JP2009005703A - Mutated androgen receptor, cancer cell expressing the same, method for producing them and use of them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mutated androgen receptor (AR) useful for screening, etc., the preventing/treating drugs of androgen non-dependent cancers. <P>SOLUTION: The mutated AR, DNA, etc., encoding the AR, method for producing the mutated AR-expressing cancer cell lines in vitro, method for screening anti-androgen agents by using the mutated AR-expressing cancer cell lines obtained by the above method, method for classifying the anti-androgen agents, and cocktail type treating agents, etc., obtained by combining the anti-androgen agents classified by the above method are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a mutant androgen receptor derived from human prostate cancer cells, a human prostate cancer cell line expressing it, a method for producing them, and an anti-androgen-independent prostate cancer using the anti-proliferative activity of the prostate cancer cells. The present invention relates to a screening method for androgenic agents.

  The global standard for androgen-inhibited endocrine therapy is standard for advanced stage prostate cancer with metastasis. In localized prostate cancer without metastasis, it has been proven that endocrine therapy with antiandrogens after radical prostatectomy and radiation therapy delays the progression of prostate cancer. Endocrine therapy is also used for localized prostate cancer. Is spreading.

  Androgen is a general term for steroid hormones that have androgenic action, and includes testosterone synthesized in testis, dehydroepiandrosterone, androstenedione synthesized in adrenal cortex. Since about 95% of androgens are secreted from the testis, surgical and eradication techniques to block testosterone-derived testosterone or drugs (estrogens, LHRH agonists, etc.) are frequently used as androgen-suppressing endocrine therapy . However, since the adrenal cortex-derived testosterone precursor is converted into highly active dihydrotestosterone (DHT) in prostate cells, it appears to be present in a proportion of prostate cancer lesions even if it is only a few percent It may not be ignored as a growth stimulus for androgen-sensitive cancer cells. Therefore, MAB (Maximal Androgen Blockade) therapy in which an anti-androgen agent (particularly a nonsteroidal drug) that antagonistically inhibits the binding of androgen to the receptor in the prostate is also used in combination with castration.

  On the other hand, an effective prevention and treatment method for androgen-independent relapsed cancer which has been faced sooner or later in most advanced cases treated with endocrine therapy has not yet been developed. Although the mechanism of relapsed cancer for endocrine therapy has not yet been elucidated, substances other than androgen due to the emergence / increase of androgen hypersensitivity due to amplification or overexpression of the androgen receptor (AR) gene, or AR mutations ( For example, a plurality of factors are becoming clear, such as glucocorticoids and antiandrogen agents themselves) acting as agonists.

  For example, some clinical findings have been reported regarding the relationship between relapsed cancer and AR mutation after administration of an antiandrogen. That is, AR mutation was observed in 5 out of 17 patients whose prostate cancer relapsed after endocrine therapy with combined use of flutamide and castration, and all of them were at the 877th amino acid (amino acid number 882 in the amino acid sequence of SEQ ID NO: 2). Equivalent) missense mutation (Taplin et al., Cancer Res., 59: 2511-2515, 1999). Some antiandrogens including flutamide against this type of mutant AR, on the contrary, showed an effect of stimulating cancer cell growth (Veldscholte et al., Biochem. Biophys. Res. Commun., 173). : 534-540, 1990). In addition, AR missense mutations were confirmed in 3 out of 11 biopsy samples from patients whose prostate cancer relapsed after endocrine therapy combined with bicalutamide and surgical castration. All of these mutation sites were flutamide-resistant relapses. It was different from the 877th amino acid which is prominent in cancer (Haapala et al., Lab. Invest., 81: 1647-1651, 2001).

In developing a drug useful for the prevention and treatment of androgen-independent cancer based on an AR mutation, a cancer cell line that expresses the mutant AR is a very useful research tool. However, all of the mutant AR-expressing cancer cells reported so far are clinically obtained from relapsed cancer or prostate cancer before treatment, and the mutation is induced in vitro in cancer cells expressing AR. There is no example of establishing a cancer cell line that expresses AR having a mutation similar to that of androgen-independent cancer-derived mutant AR found clinically. In addition, most of the mutations AR found clinically from androgen-independent cancers have a single mutation at the amino acid level, and there are no reports of ARs having mutations at two or more amino acids. It is rare.
Accordingly, an object of the present invention is to generate in vitro a cancer cell line that expresses an AR having a mutation similar to that of a clinically found androgen-independent cancer-derived mutant AR, and the mutant AR obtained by the method. It is to provide a cancer cell line that expresses the same, and to provide a method for screening a drug effective for the prevention and treatment of androgen-independent relapse cancer for endocrine therapy using them.

  As a result of intensive studies to achieve the above object, the present inventors have cultivated human prostate cancer cells sensitive to a predetermined antiandrogen agent for a long time in the presence of the antiandrogen agent. It was found that mutations that significantly reduce the potency of the drug or that exhibit agonist-like effects are induced in AR, resulting in antiandrogen resistance, ie, androgen-independent cancer, and start to grow. . Furthermore, as a result of isolating AR cDNA from cancer cells showing proliferation and analyzing the entire nucleotide sequence, AR mutation sites were obtained clinically from relapsed cancer in patients who had been subjected to endocrine therapy with antiandrogens. It was found to be in good agreement with that of the mutant AR. As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] Amino acid sequence of the following (a) or (b):
(A) an amino acid sequence in which the tryptophan of amino acid number 746 is replaced by leucine in the amino acid sequence represented by SEQ ID NO: 2;
(B) an amino acid sequence represented by SEQ ID NO: 2, wherein tryptophan of amino acid number 746 is replaced with leucine or cysteine and threonine of amino acid number 882 is replaced with alanine;
A protein or salt thereof containing the same or substantially the same amino acid sequence as
[2] A partial peptide of the protein according to [1] above, which contains at least a partial amino acid sequence corresponding to a region necessary for binding to an androgen of a normal androgen receptor, or an amide or ester thereof, or Its salt,
[3] A polynucleotide containing a polynucleotide encoding the protein according to [1] above or the partial peptide according to [2] above,
[4] The polynucleotide according to [3], which is DNA,
[5] The following base sequence (a) or (b):
(A) a base sequence in which the base of base number 2237 is substituted with thymine in the base sequence represented by SEQ ID NO: 1;
(B) a base sequence represented by SEQ ID NO: 1, wherein the base of base number 2237 or 2238 is substituted with thymine and the base of base number 2644 is substituted with guanine;
The polynucleotide of [3] above, comprising
[6] A diagnostic agent containing the polynucleotide according to [3] above,
[7] The diagnostic agent according to the above [6], which is for diagnosis of hormone-independent cancer,
[8] A recombinant vector containing the polynucleotide according to [3] above,
[9] A transformant transformed with the recombinant vector according to [8] above,
[10] An animal cell having the ability to produce the protein of [1] above,
[11] The animal cell according to [10], wherein the animal cell is a cancer cell,
[12] The animal cell according to [11], wherein the cancer cell is a cell derived from prostate cancer,
[13] The transformant according to [9] or the animal cell according to [10] is cultured to produce the protein according to [1] or the partial peptide according to [2]. [1] a method for producing the protein or salt thereof according to [1], or the partial peptide or amide or ester thereof or salt thereof according to [2] above;
[14] An antibody against the protein or salt thereof according to [1] above or the partial peptide or amide or ester thereof or salt thereof according to [2] above, wherein the antibody does not recognize normal androgen receptor protein or salt thereof,
[15] An antibody against a protein or salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence in which the tryptophan of amino acid number 746 is replaced by leucine or cysteine in the amino acid sequence represented by SEQ ID NO: 2, An antibody that does not recognize a protein or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence in which the threonine of amino acid number 882 is substituted with alanine in the amino acid sequence represented by SEQ ID NO: 2;
[16] An antibody against a protein or salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence in which the threonine of amino acid number 882 is substituted with alanine in the amino acid sequence represented by SEQ ID NO: 2, An antibody that does not recognize a protein containing the same or substantially the same amino acid sequence as the amino acid sequence in which the tryptophan of amino acid number 746 is replaced by leucine or cysteine in the amino acid sequence represented by 2, or a salt thereof,
[17] The antibody according to [14], which is a neutralizing antibody that suppresses the transcription factor activity of the protein according to [1],
[18] A diagnostic agent comprising the antibody according to any one of [14] to [16] above,
[19] The diagnostic agent according to [18] above, which is used for diagnosis of androgen-independent phase transition of hormone-sensitive cancer,
[20] An androgen and a protein according to [1] above or a salt thereof, characterized by using the protein according to [1] above or a salt thereof or the partial peptide according to [2] above or an amide or ester thereof or a salt thereof. A method for screening a salt, a partial peptide according to the above [2], a amide or ester thereof, a compound that changes the binding property with a salt thereof, or a salt thereof,
[21] An androgen and a protein according to [1] above, which comprises the protein according to [1] or a salt thereof, or the partial peptide according to [2] above or an amide or ester thereof or a salt thereof. A kit for screening a salt or a compound that changes the binding property to the partial peptide of the above [2] or an amide or ester thereof or a salt thereof, or a salt thereof;
[22] Androgen and the protein or salt thereof described in [1] or the partial peptide described in [2] above, which can be obtained using the screening method described in [20] or the screening kit described in [21] A compound or a salt thereof that changes the binding property with the amide or the ester or the salt thereof,
[23] A medicament comprising the compound or salt thereof according to [22] above,
[24] The medicament of the above-mentioned [23], which is a prophylactic / therapeutic agent for hormone-sensitive cancer in androgen-dependent and androgen-independent periods,
[25] A method for quantifying mRNA encoding the protein according to [1], wherein the polynucleotide according to [3] or a part thereof is used.
[26] The protein according to [1] above or the salt thereof, or the partial peptide according to [2] above, or an amide thereof, or a method thereof, wherein the antibody according to any one of [14] to [16] above is used. Quantification method of ester or its salt,
[27] A method for diagnosing androgen-independent phase transition of hormone-sensitive cancer, characterized by using the quantification method described in [25] or [26] above,
[28] Cancer cells sensitive to a predetermined anti-androgen agent are cultured in the presence of the anti-androgen agent, and a cancer cell line in which proliferation is observed is selected, and the androgen receptor gene 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 base sequence and selecting a strain having a mutation in the sequence;
[29] The method according to [28] above, wherein the mutation site coincides with a clinical mutation site that appears by administration of the antiandrogen agent,
[30] A cancer cell line that expresses a mutant androgen receptor and is sensitive to a predetermined antiandrogen agent is cultured in the presence of the antiandrogen agent to select a cancer cell line that has been observed to grow, The anti-androgen-resistant cancer expressing a multi-mutant androgen receptor, characterized by analyzing a base sequence of a mutant androgen receptor gene in a cancer cell line and selecting a strain having another mutation in the sequence Cell line production method,
[31] The method according to any one of [28] to [30] above, wherein the antiandrogen is flutamide or an analog thereof, or bicalutamide or an analog thereof,
[32] A cancer cell line expressing a mutant androgen receptor obtained by the method of [28] above,
[33] A cancer cell line expressing a multiple mutant androgen receptor obtained by the method described in [30] above,
[34] The cancer cell line of [32] or [33] above, further comprising a gene capable of expression analysis under the control of a promoter responsive to androgen,
[35] The cancer cell line according to [34], wherein the gene is a prostate-specific antigen gene or a foreign reporter gene,
[36] A cancer cell line described in [32] or [33] above, which is cultured in the presence of a test substance, and exhibits an antagonistic action against a mutant androgen receptor expressed in the cancer cell line Androgen screening method,
[37] The cancer cell line described in [34] above is cultured in the presence of a test substance, and the expression of a gene under the control of a promoter responsive to androgen in the cancer cell is analyzed. A screening method for an anti-androgen that shows an antagonistic action against a mutant androgen receptor expressed in a cell line,
[38] An anti-androgen that exhibits an antagonistic action against a mutant androgen receptor selected by the method of [36] or [37] above,
[39] A resistant cancer non-inducing or culturing characterized by culturing cells of androgen-sensitive cancer in the presence of a test substance, and examining over time whether or not a cancer cell line capable of growing under these conditions appears. A screening method for difficult-androgenic antiandrogens,
[40] A resistance cancer non-inducible or difficult-to-inducible antiandrogen selected by the method of [39] above,
[41] The antiandrogen according to the above [40], which is non-mutation-inducing or difficult-to-induce androgen receptor,
[42] A prophylactic / therapeutic agent for androgen-dependent or androgen-independent hormone-sensitive cancer, comprising the antiandrogen agent according to [38] or [40] above,
[43] Prevention of hormone-sensitive cancer in androgen-dependent or androgen-independent phase in mammals, comprising administering an effective amount of the anti-androgen agent described in [38] or [40] to the mammals Method of treatment,
[44] Use of the anti-androgen agent according to the above [38] or [40] for the manufacture of a prophylactic / therapeutic agent for hormone-sensitive cancer in androgen-dependent or androgen-independent phase,
[45] A prophylactic / therapeutic agent for hormone-sensitive cancers in androgen-dependent and androgen-independent phases, comprising a combination of two or more antiandrogens that exhibit antiandrogenic activity against different mutant androgen receptors,
[46] The agent described in [45] above, wherein one of the two or more antiandrogenic agents is selected by the method described in [36] or [37] above,
[47] An androgen-dependent phase or androgen-independent in the mammal, comprising administering to the mammal each effective amount of two or more anti-androgen agents that exhibit antiandrogen activity against different mutant androgen receptors Prevention and treatment methods for hormone-sensitive cancer in the childhood,
[48] The method according to [47] above, wherein one of the two or more anti-androgens is selected by the method according to [36] or [37] above,
[49] Use of two or more anti-androgen agents exhibiting anti-androgenic activity against different mutant androgen receptors for the manufacture of a prophylactic / therapeutic agent for hormone-sensitive cancer in androgen-dependent or androgen-independent phase,
[50] The use according to [49] above, wherein one of the two or more antiandrogens is selected by the method according to [36] or [37] above,
[51] A resistant cancer that expresses another mutant androgen receptor, taking as a group anti-androgen agents that can give rise to resistant cancer cell lines that express the same mutant androgen receptor by the method described in [28] above A method of classifying antiandrogens, characterized in that they are separated from the group consisting of antiandrogens that can give rise to cell lines,
[52] A prophylactic / therapeutic agent for androgen-dependent and androgen-independent hormone-sensitive cancer, comprising a combination of two or more antiandrogen agents classified into different groups by the method described in [51] above,
[53] An androgen-dependent period or non-androgen in the mammal, wherein each effective amount of two or more antiandrogen agents classified into different groups according to the method described in [51] is administered to the mammal Prevention and treatment methods for hormonally sensitive cancer in the dependent period,
[54] Use of two or more anti-androgen agents classified into different groups according to the method of [51] for the manufacture of a prophylactic / therapeutic agent for hormone-sensitive cancer in androgen-dependent or androgen-independent phase, And [55] A mammal in which a resistant cancer against the antiandrogen has emerged by long-term administration of an effective amount of the antiandrogen classified into a group by the method described in [51] above, is classified into another group. A method for preventing and treating hormone-sensitive cancer in an androgen-independent period, which comprises administering an effective amount of an antiandrogen agent,
[56] The cancer cell line according to [34] or [35] above, wherein the promoter responsive to androgen is a PSA promoter,
[57] The screening method according to [37] above, wherein the promoter responsive to androgen is a PSA promoter,
[58] An anti-androgen receptor responsiveness evaluation kit for androgen receptor transcription factor activity or an androgen receptor modulator screening kit, comprising a mammalian cell containing a gene capable of expression analysis under the control of a PSA promoter,
[59] The kit according to [58], wherein the mammalian cell further expresses a predetermined androgen receptor,
[60] The kit according to [58] above, wherein a plurality of PSA promoters are linked in tandem,
[61] The kit according to [58] above, wherein the gene whose expression can be analyzed is a prostate-specific antigen gene or a foreign reporter gene,
[62] The androgen characterized in that an androgen receptor and a predetermined antiandrogen agent are contacted with a mammalian cell containing a gene capable of expression analysis under the control of a PSA promoter, and the expression of the gene is analyzed A method for evaluating the antiandrogen responsiveness of a transcription factor activity of a receptor;
[63] The androgen receptor characterized by contacting a predetermined androgen receptor and a test substance with a mammalian cell containing a gene capable of expression analysis under the control of a PSA promoter, and analyzing the expression of the gene Body modulator screening method,
[64] The method of the above-mentioned [62] or [63], wherein the contact with the androgen receptor is made by expressing the receptor in a mammalian cell,
[65] The method according to [62] or [63] above, wherein the plurality of PSA promoters are linked in tandem, and [66] the gene whose expression can be analyzed is a prostate-specific antigen gene or a foreign reporter gene The method according to the above [62] or [63] is provided.

