JP5292608B2 - Method for screening active ingredient of adult T cell leukemia / lymphoma onset inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening a sideration inhibitor of adult T cell leukemia/lymphoma (ATLL) having an effect for delaying or preventing the sideration of ALTT by clearing the sideration mechanism of ATLL and using the inhibition of the sideration mechanism of ATLL, and the sideration inhibitor of ATLL based on the sideration mechanism of ATLL. <P>SOLUTION: The screening method can be executed by using the bonding inhibition of Tax and the CBD region of Cdc37 as an index. The sideration inhibitor of ATLL contains a substance, which has action for inhibiting the bonding of Tax and the CBD region of CDc37, as an effective component. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

The present invention provides new knowledge about the onset mechanism of adult T-cell leukemia / lymphoma (hereinafter also simply referred to as “ATLL”). The present invention also relates to a method for screening an active ingredient of a drug that suppresses the onset of ATLL ( ATLL onset inhibitor) based on such findings.

  Adult T-cell leukemia / lymphoma (ATLL) is a very high-grade leukemia / lymphoma that develops in some infected individuals with human T-cell leukemia virus (HTLV-1), a type of retrovirus. There are about 20 million HTLV-1 carriers worldwide, and it has been epidemiologically revealed that about 5% of them develop ATLL (Non-Patent Documents 1 to 3). In Japan, there are many in Kyushu and Shikoku, the number of carriers nationwide is said to be about 1 million, and the incidence of ATLL is said to be about 700 cases a year (Non-patent Document 4). The incubation period is as long as 40 to 60 years, and it is difficult to predict the onset. In addition, the prognosis of those with onset is poor, and at present, infection prevention is considered the best way to prevent ATLL.

  The onset of ATLL is caused by Tax, which is an oncogene product of HTLV-1 (Non-patent Document 5). Tax activates transcription factors such as NF-κB and AP-1 that are involved in cell growth and apoptosis suppression, and can inhibit tumor suppressor gene products such as p53, Rb, and Mad1, DNA repair, and chromosome distribution check function. Known (see FIG. 1). Among them, activation of NF-κB is essential for immortalization of HTLV-1-infected T cells in the early stage of ATLL development. Accumulation of gene mutations in host chromosomal DNA and abnormal chromosome distribution due to activation of NF-κB It is known that this will eventually lead to the development of ATLL. Therefore, it is possible to prevent the onset and delay of ATLL by suppressing NF-κB activation (NF-κB enhancement) induced by Tax early after HTLV-1 infection. (Non-Patent Documents 6 and 7).

  NF-κB is a transcription factor involved in immune responses. In vivo, the expression is usually strictly controlled, but it is subject to stress-induced external stimuli such as inflammatory cytokines, bacterial and viral infections (including malignant oncogenic viruses), ultraviolet rays, and diesel powder particles. It is activated and translocated into the nucleus, and induces the expression of the gene under its control, resulting in a biological defense mechanism that avoids immune response and cell death (Non-Patent Documents 8 to 10). These reactions are usually transient, but some special stimuli caused by chemicals such as viruses, UV rays, and allergens cause constitutive or excessive activation (enhancement) of NF-κB, and allergies This is one of the causes for inducing diseases and malignant tumors (see FIG. 2).

  Currently, the treatment of ATLL is mainly multi-drug chemotherapy mLSG15, which is an improved version of CHOP therapy using mainly DNA synthesis inhibitors and cytoskeletal polymerization inhibitors. However, since this multi-drug combination chemotherapy is based on cytotoxicity (tumor killing cell activity), there is a problem that the burden on the patient due to side effects is large and the QOL of the patient is significantly reduced. Furthermore, even if mLSG15 is used, the 2-year survival rate of patients is less than 30% (Non-patent Documents 11 and 12). Under these circumstances, the activity of molecular target drugs that have a different mechanism of action from existing anti-tumor drugs and are less burdensome on patients, such as the Bcr-Abl chimeric protein that is the cause of the development of chronic myeloma in recent cases, Antitumor drugs such as Imatinib (trade name Gleevec, manufactured by Novartis) that are specifically suppressed are expected to appear.

