCCCCAGGAGC-3' (SEQ ID NO: 6) were used as primers. PCR reaction using Was done.
- the DNA fragment is mixed with pGL3-Basic vector (Promega) digested with Hind III and Sac I and ligated using Ligation high (T0Y0B0) to transform E. coli DH5 ⁇ competent cells.
- pGL3-PSA-Luc in which a luciferase gene was linked downstream of the PSA promoter was obtained.
- pGL3-PSA-Luc was digested with HindIII, blunted with a Blunting kit (TaKaRa), and then digested with KpnI (TaKaRa) to collect a fragment containing the PSA promoter overnight.
- the method of the present invention provides an effective predictor for androgen-independent relapsed prostate cancer.
- Anti-therapeutic drugs can be screened.
- the appearance (relapse) of androgen-independent cancer can be detected at an early stage.
- by applying the novel endocrine therapy derived according to the present invention it is expected that the survival rate and duration of prostate cancer will be significantly improved.
- the present invention is based on Japanese Patent Application Nos. 2002-162206 and 2002-255612 filed in Japan, the contents of which are incorporated in full herein.
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP03730778A EP1550720B1 (en) | 2002-06-03 | 2003-06-02 | Mutated androgen receptor, cancer cells expressing the same, method of constructing the same and use thereof |
DE60331934T DE60331934D1 (de) | 2002-06-03 | 2003-06-02 | Mutierter androgenrezeptor, diesen exprimierende krebszellen, verfahren zur konstruktion davon und verwendung davon |
AT03730778T ATE462724T1 (de) | 2002-06-03 | 2003-06-02 | Mutierter androgenrezeptor, diesen exprimierende krebszellen, verfahren zur konstruktion davon und verwendung davon |
AU2003241738A AU2003241738A1 (en) | 2002-06-03 | 2003-06-02 | Mutated androgen receptor, cancer cells expressing the same, method of constructing the same and use thereof |
US10/516,705 US20050181462A1 (en) | 2002-06-03 | 2003-06-02 | Mutant androgen receptor, cancer cells expressing the same, a method of producing them and use thereof |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002-162206 | 2002-06-03 | ||
JP2002162206 | 2002-06-03 | ||
JP2002255612A JP4447826B2 (ja) | 2002-06-03 | 2002-08-30 | 変異アンドロゲン受容体、それを発現する癌細胞、それらの作出方法およびそれらの用途 |
JP2002-255612 | 2002-08-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2003102188A1 true WO2003102188A1 (fr) | 2003-12-11 |
Family
ID=29714332
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2003/006942 WO2003102188A1 (fr) | 2002-06-03 | 2003-06-02 | Recepteur d'androgene mute, cellules cancereuses l'exprimant, procede de construction de ce recepteur et utilisations correspondantes |
Country Status (7)
Country | Link |
---|---|
US (1) | US20050181462A1 (ja) |
EP (2) | EP2090656A1 (ja) |
JP (1) | JP4447826B2 (ja) |
AT (1) | ATE462724T1 (ja) |
AU (1) | AU2003241738A1 (ja) |
DE (1) | DE60331934D1 (ja) |
WO (1) | WO2003102188A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7649001B2 (en) | 2002-08-12 | 2010-01-19 | Takeda Pharmaceutical Company Limited | Fused benzene derivative and use |
WO2011040421A1 (ja) | 2009-09-29 | 2011-04-07 | 武田薬品工業株式会社 | スクリーニング方法 |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8841422B2 (en) | 2008-09-17 | 2014-09-23 | University Of Maryland, Baltimore | Human androgen receptor alternative splice variants |
US8133724B2 (en) * | 2008-09-17 | 2012-03-13 | University Of Maryland, Baltimore | Human androgen receptor alternative splice variants as biomarkers and therapeutic targets |
EP2912194B1 (en) * | 2012-10-26 | 2019-05-08 | Memorial Sloan-Kettering Cancer Center | Androgen receptor variants and methods for making and using |
CN117363621B (zh) * | 2023-10-11 | 2024-09-13 | 南方医科大学南方医院 | 一种雄激素受体ar的n端体细胞基因突变体在构建抗肝癌药物筛选模型中的应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6291194B1 (en) * | 2000-07-28 | 2001-09-18 | Taneli Raivio | Assay for determination of androgenic or anti-androgenic activity of a serum sample or a test compound |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6994992B1 (en) * | 1999-02-24 | 2006-02-07 | Trustees Of Tufts College | Androgen-induced suppressor of cell proliferation and uses thereof |
GB0005689D0 (en) * | 2000-03-09 | 2000-05-03 | Schering Ag | Crystal |
US20050101657A1 (en) * | 2001-12-28 | 2005-05-12 | Takeda Chemical Industries Ltd. | Androgen receptor antagonists |
-
2002
- 2002-08-30 JP JP2002255612A patent/JP4447826B2/ja not_active Expired - Fee Related
-
2003
- 2003-06-02 EP EP09075054A patent/EP2090656A1/en not_active Withdrawn
- 2003-06-02 EP EP03730778A patent/EP1550720B1/en not_active Expired - Lifetime
- 2003-06-02 DE DE60331934T patent/DE60331934D1/de not_active Expired - Lifetime
- 2003-06-02 AT AT03730778T patent/ATE462724T1/de not_active IP Right Cessation
- 2003-06-02 AU AU2003241738A patent/AU2003241738A1/en not_active Abandoned
- 2003-06-02 WO PCT/JP2003/006942 patent/WO2003102188A1/ja active Application Filing
- 2003-06-02 US US10/516,705 patent/US20050181462A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6291194B1 (en) * | 2000-07-28 | 2001-09-18 | Taneli Raivio | Assay for determination of androgenic or anti-androgenic activity of a serum sample or a test compound |
Non-Patent Citations (11)
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7649001B2 (en) | 2002-08-12 | 2010-01-19 | Takeda Pharmaceutical Company Limited | Fused benzene derivative and use |
WO2011040421A1 (ja) | 2009-09-29 | 2011-04-07 | 武田薬品工業株式会社 | スクリーニング方法 |
Also Published As
Publication number | Publication date |
---|---|
US20050181462A1 (en) | 2005-08-18 |
EP1550720B1 (en) | 2010-03-31 |
ATE462724T1 (de) | 2010-04-15 |
EP1550720A1 (en) | 2005-07-06 |
AU2003241738A1 (en) | 2003-12-19 |
EP2090656A1 (en) | 2009-08-19 |
EP1550720A4 (en) | 2006-07-26 |
JP4447826B2 (ja) | 2010-04-07 |
JP2004057180A (ja) | 2004-02-26 |
DE60331934D1 (de) | 2010-05-12 |
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