  By the method of the present invention, an effective prophylactic / therapeutic agent for androgen-independent relapsed prostate cancer can be screened. In addition, the appearance (relapse) of androgen-independent cancer can be detected early. Furthermore, applying the novel endocrine therapy derived by the present invention is expected to greatly improve the survival rate and duration of prostate cancer.

The mutant AR of the present invention has an amino acid sequence ("", in which the tryptophan of amino acid number 746 is replaced with leucine in the amino acid sequence represented by SEQ ID NO: 2 (Proc. Natl. Acad. Sci. USA, 85: 7211-7215, 1988). Or an amino acid sequence represented by SEQ ID NO: 2 in which tryptophan of amino acid number 746 is replaced with leucine or cysteine and threonine of amino acid number 882 is replaced with alanine ("" W746L "). W746L (C) + T882A ”(sometimes abbreviated as“ W746L (C) + T882A ”).
The mutant AR of the present invention is, for example, any cell (eg, spleen cell, nerve cell, glial cell, pancreatic β) of a mammal (eg, human, guinea pig, rat, mouse, rabbit, pig, sheep, cow, monkey, etc.). Cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, Mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes or stromal cells, Or progenitor cells of these cells, stem cells, cancer cells, etc., blood cells, or any tissue in which these cells exist, such as the brain, each part of the brain (eg, olfactory bulb, buccal nucleus, cerebral base) Sphere, hippocampus, thalamus, hypothalamus, hypothalamic nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen, caudate nucleus, brain dye, nigro), spinal cord, pituitary gland, stomach, Pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, It may be a protein derived from prostate, testis, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. (especially prostate), or may be a synthetic protein.

Examples of the amino acid sequence substantially identical to W746L or W746L (C) + T882A include, for example, about 50% or more, preferably about 70% or more, more preferably about 80% or more, and more preferably about each amino acid sequence. Examples thereof include amino acid sequences having 90% or more, most preferably about 95% or more identity.
As a protein containing substantially the same amino acid sequence as W746L or W746L (C) + T882A, for example, it has an amino acid sequence substantially the same as W746L or W746L (C) + T882A, A protein having substantially the same activity is preferable.
The substantially homogeneous activity includes, for example, ligand (including other endogenous steroid hormones and agonists and antagonists such as antiandrogens), cell proliferation activity, PSA (prostate specific) An action of increasing the expression of a gene under the control of a promoter that responds to an androgen such as an antigen). “Substantially homogeneous” means that their activities are homogeneous in nature. Therefore, the activities such as ligand binding activity, cell proliferation activity, and gene expression increasing action under the control of a promoter responsive to androgen are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more Preferably, it is about 0.5 to 2 times), but 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, and can be measured, for example, according to a screening method described later.
More specifically, the activity substantially the same as that of W746L includes that bicalutamide or an analog thereof exhibits agonist activity, that is, cell proliferation activity and PSA expression inducing activity in the presence of bicalutamide. In addition to the bicalutamide response similar to W746L, the activity substantially the same as W746L (C) + T882A is that flutamide or an analog thereof exhibits agonist activity, that is, cell proliferation activity and PSA in the presence of flutamide. Examples thereof include expression inducing activity.

  The mutant AR of the present invention includes (1) one or more of W746L or W746L (C) + T882A (preferably about 1 to 30, more preferably about 1 to 10, more preferably Amino acid sequence in which several (1-5) amino acids are deleted, (2) 1 or 2 (preferably about 1-30, more preferably 1 to W746L or W746L (C) + T882A An amino acid sequence to which about 10 amino acids, more preferably several (1 to 5) amino acids are added, (3) one or more (preferably 1 to 30) in W746L or W746L (C) + T882A About, more preferably about 1 to 10, more preferably several (1 to 5) amino acids are substituted with other amino acids, or (4) an amino acid sequence combining them (however, 746 Th (and 882) amino acid is conserved Such as proteins containing yl) is also used.

The protein in the present specification has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. In the mutant AR of the present invention including W746L or W746L (C) + T882A, the C-terminus is usually a carboxyl group (—COOH), carboxylate (—COO ⁻ ), amide (—CONH ₂ ) or ester (—COOR). ).
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, for example, phenyl C _6-12 aryl groups such as α-naphthyl, C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl _In addition to the ₁₄ aralkyl group, a pivaloyloxymethyl group, which is widely used as an oral ester, is used.
When the mutant AR of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the mutant AR of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Furthermore, in the mutant AR of the present invention, the amino group of the N-terminal methionine residue is a protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl) in the above-described protein. In which the N-terminal side is cleaved in vivo and the resulting glutamyl group is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH, amino group) , An imidazole group, an indole group, a guanidino group, etc.) protected with an appropriate protecting group (for example, a C _1-6 acyl group such as a C _2-6 alkanoyl group such as formyl group or acetyl), or a sugar Also included are complex proteins such as so-called glycoproteins with chains attached.
Specific examples of the mutant AR of the present invention include human-derived AR (more preferably derived from human prostate cancer) containing W746L or W746L (C) + T882A.

As the partial peptide of the mutant AR of the present invention (hereinafter sometimes abbreviated as the partial peptide of the present invention), for example, among the mutant AR molecules of the present invention, the normal AR (the amino acid sequence represented by SEQ ID NO: 2 and its A polymorphism) having a partial amino acid sequence corresponding to a region necessary for binding to androgen is used. Like other nuclear receptors, AR is arranged from the N-terminal side in the order of transcription activation domain, DNA binding domain, hinge region, ligand binding domain, and the ligand binding domain is an amino acid sequence represented by SEQ ID NO: 2. Is an amino acid sequence represented by amino acid numbers 672 to 924.
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 necessary for binding to a normal AR androgen, but at least 20 or more, preferably 50 or more, more preferably Is preferably a peptide having a sequence of 100 or more amino acids.
The amino acid sequence substantially the same as the partial peptide of the present invention is about 50% or more, preferably about 70% or more, more preferably about 80% or more, more preferably about 90% or more, most preferably, these amino acid sequences. Indicates an amino acid sequence having a homology of about 95% or more.
Here, “substantially the same quality of activity” has the same meaning as described above. The “substantially equivalent activity” can be measured in the same manner as described above.

In the partial peptide of the present invention, one or two or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the amino acid sequence are deleted, or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are added to the amino acid sequence, or the amino acids One or more amino acids in the sequence (preferably about 1 to 10, more preferably about several, and more preferably about 1 to 5) may be substituted with other amino acids (however, 746) (And the 882nd amino acid is conserved).
In the partial peptide of the present invention, the C-terminus may be usually a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ) or an ester (—COOR). Here, R in the ester has the same meaning as described above.
When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Furthermore, in the partial peptide of the present invention, as in the mutant AR of the present invention described above, the amino group of the N-terminal methionine residue was protected with a protective group, and the N-terminal side was cleaved in vivo. Examples include those obtained by pyroglutamine oxidation of Gln, those in which a substituent on the side chain of an amino acid in the molecule is protected with an appropriate protecting group, or complex peptides such as so-called glycopeptides to which sugar chains are bound.
Examples of the salt of the mutant AR or partial peptide thereof of the present invention include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable. Examples of such salts include salts with inorganic acids (for example, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (for example, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). , Salts with tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid).

The mutant AR or a salt thereof of the present invention can be produced by isolating and purifying from an animal cell having the ability to produce the mutant AR using a known method for purifying receptor proteins. For example, after homogenizing human or mammalian tissue or cells, extraction with acid or the like can be performed, and the extract can be purified and isolated by combining chromatography such as reverse phase chromatography or ion exchange chromatography. . Animal cells having the ability to produce the mutant AR of the present invention include relapse of prostate cancer patients to which the above-mentioned human or mammalian cells or tissues (particularly antiandrogen agents (eg, bicalutamide or bicalutamide and flutamide) are administered). Cancer cell line) or a cancer cell line expressing normal AR or AR having T882A mutation (eg LNCaP cell line) in the presence of an antiandrogen (eg bicalutamide or an analog thereof) Examples include an anti-androgen-resistant cancer cell line selected as an index.
The mutant AR of the present invention or a salt thereof can also be produced by culturing a transformant transformed with a recombinant vector containing a DNA encoding the mutant AR of the present invention described later. Alternatively, based on the amino acid sequence of W746L or W746L (C) + T882A, it can also be produced by a protein synthesis method described later or a method analogous thereto.