The activation of NF-κB induced by the inflammatory cytokine TNF-α is due to the molecular chaperone Hsp90 inhibitor geldanamycin (GA) inhibiting the function of NF-κB-activating kinase IKK. It is reported to be suppressed (Non-patent Document 13). Recently, 17-AAG (17- (Allylamino) -17-demethoxygeldanamycin), a derivative of geldanamycin, which is also an Hsp90 inhibitor, has been reported to suppress the growth of ATL cells. Usefulness has been suggested (Non-Patent Document 14).
Takatsuki, K. (1995) “Adult T-cell leukemia.” Intern Med. 34 (10), 947-952. Tajima Kazuo (2004) Epidemiology of adult T-cell leukemia / lymphoma, Integrated Clinical, 53 (7), 2038-2047. Proietti, FA, Carneiro-Proietti, AB, Catalan-Soares, BC, Murphy, EL (2005) “Global epidemiology of HTLV-I infection and associated diseases.” Oncogene. 24 (39): 6058-68. Shigeo Hino (2002) Adult T-cell leukemia, IDWR, 4. Jeang, KT (2001) “Functional activities of the human T-cell leukemia virus type I Tax oncoprotein: cellular signaling through NF-κB.” Cytokine Growth Factor Rev. 12 (2-3), 207-217. Iha, H., Kasai, T., Kibler, KV, Iwanaga, Y., Tsurugi, K., Jeang, KT (2001) “Pleiotropic effects of HTLV type 1 Tax protein on cellular metabolism: mitotic checkpoint abrogation and NF-κB activation. ”AIDS Res Hum Retroviruses. 16 (16), 1633-1638. Iha, H., Kibler, KV, Yedavalli, VR, Peloponese, JM, Haller, K., Miyazato, A., Kasai, T., Jeang, KT (2003) “Segregation of NF-kB activation through NEMO / IKKg by Tax and TNF-a: implications for stimulus-specific interruption of oncogenic signaling. ”Oncogene. 22 (55): 8912-8923. Karin, M., Cao, Y., Greten, FR, Li, ZW (2002) “NF-κB in cancer: from innocent bystander to major culprit.” Nat. Rev. Cancer. 2 (4), 301-310. Li, Q., Verma, IM (2002) “NF-κB regulation in the immune system.” Nat. Rev. Immunol. 2 (10), 725-734. Karin, M., Greten, FR (2005) “NF-κB: linking inflammation and immunity to cancer development and progression.” Nat. Rev. Immunol. 5 (10), 749-759. Kazunari Yamaguchi (2004) ATL / L multi-drug chemotherapy, combined clinical, 53 (7), 2137-2143. Taguchi Hirokuni (2007) ATL chemotherapy, HTLV-1 and disease, Watanabe, Kamihira, Yamaguchi, Bunkodo 42-53. Chen, G., Cao, P., Goeddel, DV (2002) “TNF-induced recruitment and activation of the IKK complex require Cdc37 and Hsp90.” Mol. Cell 9 (2): 401-410. H. Kawakami, et al., (2007) “Inhibition of heat shock protein-90 modulates multiple functions required for survival of human T-cell leukemia virus type I-infected T-cell lines and adult T-cell leukemia cells.” Int J. Cancer: 120, 1811-1820 (2007)

The present invention, adult T-cell leukemia / to lymphoma clarify pathogenesis of (ATLL), the active ingredient of the inhibition of the onset mechanism as an index, ATLL suppressive agent with or prevent the effect delaying the ATLL onset It is an object to provide a method for screening.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies, and (1) Hsp90, which is a target of a geldanamycin derivative (17-AAG) that has been suggested to be effective against ATLL. Forms a complex with the cofactor (cochaperone) Cdc37 (Hsp90 / Cdc37 complex), and Tax stabilizes by binding to the complex. (2) However, Tax stabilization is Rather than by binding to the cofactor Cdc37, more specifically, Tax binds directly to the CBD37 CBD region (181-200 region) through its molecular interaction and is thereby stabilized. I found. In addition, the present inventors (3) inhibits the binding of Tax to the Cdc37 / CBD region, thereby making Tax unstable and promoting its degradation, and (4) NF accompanying the degradation of Tax. (5) 17-DMAG (17- (dimethylaminoethylamino) -17-demethoxygeldanamycin) (references 1 to 3) is a human cultured cell into which a Tax expression vector has been introduced. Reduce NF-κB activation and other canceration signals (HTLV-1-LTR transcriptional activation that promotes HTLV-1 replication), inhibit proliferation of ATL patient-derived cells and induce apoptosis, and It was confirmed that oral administration to an ATL-onset model mouse (Reference Document 4) also exerts an inhibitory effect on multiorgan invasion of ATL cells (Experimental Example 6).