For the synthesis of the mutant AR of the present invention or a partial peptide thereof, a salt thereof or an amide thereof, a commercially available protein synthesis resin can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, etc. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various condensation methods known per se according to the sequence of the target protein. At the end of the reaction, the protein is cut out from the resin, and at the same time, various protecting groups are removed, and further, intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or its amide.
For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOOBt), or the protected amino acid is activated in advance as a symmetric anhydride, HOBt ester or HOOBt ester. It can later be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be usable for protein bond forming reaction, and is usually selected appropriately from a range of about −20 ° C. to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

Examples of the protecting group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group may be, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of the group suitable for esterification include a lower alkanoyl 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 and an ethoxycarbonyl group. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of groups for protecting the phenolic hydroxyl group of tyrosine, for example, Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl are used.
As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.
Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, 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 acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Addition of a cation scavenger such as butanedithiol or 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine was removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan was the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the removal of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
As another method for obtaining a protein amide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (protein) chain is extended to the desired chain length on the amino group side. Producing a protein from which only the protecting group of the α-amino group at the N-terminal of the peptide chain is removed and a protein from which only the protecting group of the carboxyl group at the C-terminal has been removed are prepared. To condense. The 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-described method to obtain the desired crude protein. This crude protein can be purified using various known purification means, and the main fraction can be freeze-dried to obtain an amide form of the desired protein.
To obtain a protein ester, for example, the α-carboxyl group of the carboxy terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired protein ester is obtained in the same manner as the protein amide. be able to.

The mutant AR of the present invention or the partial peptide of the present invention (hereinafter referred to as “the partial peptide of the present invention or its amide or ester or salt thereof” is simply referred to as “the partial peptide of the present invention” and “the mutant AR of the present invention or salt thereof. Is simply referred to as “mutant AR of the present invention”), 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. Can be manufactured. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the partial peptide or amino acid that can constitute the mutant AR of the present invention or the partial peptide of the present invention is condensed with the remaining part, and when the product has a protective group, the target peptide is removed by removing the protective group. Can be manufactured. Examples of known condensation methods and protecting group elimination include the methods described in the following (1) to (5).
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966);
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965);
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975);
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977);
(5) Supervised by Haruaki Yajima, Development of follow-up medicines Vol.14 Peptide synthesis Hirokawa Shoten.
In addition, after the reaction, the mutant AR of the present invention or the partial peptide of the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. . When the mutant AR of the present invention obtained by the above method or the partial peptide of the present invention is a free form, it can be converted into an appropriate salt by a known method. It can be converted into a free form by the method.

The polynucleotide encoding the mutant AR of the present invention may be any polynucleotide as long as it contains the base sequence (DNA or RNA, preferably DNA) encoding the mutant AR of the present invention described above. 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 a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
Using the polynucleotide encoding the mutant AR of the present invention, the mutation of the present invention can be performed, for example, by the method described in the known experimental medicine special edition “New PCR and its application” 15 (7), 1997 or the like or a method analogous thereto. AR mRNA 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 cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Moreover, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells / tissues described above.
Specifically, the DNA encoding the mutant AR of the present invention includes, for example, a base sequence (G2237T) in which the base of base number 2237 is substituted with thymine in the base sequence represented by SEQ ID NO: 1, or SEQ ID NO: 1. DNA containing a nucleotide sequence (G2237 (2238) T + A2644G) in which the nucleotide of base number 2237 or 2238 is substituted with thymine and the base of nucleotide number 2644 is substituted with guanine in the represented nucleotide sequence, or the nucleotide sequence Has a base sequence that hybridizes under highly stringent conditions, and has substantially the same activity as the mutant AR of the present invention (eg, ligand binding activity, cell proliferation activity, PSA (prostate specific antigen) increasing action, etc.) Any DNA may be used as long as it encodes a mutant AR having).
Examples of DNA that can hybridize 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% with the respective base sequences. A DNA containing a base sequence having the above identity is used.

Hybridization can be performed according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). it can. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
The nucleotide containing a part of the base sequence of the DNA encoding the mutant AR of the present invention or a part of the base sequence complementary to the DNA includes DNA encoding the following partial peptide of the present invention. It is used to include not only RNA but also RNA.
According to the present invention, the nucleotide sequence information of the DNA encoding the mutant AR of the present invention, wherein an 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 synthesis based on Such nucleotides (nucleic acids) can specifically hybridize with the RNA of the mutant AR gene of the present invention by precisely controlling temperature conditions, etc., and can inhibit the synthesis or function of the RNA, or 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. The polynucleotide complementary to the selected sequence of the AR-related RNA of the present invention and the nucleotide capable of specifically hybridizing with the mutant AR-related RNA of the present invention are the mutant AR of the present invention in vivo and in vitro. It is useful for regulating and controlling gene expression, and for treatment of diseases, especially hormone-sensitive cancers (eg prostate cancer) in androgen-dependent and androgen-independent phases or transition to androgen-independent phases (relapse) Useful for diagnosis.
The term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between nucleotide, base sequence or nucleic acid and peptide (protein) usually refers to the amino acid of the peptide (protein) in the instruction derived from the nucleotide (nucleic acid) sequence or its complement. . 5 'end hairpin loop, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3' end untranslated of the mutant AR gene of the present invention The region, the 3 ′ end palindromic region, and the 3 ′ end hairpin loop can be selected as preferred target regions, but any region within the mutant AR gene can be selected as the target.

  The relationship between the target nucleic acid and a nucleotide complementary to at least a part of the region of interest, that is, the relationship between the nucleotide that can hybridize to the object, can be said to be “antisense”. Antisense nucleotides include 2-deoxy-D-ribose-containing deoxynucleotides, D-ribose-containing nucleotides, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, or non-nucleotides Other polymers with backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a pair of bases as found in DNA or RNA And a nucleotide having a configuration allowing ring attachment and base attachment). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and unmodified polynucleotides (or unmodified oligonucleotides), and more publicly known Modified, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), For example, proteins (nucleases, nuclease inhibitors, toxins, , Signal peptides, poly-L-lysine, etc.) and sugars (for example, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (For example, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, and those with modified bonds (eg, α-anomeric nucleic acids) There may be. Here, “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other heterocyclic bases modified. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens or aliphatic groups, or converted to functional groups such as ethers or amines. May have been.

The antisense nucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and to be antitoxic if toxic Make sense nucleic acids less toxic.
Many modifications are known in the art. For example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed., Antisense Research and Applications, CRC Press, 1993, etc. There is a disclosure.
The antisense nucleic acid of the present invention may be altered or contain modified sugars, bases and bonds, provided in special forms such as liposomes, microspheres, applied by gene therapy, It can be given in an added form. In this way, the additional form can be used as a polycationic substance such as polylysine that works to neutralize the charge of the phosphate group skeleton, or a lipid that enhances the interaction with the cell membrane or increases the uptake of nucleic acids ( Examples thereof include crude water-based products such as phospholipid and cholesterol. Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 ′ end or 5 ′ end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of 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 as long as it contains the base sequence encoding the partial peptide of the present invention described above. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. In addition, it can be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using an mRNA fraction prepared from the cells / tissues described above.
Specifically, examples of the DNA encoding the partial peptide of the present invention include (1) DNA having a partial base sequence of DNA having G2237T or G2237 (2238) T + A2644G, or (2) each base sequence And has a base sequence that hybridizes under highly stringent conditions, and has substantially the same activity as the partial peptide of the present invention (eg, ligand binding activity, cell proliferation activity, PSA (prostate cancer specific antigen) increasing action) DNA having a partial base sequence of DNA encoding a mutant AR having
Examples of DNA that can hybridize 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% with the respective base sequences. A DNA containing a base sequence having the above homology is used.

  As a means for cloning DNA that completely encodes the mutant AR of the present invention or a partial peptide thereof (hereinafter sometimes simply referred to as the mutant AR of the present invention), a partial base sequence of the mutant AR of the present invention is used. Amplified by a PCR method using a synthetic DNA primer, or a DNA that is incorporated into an appropriate vector and labeled with a DNA fragment or a synthetic DNA that encodes part or all of the mutant AR of the present invention It can be selected by hybridization. The method of hybridization can be performed according to the method described in Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989), for example. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.

The DNA base sequence is converted using a known kit such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc. The method can be carried out according to a method known per se, such as a method or a Kunkel method, or a method analogous thereto.
The cloned DNA encoding the mutant AR can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
The mutant AR expression vector of the present invention is, for example, (a) excising the target DNA fragment from the DNA encoding the mutant AR of the present invention; It can manufacture by connecting.

Examples of vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when animal cells are used as the host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Among these, it is preferable to use a CMV promoter, an SRα promoter, or the like. When the host is Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, etc. When the host is Bacillus, SPO1 promoter, SPO2 promoter and penP promoter, the host is In the case of yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ^r ), neomycin resistance gene (hereinafter sometimes abbreviated as Neo ^r, G418 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr ⁻ ) cells, the target gene can be selected even in a medium not containing thymidine.
If necessary, a signal sequence suitable for the host is added to the N terminal side of the AR of the present invention. When the host is Escherichia, the PhoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc. In some cases, MFα • signal sequence, SUC2 • signal sequence, etc. When the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used.
A transformant can be produced using the vector containing the DNA encoding the mutant AR of the present invention thus constructed.

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells and the like are used.
As a specific example of the genus Escherichia, Escherichia coli K12 / DH1 [Procedures of the National Academy of Sciences of the USA (Proc. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology. )], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. Used.
Examples of the genus Bacillus include Bacillus subtilis MI114 [Gen, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984). )] Etc. are used.
Examples of yeast include Saccharomyces cerevisiae ^{(Saccharomyces cerevisiae) AH22, AH22R -} , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe (Schizosaccharomyces pombe) NCYC1913, NCYC2036, such as Pichia pastoris (Pichia pastoris) is employed It is done.

As insect cells, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), Trichoplusia ni midgut-derived MG1 cells, Trichoplusia ni egg-derived High Five ^™ Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, sputum-derived cell lines (Bombyx mori N; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)).
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr ⁻ ) cells), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells and the like are used.

For transforming Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (Proc. Natl. Acad. Sci. USA) 1972), Gene, Vol. 17, 107 (1982), and the like.
Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979).
To transform yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Proceedings of the National Academy of Sciences of The method can be carried out according to the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
In order to transform animal cells, for example, cell engineering separate volume 8 new cell engineering experiment protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973).
In this way, a transformant transformed with an expression vector containing DNA encoding AR is obtained.
When cultivating a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, and potato extract. Examples of the inorganic or organic substance and inorganic substance include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics] 431-433, Cold Spring Harbor Laboratory, New York 1972]. In order to make the promoter work efficiently as necessary, a drug such as 3β-indolylacrylic acid can be added. When the host is an Escherichia bacterium, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
When the host is a genus Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
When cultivating a transformant whose host is yeast, examples of the medium include a Burkholder minimum medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science. USA, Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National -Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.

When culturing a transformant whose host is an insect cell or an insect, a medium such as 10% bovine serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) is added. What added the thing suitably is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)] is used. The pH is preferably about 6-8. Culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.
As described above, the mutant AR of the present invention can be generated inside or outside the transformant.

Separation and purification of the mutant AR of the present invention from the culture can be performed, for example, by the following method.
When the mutant AR of the present invention is extracted from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw. For example, a method of obtaining a crude extract of the mutant AR of the present invention by centrifugation or filtration after destroying cells or cells by, for example, 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 ^™ . When the mutant AR of the present invention is secreted into the culture solution, the cells or cells are separated from the supernatant by a method known per se after completion of the culture, and the supernatant is collected.
Purification of the AR of the present invention contained in the thus obtained culture supernatant or extract can be carried out by appropriately combining known separation / purification methods. These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as a method, isoelectric focusing method, or the like is used.

When the mutant AR of the present invention thus obtained is obtained as a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and conversely, when obtained as a salt, a method known per se Alternatively, it can be converted into a free form or other salt by a method similar thereto.
The mutant AR of the present invention produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein-modifying enzyme to act before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The activity of the mutant AR of the present invention or a salt thereof thus produced can be measured by a binding experiment with a labeled ligand and an enzyme immunoassay using the antibody of the present invention (detailed below).

The antibody against the mutant AR of the present invention may be either a polyclonal antibody or 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 according to a method for producing an antibody or antiserum known per se, using the mutant AR of the present invention as an antigen.

The antibody of the present invention is characterized by specifically recognizing the mutant AR of the present invention but not recognizing normal AR. Therefore, as an antigen to be used, a peptide comprising amino acid number 746 of SEQ ID NO: 2 and a partial amino acid sequence in the vicinity thereof and / or a peptide comprising amino acid number 882 and a partial amino acid sequence in the vicinity thereof is preferable.
Another antibody of the present invention contains the same or substantially the same amino acid sequence as the amino acid sequence (W746L (C)) in which the tryptophan of amino acid number 746 is replaced by leucine or cysteine in the amino acid sequence represented by SEQ ID NO: 2. Which specifically recognizes the protein or salt thereof, but contains the same or substantially the same amino acid sequence as the amino acid sequence (T882A) in which the threonine of amino acid number 882 is replaced by alanine in the amino acid sequence represented by SEQ ID NO: 2 It does not recognize the protein or its salt. Yet another antibody of the present invention specifically recognizes a protein containing the same or substantially the same amino acid sequence as T882A or a salt thereof, but has the same or substantially the same amino acid sequence as W746L (C). It is an antibody that does not recognize the contained protein or its salt. These antibodies can be prepared using the mutation site of mutation AR recognized by each antibody and a partial amino acid sequence in the vicinity thereof as an antigen.

[Production of monoclonal antibodies]
(A) Production of Monoclonal Antibody-Producing Cells The mutant AR of the present invention is administered to a mammal at a site where antibody production is possible by administration, or together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, 2 to 10 times in total. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer from a mouse, collect spleen or lymph nodes 2 to 5 days after the final immunization, A monoclonal antibody-producing hybridoma can be prepared by fusing the antibody-producing cells contained with myeloma cells. 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 the 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, 256, 495 (1975)). Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.
Examples of myeloma cells include NS-1, P3U1, SP2 / 0, etc., 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 PEG (preferably PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80%. The cell fusion can be efficiently carried out by incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.

Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which the antigen of the mutant AR of the present invention is adsorbed directly or together with a carrier, Next, add an anti-immunoglobulin antibody labeled with a radioactive substance or enzyme (if the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and detect the monoclonal antibody bound to the solid phase. A method in which a hybridoma culture supernatant is added to a solid phase on which an anti-immunoglobulin antibody or protein A is adsorbed, a mutant AR labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected. Is given.
Selection of the monoclonal antibody can be carried out according to a method known per se or a method analogous thereto, but can usually be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As the selection and breeding medium, any medium may be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1-10% fetal calf serum, or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.

(B) Purification of monoclonal antibody Separation and purification of monoclonal antibodies are carried out in the same manner as in the separation and purification of ordinary polyclonal antibodies (eg, salting-out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis). Method, adsorption / desorption method using ion exchanger (eg DEAE), ultracentrifugation method, gel filtration method, antigen-binding solid phase or active adsorbent such as protein A or protein G, and collecting the antibody to dissociate Specific purification method for obtaining antibody] can be performed.

[Preparation of polyclonal antibody]
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 (antigen of the mutant AR of the present invention) and a carrier protein is prepared, a mammal is immunized in the same manner as the above-described monoclonal antibody production method, and the immunized animal is immunized against the mutant AR of the present invention. It can be produced by collecting the antibody-containing material and separating and purifying the antibody.
Regarding the complex of immune antigen and carrier protein used to immunize mammals, the type of carrier protein and the mixing ratio of carrier and hapten should be such that an antibody can be efficiently produced against a hapten immunized by crosslinking to a carrier. Any ratio may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, keyhole limpet hemocyanin and the like are about 0.1 to 20 in weight ratio with respect to hapten 1. Preferably, a method of coupling at a ratio of about 1 to 5 is used.
Various coupling agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
The condensation product is administered to a warm-blooded animal by itself, together with a carrier and a 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 antibody production ability upon administration. The administration can usually be carried out about once every 2 to 6 weeks, about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc. of mammals immunized by the above method, preferably blood.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the above-described measurement of the antibody titer in the serum. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

The DNA encoding the mutant AR of the present invention, a salt thereof, a partial peptide thereof, an amide thereof, an ester thereof, or a salt thereof is (1) a preventive and / or therapeutic agent for a disease that can be improved by overexpression of the mutant AR of the present invention. , (2) a genetic diagnostic agent, (3) a screening method for a compound or its salt that changes the expression level of the mutant AR of the present invention, and (4) various diseases containing a compound that changes the expression level of the mutant AR of the present invention. (5) a method for screening a compound (agonist, antagonist, etc.) that changes the binding property between the mutant AR of the present invention and a ligand, (6) the binding property between the mutant AR of the present invention and a ligand A prophylactic and / or therapeutic agent for various diseases containing a compound (agonist or antagonist) that alters the activity, (7) the mutation of the present invention R quantification pharmaceutical comprising the antibody of the present invention (8), medicine containing (9) Antisense DNA, can be used for such preparation of (10) DNA transferred animal.
In particular, by using a receptor binding assay system using an animal cell capable of expressing the mutant AR of the present invention or an expression system of a recombinant mutant AR, the binding of a ligand to a receptor specific to humans and mammals is changed. Compounds (eg, agonists, antagonists, etc.) to be screened can be screened, and the agonists or antagonists can be used as prophylactic / therapeutic agents for 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 sometimes abbreviated as the antibody of the present invention). ) Will be specifically described below.

(1) Preventive and / or therapeutic agent for diseases that can be improved by overexpression of mutant AR of the present invention The mutant AR of the present invention is characterized in that bicalutamide or an analog thereof, or further flutamide or an analog thereof acts as an agonist. To do. In prostate cancer, it is known that the growth of AR is excessively suppressed when it is excessively stimulated. Therefore, (i) the mutant AR of the present invention or (ii) the DNA of the present invention is locally delivered to cancer cells. Therefore, it is possible to suppress the proliferation of anti-androgen-resistant cancer cells.
When the mutant AR of the present invention is used as the prophylactic / therapeutic agent, it can be formulated according to conventional means.
On the other hand, when the DNA of the present invention is used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention is used alone or after being inserted into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc. It can be administered according to means. The DNA of the present invention can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter.
For example, (i) the mutant AR of the present invention or (ii) the DNA of the present invention is orally administered as a tablet, capsule, elixir, microcapsule, etc., sugar-coated as necessary, or water or other It can be used parenterally in the form of an injectable solution such as a sterile solution with a pharmaceutically acceptable liquid or a suspension. For example, (i) the mutant AR of the present invention or (ii) the DNA of the present invention is generally recognized together with known physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. Can be prepared by mixing in the unit dosage form required for the practice of the formulation. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). .
The dose of the mutant AR of the present invention varies depending on the administration subject, target organ, symptom, administration method, etc., but in the case of oral administration, for example, generally in cancer patients (60 kg), it is about The amount is 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.
The dose of the DNA of the present invention varies depending on the administration subject, target organ, symptom, administration method, and the like, but in the case of oral administration, for example, generally about 0 per day for cancer patients (60 kg). 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

(2) Gene diagnostic agent The DNA of the present invention can be used as a probe to transform the mutant AR of the present invention in mammals (eg, humans, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Cancer cells that have acquired resistance to genetic diagnostic agents for cancer, particularly antiandrogens (eg, bicalutamide, flutamide, etc.) because the encoded DNA or mRNA (ie, abnormal normal AR gene or mRNA) can be detected It is useful as a genetic diagnostic agent for the appearance and increase of
The above gene diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processings of. The National Academy of Sciences of USA (Proceedings of the National Academy of Sciences of the United States of America), Vol. 86, 2766-2770 (1989)).

(3) Screening method for compound that changes expression level of mutant AR of the present invention The DNA of the present invention can be used as a probe for screening compounds that change the expression level of mutant AR of the present invention.
That is, the present invention provides, for example, (i) (I) blood of a non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.), (II) a specific organ, (III) A method for screening a compound that changes the expression level of the mutant AR of the present invention by measuring the amount of mRNA of the mutant AR of the present invention contained in a tissue or cell isolated from an organ, or (ii) a transformant or the like. provide.

Specifically, the amount of mRNA of the mutant AR of the present invention is measured as follows.
(I) Drugs or physics against non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) having cells expressing normal AR or mutant AR of the present invention After a certain period of time is applied, blood, or a specific organ, or a tissue or cell isolated from the organ is obtained. 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 by a conventional method and using a technique such as TaqMan PCR. It is also possible to analyze by performing Northern blotting.
(Ii) 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 method described above, and the AR of the present invention contained in the transformant or the animal cell is prepared. mRNA can be quantified and analyzed in the same manner.

Screening for compounds that change the expression level of the mutant AR of the present invention,
(I) A certain period of time (30 to 24 hours, preferably 30 minutes before applying a drug or physical stress to a non-human mammal having cells expressing a normal AR or a mutant AR of the present invention. Or after 12 hours, more preferably from 1 hour to 6 hours) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or A test compound is administered simultaneously with a drug or physical stress, and after a certain period of time has elapsed after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), cells Can be performed by quantifying and analyzing the amount of mRNA of the mutant AR of the present invention contained in
(Ii) When cultivating a transformant or an animal cell having the ability to express the mutant AR of the present invention according to a conventional method, a test compound is mixed in a medium and cultured for a certain time (after 1 to 7 days, preferably 1 day to 3 days later, more preferably 2 days to 3 days later), the amount of mRNA of the mutant AR of the present invention contained in the transformant or an animal cell capable of expressing the mutant AR of the present invention is quantified and analyzed This can be done.

The compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of the mutant AR of the present invention. Specifically, (a) the expression level of the mutant AR of the present invention. A compound that excessively enhances cell stimulating activity (for example, cell proliferation stimulation, PSA expression inducing activity, etc.) via the mutant AR of the present invention, and (b) decreases the expression level of the mutant AR of the present invention. Thus, it is a compound that attenuates the cell stimulating activity.
Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products and the like, and 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, in combination with an anti-androgen agent, and can suppress the growth of anti-androgen-resistant cancer (eg, prostate cancer). Etc.).
Since the compound that attenuates the cell stimulating activity decreases the physiological activity of the mutant AR of the present invention, it is useful as a prophylactic / therapeutic agent for androgen-independent hormone-sensitive cancer (eg, prostate cancer).

When a compound obtained by using the screening method of the present invention or a salt thereof is used as a pharmaceutical composition, it can be used in accordance with conventional means. For example, in the same manner as the medicament containing the mutant AR of the present invention described above, a tablet, a capsule, an elixir, a microcapsule, a sterile solution, a suspension, and the like can be obtained.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, 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. However, in the case of oral administration, for example, generally in cancer patients (60 kg) The amount is 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

(4) Preventive and / or therapeutic agents for various diseases containing the compound that changes the expression level of the mutant AR of the present invention The compound that changes the expression level of the mutant AR of the present invention is a hormone-dependent and hormone-independent compound. It can be used as a prophylactic / therapeutic agent for cancer (eg, prostate cancer). The compound can be formulated according to conventional means.
For example, the compound may be used as a tablet, capsule, elixir, microcapsule or the like, sugar-coated as needed, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of an injection such as a suspension. For example, mixing the compound with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in a unit dosage form as required for generally accepted formulation practice. Can be manufactured by. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The adjusted injection solution is usually filled into a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, 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. However, in the case of oral administration, for example, generally in cancer patients (60 kg) The amount is 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

(5) Screening method for compounds (agonists, antagonists, etc.) that change the binding property between the mutant AR of the present invention and a ligand Using the mutant AR of the present invention or constructing an expression system for a recombinant mutant AR, A compound (for example, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, etc.) that changes the binding between the ligand and the mutant AR of the present invention by using a receptor binding assay system using an expression system, or The salt can be screened efficiently.
Such compounds include (a) a compound having an activity of promoting cell stimulating activity (for example, cell proliferation activity, PSA expression induction activity, etc.) via the mutant AR of the present invention (so-called mutant AR of the present invention). Agonist), (b) a compound that does not have the cell stimulating activity (so-called antagonist to the mutant AR of the present invention), (c) a compound that enhances the binding force between the ligand and the mutant AR of the present invention, or (d) a ligand. And a compound that decreases the binding force between the mutant AR of the present invention and the like.
That is, the present invention compares (i) the case where the mutant AR of the present invention is contacted with a ligand and (ii) the case where the mutant AR of the present invention is contacted with a ligand and a test compound. A method for screening a compound or a salt thereof that changes the binding property between the ligand characterized by the above and the mutant AR of the present invention is provided.
In the screening method of the present invention, in the cases of (i) and (ii), for example, the binding amount of the ligand (androgen) to the AR of the present invention, cell stimulating activity (eg, cell proliferation activity, PSA expression inducing activity), etc. Are measured and compared.

More specifically, the present invention provides:
(I) The amount of labeled ligand binding to the mutant AR when the labeled ligand is brought into contact with the mutant AR of the present invention and when the labeled ligand and the test compound are brought into contact with the mutant AR of the present invention. A method for screening a compound or a salt thereof that changes the binding property between the ligand of the present invention and the mutant AR of the present invention, characterized by measuring and comparing;
(II) When the labeled ligand is brought into contact with a cell containing the mutant AR of the present invention (such as an animal cell capable of expressing the mutant AR), the labeled ligand and the test compound are transformed with the mutant AR of the present invention. The amount of binding of the labeled ligand to the cell when contacted with a cell containing a cell (such as an animal cell having the ability to express a mutant AR) and the ligand of the present invention A method for screening a compound or a salt thereof that changes the binding property to the mutant AR;
(III) When the labeled ligand is brought into contact with the mutant AR expressed by culturing the transformant containing the DNA of the present invention, and the labeled ligand and the test compound are transformed containing the DNA of the present invention. A ligand characterized by measuring and comparing the binding amount of a labeled ligand to the mutant AR when contacted with the mutant AR of the present invention expressed by culturing the body, and the mutant AR of the present invention A method for screening a compound that changes binding or a salt thereof, and (IV) a compound that activates the mutant AR of the present invention (for example, an androgen, bicalutamide, antiandrogen such as flutamide, etc.) and the like, containing the mutant AR of the present invention When the cell is contacted with a cell (such as an animal cell capable of expressing the mutant AR), and the mutation of the present invention When a compound that activates R and a test compound are contacted with a cell containing the mutant AR of the present invention (such as an animal cell capable of expressing the mutant AR), cell-stimulating activity via a receptor (for example, The present invention provides a method for screening a compound or a salt thereof that changes the binding property between a ligand and a mutant AR of the present invention, characterized by measuring and comparing cell proliferation activity, PSA expression-inducing activity, and the like.