  These findings indicate that a substance that has an effect of inhibiting the binding of Cdc37 to Cdc37, particularly the binding of Cdc37 to the CBD region (181-200 region) (Csd / CBD region), destabilizes and degrades Tax. It suppresses the activation of NF-κB and, as a result, suppresses the onset of ATLL, which means that it is an active ingredient of a drug that delays or prevents the onset of ATLL (ATLL onset inhibitor) .

  The present invention has been completed based on such knowledge, and has the following configuration.

(I) Method for screening active ingredient of ATLL onset inhibitor (I-1) Method for screening active ingredient of ATLL onset inhibitor characterized by using binding inhibition of Tax and Cdc37 / CBD region as an index .
(I-2) A method for screening an active ingredient of an ATLL onset inhibitor comprising the following steps (a) to (d):
(A) contacting a polypeptide containing at least 181 to 200 of the amino acid sequence of Cdc37 with Tax in the presence of a test substance;
(B) measuring the amount of binding between the polypeptide and Tax,
(C) The amount of binding obtained above is obtained by contacting a polypeptide containing at least positions 181 to 200 of the amino acid sequence of Cdc37 with Tax in the absence of the test substance (control binding). Based on the results of steps (d) and (d) and (c), a substance that decreases the binding level compared to the control binding level is selected from the test substances as the active ingredient of the ATLL development inhibitor. Process.

(II) Reagent kit for screening an active ingredient of an ATLL onset inhibitor A reagent kit for screening an active ingredient of an ATLL onset inhibitor comprising the following (i) to (iii):
(I) a polypeptide comprising at least positions 181 to 200 of the amino acid sequence of Cdc37,
(Ii) Tax,
(Iii) A reagent for detecting the binding between the polypeptide and Tax.

(III) ATLL onset inhibitor (III-1) An ATLL onset inhibitor comprising as an active ingredient a substance having an action of inhibiting the binding between Tax and the CBD region of Cdc37.
(III-2) The ATLL onset inhibitor according to claim 4, wherein the active ingredient is an antibody that recognizes and binds to at least the CBD region of Cdc37, or an anti-Tax antibody.

  According to the screening method of the present invention, it is possible to obtain a candidate substance that can be an active ingredient of an ATLL onset inhibitor capable of delaying the onset of ATLL that is difficult to treat once it develops or preventing the onset itself. That is, the substance selected by such a method has an action of inhibiting the binding between Tax and Cds37, and promotes the degradation of Tax to suppress the activation of NF-κB. It can be used as an active ingredient of an ATLL onset inhibitor that can prevent the onset.

(I) Method for Screening Active Component of ATLL Development Inhibitor As described above, the present invention can be used to stabilize Tax in the cell that induces the development of ATLL. Binding, especially the binding of Cdc37 to the CBD region, and conversely, Tax can be destabilized (induced degradation) by inhibiting the binding of Tax to the CBD37 CBD region, As a result, based on the finding that the onset of ATLL can be prevented.

  That is, the screening method of the present invention uses, as an index, inhibition of binding between Tax and Cdc37, particularly inhibition of binding between the 181st to 200th regions (CBD region) in the amino acid sequence of Tax and Cdc37 among test substances. In this method, a substance having a binding inhibitory action is selected and acquired as an active ingredient of an ATLL onset inhibitor, in other words, a drug that prevents the onset delay or onset of ATLL.

  Here, as described above, Tax activates transcription factors such as NF-κB and AP-1 involved in cell proliferation and apoptosis suppression, and includes tumor suppressor gene products such as p53, Rb, and Mad1, DNA repair, and chromosomes. It is a protein known to inhibit the distribution check function (see FIG. 1) (see Non-Patent Document 5). The amino acid sequence (GenBank Accession NO. NP_057864) and the base sequence of the gene encoding it (GenBank Accession NO. AF033817) are also known.