A specific description of the screening method of the present invention will be given below.
First, the mutant AR of the present invention used in the screening method of the present invention may be any one as long as it contains the above-described mutant AR of the present invention. Animal cells having such ability are suitable.

In order to produce the mutant AR of the present invention, the above-described method is used, and it is preferable to express the DNA of the present invention in mammalian cells or insect cells. Complementary DNA is used for the DNA fragment encoding the target protein portion, but is not necessarily limited thereto. For example, gene fragments or synthetic DNA may be used. In order to introduce a DNA fragment encoding the AR of the present invention into a host animal cell and to express them efficiently, the polyhedrosis virus (nuclear polyhedrosis virus belonging to baculovirus using an insect as a host) NPV) polyhedrin promoter, SV40-derived promoter, retrovirus promoter, metallothionein promoter, human heat shock promoter, cytomegalovirus promoter, SRα promoter and the like are preferably incorporated downstream. The amount and quality of the expressed receptor can be examined by a method known per se. For example, the literature [Nambi, P. et al. Et al., The Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992].
Accordingly, in the screening method of the present invention, 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. .

In the screening method of the present invention, when cells containing the mutant AR of the present invention are used, the cells may be fixed with glutaraldehyde, 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 expresses the mutant AR, and preferred host cells include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like.
The amount of the mutant AR in the cell containing the mutant AR is preferably 10 ³ to 10 ⁸ molecules per cell, and more preferably 10 ⁵ to 10 ⁷ molecules. In addition, as the expression level increases, the ligand binding activity (specific activity) increases, and it becomes possible not only to construct a highly sensitive screening system, but also to measure a large amount of samples in the same lot.

In order to carry out the above (I) to (III) for screening a compound that changes the binding property between the ligand and the mutant AR of the present invention, for example, an appropriate mutant AR fraction and a labeled ligand are required. is there.
As 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 to that is desirable. Here, equivalent activity refers to equivalent ligand binding activity, cell stimulation activity, and the like.
As the labeled ligand, a labeled ligand (androgen), a labeled ligand analog compound and the like are used. For example, testosterone or DHT labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] or the like is used.
Specifically, in order to screen for a compound that changes the binding property between the ligand and the mutant AR of the present invention, cells containing the mutant AR of the present invention are first suspended in a buffer suitable for screening. A mutant AR preparation is prepared. The buffer may be any buffer that does not inhibit the binding between the ligand and the mutant AR, such as a phosphate buffer at pH 4 to 10 (preferably pH 6 to 8), Tris-HCl buffer, and the like. In addition, for the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ^™ (Kao-Atlas), digitonin, deoxycholate and the like can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leupeptin, E-64 (manufactured by Peptide Institute) and pepstatin can be added for the purpose of suppressing the degradation of receptors and ligands by protease. A fixed amount (5000 cpm to 500,000 cpm) of a labeled ligand is added to 0.01 ml to ¹⁰ ml of the receptor solution, and 10 ⁻⁴ M to ^{10 −10} M of a test compound is allowed to coexist at the same time. In order to know the amount of non-specific binding (NSB), a reaction tube to which a large excess of unlabeled ligand is added is also prepared. The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours. After the reaction, the solution is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and then the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or a γ-counter. When the count (B ₀ -NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B ₀ ) when there is no antagonistic substance is 100%, the specific binding amount (B-NSB) is, for example, , 50% or less of the test compound can be selected as a candidate substance capable of competitive inhibition.

Of the above method (IV) for screening for a compound that changes the binding property between the ligand and the mutant AR of the present invention, the most preferred embodiment will be described in detail later.
In general, first, cells containing the mutant AR of the present invention are cultured in a multiwell plate or the like. Before conducting screening, replace with a fresh medium or an appropriate buffer that is not toxic to the cells, add test compounds, etc., and incubate for a certain period of time. The supernatant is collected and the product produced is quantified according to the respective method. When the production of a substance used as an index of cell stimulating activity (for example, PSA) is difficult to assay with a degrading enzyme contained in cells, an assay may be performed by adding an inhibitor to the degrading enzyme.
In order to perform screening by measuring cell stimulating activity, cells expressing an appropriate mutant AR are required. As a cell expressing the mutant AR of the present invention, a mutant AR-expressing cell obtained by natural or artificial mutation, a cell line expressing the aforementioned recombinant mutant AR, and the like are desirable.
Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. It may be a known compound.

A kit for screening a compound or a salt thereof that changes the binding property between a ligand and the mutant AR of the present invention is a cell containing the mutant AR of the present invention or the mutant AR of the present invention (an animal having the ability to express the mutant AR). Cell etc.).
Examples of the screening kit of the present invention include the following.
1. Reagent for screening (1) Measurement buffer and washing buffer
Hanks' Balanced Salt Solution (Gibco) plus 0.05% bovine serum albumin (Sigma).
The solution may be sterilized by filtration through a 0.45 μm filter and stored at 4 ° C. or may be prepared at the time of use.
(2) Mutant AR preparation CHO cells expressing the mutant AR of the present invention are subcultured at 5 × 10 ⁵ cells / well in a 12-well plate and incubated at 37 ° C., 5% CO ₂ , 95% air for 2 days. What was cultured.
(3) Labeled ligand A commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S], etc. labeled DHT solution is stored at 4 ° C. or −20 ° C. for use. Dilute to 1 μM with measurement buffer.
(4) Ligand standard solution DHT is dissolved to 1 mM in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) and stored at -20 ° C.

2. Measurement Method (1) The mutant AR-expressing CHO cells of the present invention cultured on a 12-well tissue culture plate are washed twice with 1 ml of the measurement buffer, and then 490 μl of the measurement buffer is added to each well.
(2) After 5 μl of a test compound solution of 10 ⁻³ to 10 ⁻¹⁰ M is added, 5 μl of a labeled ligand is added and reacted at room temperature for 1 hour. In order to know the amount of non-specific binding, 5 μl of 10 ⁻³ M ligand is added instead of the test compound.
(3) Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled ligand bound to the cells is dissolved in 0.2N NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (Wako Pure Chemical Industries, Ltd.).
(4) The radioactivity is measured using a liquid scintillation counter (manufactured by Beckman), and the Percent Maximum Binding (PMB) is determined by the following equation [Equation 1].

[Equation 1]
PMB = [(B−NSB) / (B ₀ −NSB)] × 100
PMB: Percent Maximum Binding
B: Value when sample is added NSB: Non-specific binding (non-specific binding amount)
B ₀ : Maximum amount of binding

A compound obtained by using the screening method or screening kit of the present invention or a salt thereof is a compound having an action of changing the binding property between the ligand and the mutant AR of the present invention. Specifically, Compound having cell stimulating activity (for example, cell proliferation activity, PSA production, etc.) via AR of invention (so-called agonist for mutant AR of the present invention), (b) Compound not having cell stimulating activity (so-called present An antagonist for the mutant AR of the invention), (c) a compound that enhances the binding power between the ligand and the mutant AR of the present invention, or (d) a compound that decreases the binding power between the ligand and the mutant AR of the present invention.
Examples of the compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products and the like, and these compounds may be novel compounds or known compounds.
Since the antagonist to the mutant AR of the present invention can suppress the physiological activity of the ligand (androgen) and agonist (some antiandrogens such as bicalutamide and flutamide) for the mutant AR of the present invention, androgen-independent It is useful as a prophylactic / therapeutic agent for sex (antiandrogen-resistant) cancer, particularly prostate cancer.
A compound that decreases the binding force between the ligand and the mutant AR of the present invention is a safe and low-toxic drug (for example, androgen-independent (for example, androgen independent) ( It is useful as an anti-androgen drug resistant agent) for cancer, particularly for prostate cancer.

When the compound or salt thereof obtained using the screening method or 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, in the same manner as the medicament containing the mutant AR of the present invention described above, a tablet, a capsule, an elixir, a microcapsule, a sterile solution, a suspension, and the like can be obtained.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, 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. However, in the case of oral administration, for example, generally in cancer patients (60 kg) The amount is 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

(6) Preventive and / or therapeutic agents for various diseases containing a compound (agonist, antagonist) that changes the binding property between the mutant AR of the present invention and a ligand The antagonist for the mutant AR of the present invention is directed against the mutant AR of the present invention. Prevention of androgen-independent (anti-androgen-resistant) cancers, particularly prostate cancer, because the physiological activity of ligands (androgens) and agonists (some anti-androgens such as bicalutamide and flutamide) can be suppressed -It can be used as a therapeutic agent. When the compound is used for the above diseases, it can be formulated according to conventional means.
For example, the compound may be used as a tablet, capsule, elixir, microcapsule or the like, sugar-coated as needed, or aseptic solution with water or other pharmaceutically acceptable liquid, Alternatively, it can be used parenterally in the form of an injection such as a suspension. For example, mixing the compound with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in a unit dosage form as required for generally accepted formulation practice. Can be manufactured by. The amount of active ingredient in these preparations is such that an appropriate dose within the indicated range can be obtained.

Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like A swelling agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, a flavoring agent such as peppermint, red oil, or cherry are used. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above type of material. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice, such as dissolving or suspending active substances in vehicles such as water for injection, naturally occurring vegetable oils such as sesame oil, coconut oil and the like. As an aqueous solution for injection, for example, isotonic solutions (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants are used. For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 ^™ , HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

The preventive / therapeutic agent includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. The prepared injection solution is usually filled in a suitable ampoule.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, 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. However, in the case of oral administration, for example, generally in cancer patients (60 kg) The amount is 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

(7) Quantification of the mutant AR of the present invention The antibody of the present invention can specifically recognize the AR of the present invention, and therefore, the quantification of the mutant AR of the present invention in a test solution, particularly by sandwich immunoassay. It can be used for quantitative determination. That is, the present invention includes, for example, (i) the labeled antibody of the present invention bound to the antibody by competitively reacting the antibody of the present invention with the test solution and the labeled mutant AR of the present invention. A method for quantifying a mutant AR of the present invention in a test solution, which comprises measuring a ratio of the mutant AR;
(Ii) measuring the activity of the labeling agent on the insolubilized carrier after reacting the test solution with the antibody of the present invention insolubilized on the carrier and the labeled antibody of the present invention simultaneously or continuously. Provided is a method for quantitatively determining a mutant AR of the present invention in a test solution.

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 detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or F (ab ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. The measurement method using the antibody against the mutant AR of the present invention is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (for example, the amount of mutant AR) in the solution to be measured. Any measurement method may be used as long as it is a measurement method that detects the above by a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent material, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.

In the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), the labeled monoclonal antibody of the present invention is reacted (secondary reaction), and then the labeling agent on the insolubilized carrier The amount of the mutant AR of the present invention in the test solution can be quantified by measuring the activity of. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilizing method can be based on those described above.
In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
In the method for measuring the mutant AR of the present invention by the sandwich method of the present invention, 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. .

The monoclonal antibody of the present invention can be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry, and the like. In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the amount of labeling of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, a B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble one is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. Are reacted with an excess amount of labeled antibody, and then a solid phased 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 any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.

In applying these individual immunological measurement methods to the measurement method of the present invention, special conditions, operations, and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the variation | mutation AR of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation method in each method. For the details of these general technical means, it is possible to refer to reviews, books, etc. [For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continuing radioimmunoassay” (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Medical School, published in 1978), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Second Edition) (Medical School, 1982) Published by Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical School, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A )), Vol. 73 (Immunochemical Techniques (Part B)), ibid Vol. 74 (Immunochemical Techniques (Part C)), ibid Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid Vol. 92 ( Immunochemical Techniques (Part E: Monoclonal Antibodies and Gen eral Immunoassay Methods)), Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (published by Academic Press).
As described above, the AR of the present invention or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.
Furthermore, by quantifying the mutant AR of the present invention in vivo using the antibody of the present invention, diseases associated with the appearance / increase of the mutant AR of the present invention, such as androgen-independent (anti-androgen resistance) ) Can diagnose cancer, especially prostate cancer. Further, 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 tissue. In addition, production 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, analysis of the behavior of the mutant AR of the present invention in a test cell, etc. Can be used for.