Cdc37 is a cofactor of the molecular chaperone Hsp90 (co-chaperone). In addition to the amino acid region (40-110 region) called Kinase Binding Domain (KBD) that interacts with the phosphatase that is the Client, the Client Binding Domain Amino acid region (181-200 region) called (CBD), amino acid region (245-255 region) called dimerization domain (DD), amino acid region (164-170 region and 202-208 region) called Hsp90 interaction domain Roe, SM, Ali, MM, Meyer, P., Vaughan, CK, Panaretou, B., Piper, PW, Prodromou, C., Pearl, LH (2004) The Mechanism of Hsp90 regulation by the protein kinase -specific cochaperone p50 (cdc37) .Cell. 116 (1): 87-98
). The amino acid sequence (GenBank Accession NO. NP_008996) and the base sequence of the gene encoding it (GenBank Accession NO. NM_007065) are also known.

  The screening method of the present invention is not particularly limited and can be appropriately designed based on the common general knowledge of those skilled in the art. As an example, Tax and the CBD region of Cdc37 were contacted in the absence of the test substance. Examples include a screening method including comparing and evaluating the binding property (binding amount) of Tax to the CBD region between the case and the case where Tax and Cdc37 CBD are contacted in the presence of the test substance.

Specifically, the screening method can be performed by performing the following steps (a), (b), (c) and (d):
(A) contacting a polypeptide (including protein) containing at least the CBD region of Cdc37 with Tax in the presence of a test substance;
(B) measuring the amount of binding between the polypeptide and Tax,
(C) The amount of binding obtained above is obtained by bringing a polypeptide (including protein) containing at least the CBD region of Cdc37 into contact with Tax in the absence of the test substance (control binding amount). ) And (d) Based on the results of (c) above, a substance that reduces the binding amount compared to the control binding amount is selected from the test substances as the active ingredient of the ATLL development inhibitor. Process.

  Tax used in the screening method of the present invention may be a natural product, a synthetic product, or a recombinant. The Tax is preferably derived from humans, but Tax derived from mammals other than humans such as mice and other biological species can also be used.

  Examples of methods for preparing Tax include the following methods. First, based on the amino acid sequence information of Tax derived from humans or various biological species and the base sequence information encoding the same (for example, GenBank Accession No. AF033817), a probe or a primer for PCR is prepared according to a conventional method. A cDNA encoding Tax is obtained by cloning a cDNA library derived from C8166 cells derived from umbilical cord blood. These clonings can be easily performed by those skilled in the art according to a basic book such as Molecular Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).

 The prepared Tax cDNA is then inserted into a known expression vector such as pcDNA3. Thereafter, the obtained recombinant vector is introduced into a suitable host and cultured to produce a transformed cell in which a Tax (protein) encoded by the introduced Tax cDNA is expressed on the cell surface.

  In addition, as a host here, L cell, CHO cell, C127 cell, BHK21 cell, BALB / c3T3 cell (dihydrofolate reductase, thymidine kinase, etc.) which are generally widely used as host cells. COS cells, HEK293 cells, HeLa cells, and the like are preferable, but insect cells, yeast cells, bacterial cells, and the like can also be used. The Tax expression vector can be introduced into a host cell by any known introduction method, such as the calcium phosphate method (J. Virol., 52, 456-467 (1973)), LT. Examples include a method using -1 (manufactured by Panvera), a method using a lipid for gene introduction (Lipofectamine, Lipofectin; manufactured by Gibco-BRL), a method using FugenHD (manufactured by Roche), and the like. Subsequently, Tax is isolated from the obtained transformed cells according to a conventional method.

  In addition, a polypeptide having at least a CBD37 CBD region (181-200 region) (hereinafter also simply referred to as “CBD polypeptide”) used in the screening method of the present invention is also a natural product, a synthetic product, and a set. Regardless of the type of the recombinant, it can be prepared according to the method for preparing Tax. The CBD region is also preferably derived from a human, but CBD regions derived from mammals other than humans such as mice and other biological species can also be used.

  A CBD polypeptide does not necessarily have to have the entire amino acid sequence of Cdc37, but has at least a CBD region consisting of 20 amino acid residues (region of amino acid sequence 181-1200 of Cdc37) that binds to Tax. It ’s fine. That is, in the screening method of the present invention, as the CBD polypeptide, (1) a polypeptide (protein) consisting of the full-length amino acid sequence of Cdc37, (2) a polypeptide consisting of 181 to 200 residues in the amino acid sequence of Cdc37, And (3) a partial polypeptide of Cdc37 having at least 181 to 200 residues in the amino acid sequence of Cdc37 can also be used. In addition, as long as these CBD polypeptides have the amino acid sequence of the CBD region and the binding ability to Tax is maintained, a part of the amino acid sequence is deleted in other regions, or other amino acids May be substituted, added, inserted, or the like, and a polypeptide having such a modified region is also encompassed by the “CBD polypeptide” in the present invention.