(8) Medicament containing the antibody of the present invention The neutralizing activity for the mutant AR of the present invention possessed by the antibody against the mutant AR of the present invention is the activity to inactivate the cell stimulating activity involved in the mutant AR of the present invention. means. Therefore, when the antibody has neutralizing activity, cell stimulating activity (for example, cell proliferation activity, PSA production, etc.) involving the mutant AR can be inactivated. Therefore, it can be used for prevention and / or treatment of diseases caused by stimulation of cell proliferation of the mutant AR, for example, androgen-independent (anti-androgen-resistant) cancer, particularly prostate cancer.
The therapeutic / prophylactic agent for the above-mentioned diseases containing the antibody of the present invention can be used as a solution or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, Dogs, monkeys, etc.) orally or parenterally. The dose varies depending on the administration subject, target disease, symptom, administration route, etc. For example, when used for treatment / prevention of adult cancer patients, the antibody of the present invention is usually used as a single dose. About 0.01-20 mg / kg body weight, preferably about 0.1-10 mg / kg body weight, more preferably about 0.1-5 mg / kg body weight, about 1-5 times a day, preferably 1 It is convenient to administer about 3 times by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the administration comprises the above or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration.
That is, for example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (soft capsules). Syrups, emulsions, suspensions and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.

As a composition for parenteral administration, for example, an injection, a suppository and the like are used, and the injection is a dosage form such as an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, or an infusion. Is included. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above-described antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injections. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-described antibody or a salt thereof with a normal suppository base.
The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 to 100 mg for injections, Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.

(9) Medicament containing antisense DNA An antisense DNA capable of binding to the DNA of the present invention in a complementary manner and suppressing the expression of the DNA is a cell stimulating activity involving the mutant AR (for example, cell proliferation) Activity, PSA production, etc.) can be blocked at the translation level. Therefore, it can be used for prevention and / or treatment of diseases caused by stimulation of cell proliferation of the mutant AR, for example, androgen-independent (anti-androgen-resistant) cancer, particularly prostate cancer.
For example, when the antisense DNA is used, the antisense DNA can be carried out according to conventional means after being inserted alone or into a suitable vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, or the like. The antisense DNA can be formulated as it is or with a physiologically recognized carrier such as an adjuvant for promoting ingestion, and can be administered by a gene gun or a catheter such as a hydrogel catheter.
Furthermore, the antisense DNA can also be used as a diagnostic oligonucleotide probe for examining the presence and expression status of the DNA of the present invention in tissues and cells.

(10) DNA-transferred animal The present invention provides a non-human mammal having an exogenous DNA encoding the mutant AR of the present invention (hereinafter abbreviated as the exogenous DNA of the present invention).
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) An animal according to (2), wherein the rodent is a mouse or a rat, and (4) a recombinant vector containing the foreign DNA of the present invention and capable of being expressed in a mammal.
The non-human mammal having the exogenous DNA of the present invention (hereinafter abbreviated as the DNA-transferred animal of the present invention) is preferably used for unfertilized eggs, fertilized eggs, sperm and germ cells including primordial cells thereof. , At the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, aggregation method, microinjection method, It can be produced by transferring the target DNA by the particle gun method, DEAE-dextran method or the like. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc., and used for cell culture, tissue culture, etc. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a cell fusion method known per se.
Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like. Among them, rodents, especially mice (for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains, etc.) that are relatively short in ontogenesis and biological cycle from the viewpoint of creating a disease animal model system and that are easy to reproduce. As the system, B6C3F ₁ system, BDF ₁ system, B6D2F ₁ system, BALB / c system, ICR system, etc.) or rats (for example, Wistar, SD, etc.) are preferable.
Examples of the “mammal” in the recombinant vector that can be expressed in the mammal include humans and the like in addition to the above non-human mammals.

The exogenous DNA of the present invention refers to the DNA of the present invention once isolated and extracted from mammals, not the mutant AR gene inherently possessed by non-human mammals.
The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct bound downstream of a promoter that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, expression of DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology with the human DNA of the present invention. The DNA of the present invention is highly expressed by microinjecting a DNA construct (eg, vector, etc.) in which the human DNA of the present invention is bound downstream of various promoters into a fertilized egg of a target mammal, such as a mouse fertilized egg. DNA transfer mammals can be created.

Examples of AR expression vectors of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus and baculovirus. Etc. are used. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferably used.
Examples of promoters that regulate DNA expression include (i) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.), (ii) ) Promoters derived from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidity Protein, glutathione S-transferase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dyst Lofine, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I and IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongation factor 1α (EF-1α), β Actin, α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle Promoters such as α-actin, preproenkephalin A, and vasopressin are used. Among them, a cytomegalovirus promoter capable of being highly expressed throughout the body, a human polypeptide chain elongation factor 1α (EF-1α) promoter, a human and chicken β-actin promoter, and the like are preferable.
The vector preferably has a sequence (generally called a terminator) that terminates transcription of the target messenger RNA in a DNA-transferred mammal. For example, the sequence of each DNA derived from viruses and various mammals The SV40 terminator of simian virus or the like is preferably used.

In addition, the splicing signal of each DNA, enhancer region, a part of intron of eukaryotic DNA, etc. 5 ′ upstream of the promoter region, between the promoter region and the translation region or between the translation regions for the purpose of further expressing the target foreign DNA It is also possible to connect it 3 ′ downstream of the target.
The translation region of the mutant AR of the present invention is a cell derived from androgen-independent cancer of human or various mammals (eg, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), particularly prostate cancer, or Complement prepared as a whole or part of genomic DNA from DNA isolated from an anti-androgen-resistant cancer cell line obtained by the production method of the present invention, or from a cell (or cell line) -derived RNA by a known method DNA can be obtained as a raw material. Alternatively, a translation region of normal AR can be isolated according to a conventional method, and a translation region mutated by a point mutagenesis method can be prepared.

  The non-human mammal having the exogenous DNA of the present invention can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Furthermore, the target foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material. A DNA construct with a promoter can be prepared by a normal DNA engineering technique. The transfer of the foreign 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 embryo cell 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 that has inherited the foreign DNA of the present invention has the foreign DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA.

The non-human mammal having the exogenous DNA of the present invention has a high expression of the exogenous DNA of the present invention, and finally inhibits the function of the normal normal DNA, thereby finally inactivating the functional inactive form of normal AR. It may become refractory and can be used as a model animal for the disease state. For example, the exogenous DNA-transferred animal of the present invention can be used to elucidate the pathologic mechanism of androgen-independent (anti-androgen-resistant) cancer, particularly prostate cancer, and to examine the prevention / treatment method for this disease. Is possible.
Further, as other applicability of the DNA transfer animal of the present invention, for example,
(I) use as a cell source for tissue culture;
(Ii) It is specifically expressed or activated by the mutant AR of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing a polypeptide expressed by the DNA. Analysis of association with polypeptides,
(Iii) culturing cells of tissue having DNA by standard tissue culture techniques, and using these, studying the function of cells from tissues that are generally difficult to cultivate,
(Iv) Screening of drugs that enhance the function of cells by using the cells described in (iii) above, and (v) isolation and purification of the mutant AR of the present invention and production of antibodies thereof can be considered.
Furthermore, clinical symptoms of diseases related to AR, including functional inactive refractories of normal AR, etc. can be examined using the DNA-transferred animal of the present invention, and the disease model related to AR of the present invention More detailed pathological findings can be obtained in each organ of the present invention, and it can contribute to the development of a new treatment method and the research and treatment of secondary diseases caused by the disease.
Moreover, each organ can be taken out 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 with a proteolytic enzyme such as trypsin. . Furthermore, the protein production cells of the present invention can be identified, their relationship with apoptosis, differentiation or proliferation, or their signal transduction mechanisms can be investigated, and their abnormalities can be investigated. It becomes an effective research material for.
Furthermore, in order to develop therapeutic agents for diseases related to AR of the present invention, including the functionally inactive refractory of normal AR, using the DNA-transferred animal of the present invention, the above-described test methods and quantitative methods, etc. Can be used to provide an effective and rapid screening method for the therapeutic agent for the disease. Moreover, it is possible to examine and develop a DNA treatment method for a disease associated with the AR of the present invention using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention.

  The present invention also selects a cancer cell line in which growth is observed by culturing cancer cells sensitive to a predetermined antiandrogen agent in the presence of the antiandrogen agent, and the AR gene in the cancer cell line is selected. Provided is a method for producing the anti-androgen-resistant cancer cell line expressing mutant AR, which comprises analyzing a base sequence and selecting a strain having a mutation in the sequence.

  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 prostate Examples include cancer cells or cancer cells that express a mutant AR that is resistant to other antiandrogens. Here, “sensitivity (resistance) to a predetermined antiandrogen agent” means that it cannot proliferate (can proliferate) in the presence of the antiandrogen agent. Examples of cancer cells that express normal AR include biopsy samples obtained from androgen-dependent cancer patients and cancer cells derived from the removed prostate, and cells that express mutant AR are endocrine by other anti-androgen agents. Examples include cancer cells derived from patients whose androgen-independent cancer has relapsed after therapy, or LNCaP cell line (expressing a mutant AR of T882A).

The culture of cancer cells can be performed using a method used for normal culture of animal cells or with appropriate modifications. Examples of the medium include MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)]. , RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Procedure of the Society for the Biological Medicine (Proceeding of the Society for the Biological Medicine), Vol. 73, 1 (1950)]. Since androgen or other steroid contained in the serum may induce cell proliferation, it is preferable to use serum pretreated with dextran-coated charcoal. However, it is also preferred to use untreated serum-containing medium to select for AR mutations that allow other steroid hormones to act as agonists. The pH of the medium is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C., and aeration or agitation is added as necessary.
For example, cancer cells are added to any one of the above media containing 0.0001-100 μM anti-androgen (eg, flutamide, bicalutamide, nilutamide, etc.) so as to give 1-1,000,000 cells / ml. Cultivation is carried out in the incubator such as a plate under the above culture conditions. The presence or absence of cell proliferation is observed over time while changing the medium at appropriate intervals according to a conventional method. RNA is isolated from a clone in which cell proliferation has been observed. For example, AR cDNA is amplified and isolated using RT-PCR method using an oligonucleotide prepared based on a known normal AR cDNA sequence as a primer, and its nucleotide sequence is determined. By direct cloning or analysis after subcloning into an appropriate vector, clones that have actually mutated AR are selected.

  The mutation site of the mutant AR obtained as described above is characterized by the type of antiandrogen used as the selection pressure. For example, when bicalutamide is used, the amino acid number in the amino acid sequence represented by SEQ ID NO: 2 Mutations are concentrated on tryptophan of 746 (preferably substituted with leucine or cysteine), and when flutamide is used, mutations are concentrated on threonine of amino acid number 882 in the amino acid sequence represented by SEQ ID NO: 2 (preferably alanine) Is replaced). These mutation sites are in good agreement with the mutation sites clinically revealed from relapsed cancer for endocrine therapy with each antiandrogen.

  When cancer cells that express mutant AR (for example, LNCaP cells) are used as starting materials, multiple mutant AR-expressing cancer cell lines having mutations at other sites can be created. Therefore, the present invention selects a cancer cell line in which proliferation is observed by culturing cancer cells that express a mutant AR and are sensitive to a predetermined antiandrogen agent in the presence of the antiandrogen agent, Analyzing the base sequence of the mutant AR gene in the cancer cell line, and selecting a strain in which other mutations have occurred in the sequence, wherein the anti-androgen-resistant cancer cell line expressing the multiple mutant AR Provide production methods.

Since AR is a nuclear receptor having transcription factor activity that activates transcription of a specific target gene, agonist or antagonist activity against mutant AR can be easily assayed by analyzing expression of the target gene. . Examples of AR target genes include a gene group having a consensus sequence called an androgen response element (ARE) further upstream of the promoter, and PSA and kallikrein are typical examples.
If the cancer cell used is a prostate cancer cell line derived from a mammal, since the mutant AR-expressing cancer cell line produced by the method of the present invention has an endogenous PSA gene, the agonist or antagonist activity against the mutant AR is Measurement and evaluation can be performed using an anti-PSA antibody, PSA or a DNA encoding a part thereof.
Alternatively, a promoter that responds to androgen in the obtained cancer cell line (eg, a promoter having a native ARE equivalent sequence such as a PSA promoter, or an ARE sequence and SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter) It is also possible to evaluate the agonist or antagonist activity against mutant AR by introducing a DNA encoding a reporter protein under the control of a chimeric promoter combined with a promoter for animal cells, etc.) be able to. The PSA promoter is useful in that it well reflects the behavior of mutant AR in actual prostate cancer cells. When it is necessary to enhance the activity of the promoter, it is preferable to connect a plurality of (preferably 2 to 5, more preferably 2 or 3) promoters in tandem. As the reporter protein, luciferase, β-galactosidase, chloramphenicol acetyltransferase, peroxidase and the like can be used. The DNA encoding the reporter protein can be inserted into the animal cell expression vector described above and introduced using the gene introduction method described above.

  As described above, mutant AR-expressing cancer cells that are resistant to a given antiandrogen (eg, flutamide) are still sensitive to certain other antiandrogens (eg, bicalutamide). By culturing the mutant AR-expressing cancer cell line obtained in the presence of a test substance and examining the action against the mutant AR, the growth of relapsed cancer that has become resistant to the existing antiandrogen is suppressed. Other types of antiandrogens that can be obtained can be screened. Therefore, the present invention also antagonizes the mutant AR expressed in the cancer cell line, characterized by culturing the mutant AR-expressing cancer cell line obtained as described above in the presence of a test substance. Provided are screening methods for antiandrogen agents. Antagonism against the mutant AR can be detected using cell proliferation as an index, but can be detected and evaluated in a shorter time by analyzing the expression of a gene that is transcriptionally activated by the mutant AR. Examples of the gene used include a promoter that responds to the PSA gene and androgen as described above (eg, a promoter having a native ARE equivalent sequence such as a PSA promoter, or an ARE sequence and SRα promoter, SV40 promoter, LTR promoter, CMV promoter, A reporter gene under the control of a chimeric promoter combined with a promoter for animal cells such as HSV-TK promoter.

  The present invention also provides an anti-androgen that shows an antagonistic action against the mutant AR selected by the above method. For example, W746L (C) + T882A, which is the multiple mutation AR of the present invention, is resistant to both bicalutamide and flutamide, but Compound A shown in the Examples below shows that such an existing antiandrogenic agent is It also exhibits antagonistic activity against invalid mutant ARs.

  When a known antiandrogen agent (for example, bicalutamide, flutamide, etc.) is used in the method for producing a mutant AR-expressing cancer cell line of the present invention, an antiandrogen-resistant strain that expresses the mutant AR in about 3 months at the latest is established. Can be done. However, depending on the type of compound, it is predicted that cell growth will not be induced even if the culture is continued for a longer period of time. Such compounds are useful as resistant and non-inducing or non-inducing antiandrogens. Therefore, the present invention also provides a resistant cancer characterized by culturing androgen-sensitive cancer cells in the presence of a test substance and examining the presence or absence of a cancer cell line capable of growing under the conditions over time. Provided are a method for screening a non-inducible or difficult-to-inducible antiandrogen agent, and a resistant cancer non-inducible or difficult-to-inducible antiandrogen selected by the method.

The present invention provides a prophylactic / therapeutic agent for hormone-sensitive cancer, preferably prostate cancer, comprising an antiandrogen that shows an antagonistic action against the mutant AR obtained by the screening method described above. Such anti-androgen agents show androgen and antagonism against both normal and mutant AR, and thus can suppress the growth of cancer cells in both the androgen-dependent phase and the androgen-independent phase.
The present invention also provides a preventive / therapeutic agent for hormone-sensitive cancer, preferably prostate cancer, comprising an anti-androgen that is non-inducible or difficult to induce resistant cancer obtained by the screening method described above. Such antiandrogens do not cause mutations in the AR that confer resistance to the drugs, or are significantly less likely to cause changes than other antiandrogens, thus delaying the transition to androgen-independent cancer. Useful for.

The prophylactic / therapeutic agent can be produced and used according to conventional means. For example, in the same manner as the medicament containing the mutant AR of the present invention described above, a tablet, a capsule, an elixir, a microcapsule, a sterile solution, a suspension, and the like can be obtained.
Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, to mammals (eg, humans, rats, rabbits, sheep, pigs, cows, 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. However, in the case of oral administration, for example, generally in cancer patients (60 kg) The amount is 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01-30 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

The present invention also provides a cocktail therapy agent in which two or more antiandrogens that exhibit antiandrogenic activity against different mutant ARs are combined. There have been many reports on AR mutations so far, but there are very few reports of mutant ARs having two or more amino acid mutations. Therefore, if two or more antiandrogenic agents (eg, bicalutamide and flutamide) exhibiting antiandrogenic activity against different mutant ARs (eg, W746L (C) and T882A) are used in combination, cancers with either mutation Even if the cells appear, they cannot be proliferated because they are sensitive to any antiandrogen, and the transition to androgen independence may be delayed for extended periods.
As a cocktail therapy agent, each antiandrogen agent may be formulated according to the above-mentioned method, and these may be administered simultaneously, or two or more types of antiandrogen agents may be blended in one formulation.
The dose of the antiandrogen agent varies depending on the administration subject, target organ, symptom, administration method, and the like, but in the case of oral administration, for example, in general, for cancer patients (60 kg), about 0 per day. 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptom, administration method, etc. For example, in the form of an injection, for example, in cancer patients (as 60 kg), It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.

  In carrying out the cocktail therapy as described above, it is important to know what mutation AR is likely to be induced by various antiandrogens. 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, the mutant AR obtained by carrying out the method for various antiandrogens is analyzed to identify mutation sites that are likely to be induced by the antiandrogen, resulting in resistant cancer cell lines that express the same mutant AR. An anti-androgen agent (eg, flutamide, nilutamide, etc.) to be treated is classified into one group from a group consisting of an anti-androgen agent (eg: bicalutamide, etc.) that gives rise to a resistant cancer cell line expressing another mutant AR. An effective cocktail therapy can be performed by combining two or more arbitrary antiandrogens classified into different groups by such a classification method.

  The information obtained by the above-described method for classifying anti-androgens is that other anti-androgens were administered continuously until one anti-androgen was developed until resistant cancer appeared, and then other anti-androgens classified into groups different from the anti-androgens. It is also useful for carrying out sequential endocrine therapy that inhibits the growth of resistant cancers that appear after administration of drugs.

  The present invention is also characterized in that AR and a predetermined antiandrogen agent are contacted with a mammalian cell containing a gene capable of expression analysis under the control of a PSA promoter, and the expression of the gene is analyzed. A method for evaluating the response of the anti-androgen to the transcription factor activity is provided.

  As described above, the PSA promoter is useful in that it well reflects the behavior of AR in actual prostate cancer cells. Here, “PSA promoter” means at least the ARE equivalent sequence upstream of the essential promoter region such as the TATA box (AGAACAGCAAGTGCT in the case of the human PSA gene; SEQ ID NO: 3) in the 5 ′ upstream sequence of the prostate specific antigen gene. A 35 base pair portion known as an upstream androgen response region (ARR) [in the case of the human PSA gene, GTGGTGCAGGGATCAGGGAGTCTCACAATCTCCTG; SEQ ID NO: 4 (J. Biol. Chem., 271 (11): 6379 -6388, 1996)], more preferably a region containing about 600 base pairs upstream of the transcription start point. When it is necessary to enhance the activity of the PSA promoter, it is preferable to connect a plurality of (preferably 2 to 5, more preferably 2 or 3) sequences in tandem with the above region as a unit.

As the “gene capable of expression analysis”, PSA, luciferase, β-galactosidase, chloramphenicol acetyltransferase, as described above in the screening of antiandrogen using the mutant AR-expressing cancer cell line of the present invention, Examples thereof include DNA encoding a reporter protein such as peroxidase. These DNAs are inserted into the above-described animal cell expression vector (but lacking the promoter) together with the PSA promoter, and mammalian cells (eg, COS-7, Vero, CHO, CHO) using the above-described gene transfer method. (Dhfr ⁻ ), mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc.). If the gene whose expression can be analyzed is the PSA gene, the PSA gene is isolated from the genomic DNA of prostate cells in a form containing the PSA promoter using a conventional method, and this is used in an animal cell expression vector (however, the promoter is lacking) You may insert in and use. Alternatively, 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 a PSA promoter and capable of expression analysis”.

  The AR applied to the evaluation method of the present invention may be any, normal AR derived from various mammals, mutant AR derived from androgen-independent relapse cancer, derived from a cancer cell line obtained by the production method of the present invention And a recombinant AR obtained by genetic engineering techniques. These ARs can be produced by isolation and purification using a known method for purifying receptor proteins. For example, after homogenizing a mammalian cell, a resected cancer lesion, the cancer cell line of the present invention, an AR-producing host cell, etc., extraction with an acid is performed, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, etc. It can be purified and isolated by combining chromatography. In the case of recombinant AR, AR can be rapidly isolated and purified by affinity chromatography by adding a sequence encoding a tag such as a 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 whose expression can be analyzed under the control of the PSA promoter. It is preferable that it is expressed. Examples of such mammalian cells include host mammalian cells (eg, COS-7, Vero, CHO, CHO (dhfr ⁻ )), genes that can be analyzed for expression under the control of the PSA promoter and genes encoding AR. Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc.), normal mammalian prostate cells, and mutant AR-expressing cancer cell lines established from androgen-independent relapse cancer (Example: LNCaP cell line) or a transformed cell obtained by introducing a gene capable of expression analysis under the control of the PSA promoter into a mutant AR-expressing cancer cell line obtained by the production method of the present invention. In the former case, the gene encoding AR may be inserted into the animal cell expression vector and introduced into mammalian cells using the gene introduction method described above.

  As the “predetermined antiandrogen agent”, any substance (eg, bicalutamide, flutamide, etc.) exhibiting an antagonistic action against known normal or mutant AR can be used. “Antiandrogen responsiveness” means how the transcription factor activity of a test AR is affected by a predetermined antiandrogen. For example, a mammalian cell containing a gene capable of expression analysis under the control of the PSA promoter may be transformed into AR (T882A) derived from the LNCaP-FGC cell line (ATCC number: CRL-1740) or the mutant AR of the present invention (W746L or When W746L (C) + T882A) is contacted with bicalutamide, the former suppresses the expression of the gene, whereas the latter enhances the expression. That is, it is determined that T882A is sensitive to bicalutamide, whereas W746L and W746L (C) + T882A are resistant to bicalutamide.

  In the above method, the AR modulator can be screened by fixing AR to a predetermined substance and using various test substances instead of the predetermined antiandrogen. “AR modulator” means a substance that can positively or negatively regulate the transcription factor activity of AR, and includes both a substance having an agonist activity and a substance having an antagonist activity (an antiandrogenic agent) for AR. To do. Examples of the test substance include known or novel peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. When a predetermined AR and a test substance are brought into contact with a mammalian cell containing a gene whose expression can be analyzed under the control of a PSA promoter and the expression of the gene is analyzed, If the expression is suppressed, the test substance can be determined to be a negative modulator of the AR (that is, an antiandrogen).

The present invention also provides a kit suitable for use in the method for evaluating the anti-androgen responsiveness of the AR transcription factor activity or the method for screening an AR modulator. The kit includes as a component mammalian cells containing a gene capable of expression analysis under the control of the PSA promoter. As the mammalian cell, DNA encoding a reporter protein such as PSA, luciferase, β-galactosidase, chloramphenicol acetyltransferase, peroxidase, etc. is operably linked downstream of the PSA promoter, and is used for the above animal cell expression. Mammalian cells (eg COS-7, Vero, CHO, CHO (dhfr ⁻ ), mouse L cells, mouse AtT-20, inserted into a vector (but lacking a promoter) and using the above-described gene transfer method Mouse myeloma cells, rat GH3, human FL cells, etc.). When the gene whose expression can be analyzed is the PSA gene, the PSA gene is isolated from the genomic DNA of prostate cells in a form containing the PSA promoter using a conventional method, and this is used in an animal cell expression vector (however, it lacks the promoter). And inserted into mammalian cells in the same manner.

  In particular, when the kit is used for screening for a modulator of a predetermined AR, the mammalian cell may further contain an expression vector containing a DNA encoding the AR. The DNA encoding AR may be inserted into the animal cell expression vector described above and introduced into mammalian cells using the gene introduction method described above.

In the present specification and drawings, bases, amino acids, and the like are represented by abbreviations based on abbreviations by IUPAC-IUB Commission on Biochemical Nomenclature or conventional abbreviations in the field, examples of which are described below. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphate dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate

Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine Cys: Cysteine Met: Methionine Glu: Glutamic acid Asp: Aspartic acid Lys Argin P : Tryptophan Pro: proline Asn: asparagine Gln: glutamine pGlu: pyroglutamic acid Me: methyl group Et: ethyl group Bu: butyl group Ph: phenyl group TC: thiazolidine-4 (R) -carboxamide group

In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Tos: p-toluenesulfonyl CHO: formyl Bzl: benzyl Cl ₂ Bzl: 2,6-dichlorobenzyl Bom: benzyloxymethyl Z: benzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z: 2-bromobenzyl Oxycarbonyl Boc: t-butoxycarbonyl DNP: dinitrophenol Trt: trityl Bum: t-butoxymethyl Fmoc: N-9-fluorenylmethoxycarbonyl HOBt: 1-hydroxybenztriazole HOOBt: 3,4-dihydro-3-hydroxy -4-oxo-
1,2,3-benzotriazine HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
The nucleotide sequence of wild type human AR cDNA is shown.
[SEQ ID NO: 2]
The amino acid sequence of wild type human AR is shown.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but these do not limit the scope of the present invention.