  The CBD region of the CBD polypeptide consists of 20 amino acids. Therefore, the CBD polypeptide can also be synthesized according to a normal peptide synthesis method based on amino acid sequence information (GenBank Accession No .: NP_008996) of Cdc37 derived from humans or various organisms. For example, peptide synthesis is described in the literature [Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen (stock) ), 1975; Fundamentals and experiments of peptide synthesis, Maruzen Co., Ltd., 1985; Development of pharmaceuticals, Vol. 14, Peptide synthesis, Hirokawa Shoten, 1991] .

  The Tax and CBD polypeptides used in the screening method of the present invention may be isolated or further purified, but are not limited thereto. For example, instead of an isolated Tax or CBD polypeptide, cells expressing the Tax or CBD polypeptide may be used. Examples of the cells expressing Tax or CBD polypeptide include transformed cells prepared by introducing a vector having the above-described Tax or CBD polypeptide cDNA into an appropriate host and culturing. Since the cultured transformed cells express the Tax or CBD polypeptide on the cell surface, they can be used as they are in the screening method of the present invention.

Tax and CBD polypeptides may be used as they are, or those labeled with any labeling substance can be used. Examples of the labeling substance include radioisotopes (for example, ¹²⁵ I, ³ H, ¹⁴ C, ³⁵ S, etc.), fluorescent substances, chemiluminescent substances, biotin, marker proteins, peptide tags, and the like. Examples of the marker protein include conventionally known marker proteins such as alkaline phosphatase (Cell 63, 185-194 (1990)), antibody Fc region (Genbank accession number M87789), or HRP (Horse radish peroxidase). Examples of peptide tags include FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 × His or 10 × His consisting of 6 or 10 His (histidine) residues, influenza Agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck-tag, α-tubulin fragment, Known peptides such as B-tag and protein C fragments can be used.

  Test substances include, but are not limited to, nucleic acids, peptides, proteins, organic compounds, or inorganic compounds. Further, as a test substance, a low molecular weight organic compound having a molecular weight of 100 to 2000, preferably about 200 to 800, prepared using a peptide library of 2 to 50 amino acids, preferably 5 to 20 residues, or combinatorial chemistry. A library can also be used.

  Specifically, screening is performed by bringing these test substances or compositions containing them (for example, cell extracts, gene library expression products, etc.) into contact with Tax and CBD polypeptides. it can.

 In the step (a) of the screening method of the present invention, the condition for contacting the Tax and the CBD polypeptide in the presence of the test substance is a condition for binding the Tax and the CBD polypeptide in the absence of the test substance. There is no particular limitation.

  In addition, when using cells that express a Tax or CBD polypeptide instead of an isolated Tax or CBD polypeptide, the Tax or CBD polypeptide present in the cell is contacted with the CBD polypeptide or Tax, respectively. In such a case, it is sufficient if both conditions are combined. Specifically, in the case of cells, normal viable culture conditions, particularly physiological conditions such as Tris-HCl buffered saline, can be exemplified.

  Selection of the active ingredient (candidate substance) of the ATLL onset inhibitor is, for example, by contacting a Tax (including cells expressing Tax) and a CBD polypeptide (including cells expressing the CBD polypeptide) under the above conditions, This can be carried out by searching for a substance having an action of inhibiting the binding between the two.

  Specifically, the amount of binding of Tax to CBD polypeptide when Tax and CBD polypeptide are contacted in the presence of the test substance is obtained in the same manner as described above in the absence of the test substance. The test substance can be selected as a candidate substance by using as an index a decrease in the binding amount of Tax to the CBD polypeptide (control binding amount). In addition, the decrease mentioned here is not limited to the amount of control binding, and when binding of Tax to CBD polypeptide is completely inhibited and both do not bind, that is, the amount of binding of Tax to CBD polypeptide is substantially reduced. It is also included when the value becomes zero.