Example 1 Method for establishing LNCaP-cxD cell line
First culture of LNCaP-FGC (ATCC number: CRL-1740) in culture medium (RPMI1640 + 10% Dextran Charcoal (DCC) -Fetal Bovine Serum (FBS)) containing 0.1 and 1 μM bicalutamide (trade name: Casodex) Does not multiply. However, when the culture was continued for 6 to 13 weeks or more, two cell lines showing proliferation were obtained. These cells were named LNCaP-cxD11 and LNCaP-cxD2, respectively.

Example 2 Bicalutamide response of LNCaP-cxD cell line
LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC were seeded in a 24-well plate at 40000 cells / mL / well, 0.1-30 μM bicalutamide was added the next day, 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.
As a result, the growth of LNCaP-cxD11 and LNCaP-cxD2 was significantly promoted by bicalutamide [FIG. 1]. On the other hand, in the parent strain LNCaP-FGC, proliferation was significantly suppressed by bicalutamide [FIG. 1]. PSA production was also significantly enhanced by bicalutamide in LNCaP-cxD11 and LNCaP-cxD2, and significantly suppressed by bicalutamide in the parent strain [FIG. 2].

Example 3 Identification of Mutant AR
Total RNA was extracted from LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC and converted into cDNA, and the base sequence of the AR gene was analyzed by PCR direct sequencing. For LNCaP-cxD11, only the part of the androgen binding region of the AR gene was sequenced.
As a result, each of the ARs of LNCaP-cxD11 and LNCaP-cxD2 contained one gene sequence mutation accompanied by an amino acid mutation in the androgen binding region. TGG (tryptophan) of codon 746 (usually using the notation of 919 as the total number of AR codons, but corresponding to codon 741) is LNCaP-cxD11 and LNCaP-cxD2, and TTG (leucine) and TGT (cysteine), respectively. It was mutated to.

Example 4 Transcriptional promotion activity of mutant AR by bicalutamide
Cos-7 was seeded in 5,000,000 cells in a 150 cm ² flask, cultured in a culture solution (DMEM + 10% Dextran Charcoal (DCC) -Fetal Bovine Serum (FBS) +2 mM glutamine) for 24 hours, and then wild type AR or mutant AR ( Vector DNA into which W741C and W741L mutations) were inserted and vector DNA in which a luciferase gene was bound downstream of an androgen-responsive promoter were cotransfected by the liposome method. After 2 hours, the medium was changed, and after 3 hours of cultivation, 0.01 or 1 μM bicalutamide was added. After further incubation for 24 hours, the luciferase activity was measured, and the transcriptional activity of AR by bicalutamide was examined.
As a result, bicalutamide showed almost no transcriptional activity against the wild type, but bicalutamide showed transcription promoting activity against the W741C and W741L type mutant AR [FIG. 3].

Example 5 Screening of compounds showing antagonistic activity against W741C type mutant AR
A compound that inhibits the transcriptional activity of W741C mutant AR by DHT (dihydrotestosterone), that is, a compound that exhibits antagonistic activity against W741C mutant AR was searched for by a cotransfection system similar to the assay system described in Example 4. As a result, Compound A [ethyl 2,5-dimethyl-4- (4-nitrophenyl) -1H-pyrrole-3-carboxylate (manufactured by Labo Test)] was found [FIG. 4].

Reference Example 1 Construction of an expression vector containing a luciferase gene under the control of the PSA promoter
After extracting genomic DNA from LNCaP-FGC cells according to a conventional method, this was used as a template, and 5′-GGAGCTCGAATTCCACATTGTTTGCTGCACGTTGG-3 ′ (SEQ ID NO: 5) and 5′-CAAGCTTTGGGGCTGGGGAGCCTCCCCCAGGAGC-3 ′ (SEQ ID NO: 6) were used as primers. PCR reaction was performed. Using Pyrobest (TaKaRa) as a DNA polymerase, heat it at 94 ° C for 1 minute with Gene Amp PCR System 9700 (Applied Biosystems), then perform PCR reaction for 25 minutes at 98 ° C for 5 seconds, 59 ° C for 30 seconds, and 72 ° C for 1 minute. Cycling was performed to obtain an approximately 650 bp DNA fragment containing the PSA (prostate specific antigen) promoter. This fragment was digested with restriction enzymes Hind III (TaKaRa) and Sac I (TaKaRa) and then subjected to agarose gel electrophoresis to recover the DNA fragment. The DNA fragment was mixed with pGL3-Basic vector (Promega) digested with Hind III and Sac I, ligated using Ligation high (TOYOBO), and transformed into E. coli DH5α competent cells. A vector pGL3-PSA-Luc in which a luciferase gene was linked downstream of the PSA promoter was obtained. Further, pGL3-PSA-Luc was cleaved with Hind III, smoothed with a Blunting kit (TaKaRa), and then cleaved with Kpn I (TaKaRa) to recover a fragment containing the PSA promoter. The DNA fragment was cleaved with Sac I, blunted with a Blunting kit, ligated to pGL3-PSA-Luc cleaved with Kpn I, and transformed into E. coli DH5α competent cells. The connected pGL3-2PSA-Luc was obtained.

Example 6 Reporter Assay System Using PSA Promoter
Cos-7 was seeded in 5,000,000 cells in a 150 cm ² flask, cultured in a culture solution (DMEM + 10% Dextran Charcoal (DCC) -Fetal Bovine Serum (FBS) +2 mM glutamine) for 24 hours, and obtained in Reference Example 1 above. pGL3-PSA-Luc or pGL3-2PSA-Luc and vector DNA into which wild type AR had been inserted were co-transfected using SuperFect (Qiagen). After 2 hours, the medium was changed. After 3 hours of culture, 1 μM DHT (5α-dihydrotestosterone) was added, and after 24 hours of culture, luciferase activity was measured to examine transcriptional activity. The result is shown in FIG.
As is clear from FIG. 5, it was found that the effect of DHT on the transcriptional activity of AR can be measured using the PSA promoter. Furthermore, it was found that a stronger activity can be obtained by connecting the PSA promoter in tandem.

SEQ ID NO 3: ARE sequence in human PSA promoter.
SEQ ID NO: 4: ARR sequence in human PSA 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.

It is a figure which shows the effect of bicalutamide on the proliferation of LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC cells. LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC cells suspended in a medium (RPMI1640 + 10% DCC-FBS) were seeded in a 24-well plate at 40000 cells / plate, and bicalutamide was added the next day. Cells were counted 3 days after the addition of bicalutamide. Mean ± standard error, n = 3. It is a figure which shows the effect of bicalutamide on PSA production of LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC cells. LNCaP-cxD11, LNCaP-cxD2 and LNCaP-FGC cells suspended in a medium (RPMI1640 + 10% DCC-FBS) were seeded in a 24-well plate at 40000 cells / plate, and bicalutamide was added the next day. The supernatant PSA concentration 3 days after the addition of bicalutamide was measured. Mean ± standard error, n = 3. It is a figure which shows the transcription-promoting activity of wild type or mutant type (W741C, W741L) AR by bicalutamide. Vector DNA into which various ARs were inserted and vector DNA in which a luciferase gene was bound downstream of an androgen-responsive promoter were cotransfected, and luciferase activity was measured after addition of 0.01 or 1 μM bicalutamide. Mean ± standard error, n = 4. It is a figure which shows the antagonistic effect of the compound A with respect to W741C type | mold mutation AR. Co-transfect the vector DNA inserted with the W741C mutation AR and the vector DNA with the luciferase gene linked downstream of the androgen-responsive promoter, and add 40 nM DHT or 40 nM DHT and 10 μM Compound A Luciferase activity was measured. Mean ± standard error, n = 16 in DHT alone group, n = 4 in DHT + compound A group. It is a figure which shows gene transcription activation under the PSA promoter control by wild type AR activated with DHT. Co-transfect wild-type AR-inserted vector DNA with one PSA promoter or vector DNA with luciferase gene bound downstream of two PSA promoters linked to tandem, and add 1 μM DHT Later luciferase activity was measured. Mean ± standard error, n = 6.

Claims (30)

A cancer cell line in which cancer cells sensitive to a predetermined antiandrogen agent are cultured in the presence of the antiandrogen agent and proliferated is selected, and the base sequence of the androgen receptor gene in the cancer cell line is selected. A method for producing the anti-androgen-resistant cancer cell line that expresses a mutant androgen receptor, which comprises analyzing and selecting a strain in which the mutation has occurred in the sequence. The method according to claim 1, wherein the mutation site coincides with a clinical mutation site which appears by administration of the antiandrogen agent. A cancer cell line expressing a mutant androgen receptor and sensitive to a predetermined anti-androgen agent is cultured in the presence of the anti-androgen agent to select a cancer cell line, and the cancer cell line is selected. An anti-androgen-resistant cancer cell line expressing a multi-mutant androgen receptor, characterized by analyzing the nucleotide sequence of the mutant androgen receptor gene in Production method. The method according to any one of claims 1 to 3, wherein the antiandrogen is flutamide or an analog thereof or bicalutamide or an analog thereof. A cancer cell line expressing a mutant androgen receptor obtained by the method according to claim 1. A cancer cell line expressing a multiple mutant androgen receptor obtained by the method according to claim 3. The cancer cell line according to claim 5 or 6, further comprising a gene capable of expression analysis under the control of a promoter responsive to androgen. The cancer cell line according to claim 7, wherein the gene is a prostate-specific antigen gene or a foreign reporter gene. A method for screening an anti-androgen agent exhibiting an antagonistic action against a mutant androgen receptor expressed in the cancer cell line, wherein the cancer cell line according to claim 5 or 6 is cultured in the presence of a test substance. An expression in the cancer cell line characterized by culturing the cancer cell line according to claim 7 in the presence of a test substance and analyzing the expression of a gene under the control of an androgen-responsive promoter in the cancer cell. A method for screening an anti-androgen agent exhibiting an antagonistic action against a mutant androgen receptor. The antiandrogen which shows an antagonistic action with respect to the mutant androgen receptor selected by the method of Claim 9 or 10. Non-resistant or inducible resistant cancer, characterized by culturing androgen-sensitive cancer cells in the presence of a test substance and examining for the appearance of cancer cell lines that can grow under the conditions over time Screening method for antiandrogens. A resistant cancer non-inducing or non-inducing anti-androgen selected by the method according to claim 12. The antiandrogen agent according to claim 13, which is non-mutation-inducible or difficult-to-induce androgen receptor. A prophylactic / therapeutic agent for hormone-sensitive cancer in the androgen-dependent or androgen-independent phase, comprising the anti-androgen agent according to claim 11 or 13. A prophylactic / therapeutic agent for hormone-sensitive cancers in androgen-dependent and androgen-independent phases, comprising a combination of two or more antiandrogens that exhibit antiandrogenic activity against different mutant androgen receptors. The agent of claim 16, wherein one of the two or more antiandrogenic agents is selected by the method of claim 9 or 10. The anti-androgen agent capable of producing a resistant cancer cell line expressing the same mutant androgen receptor by the method according to claim 1 is used as a group to produce a resistant cancer cell line expressing another mutant androgen receptor. A method for classifying an antiandrogen agent, characterized in that the method is classified from the group consisting of antiandrogen agents that can be prepared. A prophylactic / therapeutic agent for hormone-sensitive cancer in androgen-dependent phase and androgen-independent phase, comprising a combination of two or more antiandrogen agents classified into different groups by the method according to claim 18. The cancer cell line according to claim 7 or 8, wherein the promoter responsive to androgen is a PSA promoter. The screening method according to claim 10, wherein the promoter responsive to androgen is a PSA promoter. A kit for evaluating antiandrogen response of an androgen receptor transcription factor activity or screening for an androgen receptor modulator, comprising as a constituent a mammalian cell containing a gene under the control of a PSA promoter and capable of expression analysis. The kit according to claim 22, wherein the mammalian cell further expresses a predetermined androgen receptor. The kit according to claim 22, wherein a plurality of PSA promoters are linked in tandem. The kit according to claim 22, wherein the gene whose expression can be analyzed is a prostate-specific antigen gene or a foreign reporter gene. An androgen receptor and a predetermined antiandrogen agent are contacted with a mammalian cell containing a gene capable of analyzing expression under the control of a PSA promoter, and the expression of the gene is analyzed. A method for evaluating the response of the antiandrogen to transcription factor activity. A modulator of the androgen receptor, characterized by contacting a predetermined androgen receptor and a test substance with a mammalian cell containing a gene capable of analyzing expression under the control of a PSA promoter, and analyzing the expression of the gene Screening method. 28. The method of claim 26 or 27, wherein the androgen receptor contact is made by mammalian cells expressing the receptor. 28. The method according to claim 26 or 27, wherein a plurality of PSA promoters are linked in tandem. 28. The method according to claim 26 or 27, wherein the gene whose expression can be analyzed is a prostate-specific antigen gene or a foreign reporter gene.

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