The amount of binding of Tax to CBD polypeptide can be quantified by a known method such as Western blotting using an antibody against Tax or CBD polypeptide. As an antibody used in the present invention, a commercially available antibody or an antibody contained in a commercially available kit can be used, and a monoclonal antibody or a polyclonal antibody obtained by using known means can also be used. Such a primary antibody or secondary antibody can be labeled by a known method. Examples of labeling substances include radioisotopes, enzymes, fluorescent substances, and the like. Commercially available labeling substances can be used for these labeling substances. Examples of the radioisotope include ³² P, ³³ P, ¹³¹ I, ¹²⁵ I, ³ H, ¹⁴ C, and ³⁵ S. Examples of the enzyme include alkaline phosphatase, horseradish peroxidase, β-galactosidase, β-glucosidase and the like. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC) and rhodamine.

  When using the above-mentioned labeled Tax or labeled CBD polypeptide as a Tax or CBD polypeptide, and when using the above labeled antibody as a primary antibody or secondary antibody, a measurement method (for example, radiation measurement) according to the labeling substance used The amount of Tax bound to the CDB polypeptide can also be quantified by a method, a fluorescence detection method, etc.

  Alternatively, Tax (or CBD polypeptide) is immobilized on a carrier such as a 98-well or 384-well microtiter plate, and the amount of CBD polypeptide (or Tax) bound thereto is determined with an enzyme, a fluorescent dye, or a radioisotope. It can also be measured by detecting with a labeled antibody.

  In addition, the binding between Tax and CBD polypeptide can be detected and / or measured by changing the expression level of a reporter gene that is activated in response to these bindings. As such a reporter gene, luciferase, β-galactosidase, HIS3 gene, chloramphenicol acetyltransferase (CAT), green fluorescence protein (GFP) gene and the like can be used. The peptide expressed in the cell may be a fusion peptide with another peptide. These peptides and other peptides subjected to fusion may be any peptides as long as they can be used in the screening method of the present invention, but are preferably transcriptional regulators. For example, fusion with each subunit of transcriptional regulators consisting of heterodimers known to activate transcription of reporter genes bound to DNA and to each of the Tax and CBD polypeptides that measure binding. DNAs are constructed and included in expression vectors and introduced into cells. When the test substance does not contain a compound that inhibits the binding between Tax and CBD polypeptide, Tax and CBD polypeptide form a heteromer, and the transcriptional regulator composed of the heteromer binds to DNA and the reporter gene is Activate. On the other hand, when the test substance contains a compound that inhibits the binding between Tax and CBD polypeptide, the binding between Tax and CBD polypeptide is inhibited, and as a result, the transcriptional regulatory subunit can form a heteromer. In other words, transcription of the reporter gene is not induced. By examining the change in the expression level of the reporter gene, it is possible to detect or measure a substance having a target binding inhibitory action. When examining changes in the expression level of a reporter gene in such a system, a two-hybrid system (Fields, S., and Sternglanz, R., Trends. Genet. (1994), 10, 286-292) or a three-hybrid system Can be used. The hybrid system may be constructed by a commonly used method, or a commercially available kit may be used. Commercially available two-hybrid kits include “MATCHMARKER Two-Hybrid System”, “Mammalian MATCHMARKER Two-Hybrid Assay Kit”, “MATCHMARKER One-Hybrid System” (all manufactured by Clontech), “HybriZAP Two-Hybrid Vector System” (Made by Stratagene).

  In the yeast two-hybrid method, a vector expressing a fusion protein in which either a Tax or CBD polypeptide is fused with a FLAF DNA binding region, etc., and a vector in which the other is fused with a transcriptional activation region are constructed. Is introduced into a yeast cell together with a vector encoding the reporter gene, and the compound is assayed using the reporter activity as an indicator in the presence of a sample containing the test substance. Expression of the reporter gene is induced by the binding of Tax and CBD polypeptide, but when the binding of both proteins is inhibited by the test substance, the expression of the reporter gene is suppressed. Examples of the reporter gene include, but are not limited to, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor type-1) gene in addition to HIS3 gene. As a reporter gene, a cytotoxic gene can also be expressed.

  In addition, the binding of Tax and CBD polypeptide can be performed by, for example, co-immunoprecipitation method, immunoprecipitation-immunoblot method, pull-down method using a column, method using a mass spectrometer, imaging using a fluorescent label, or combinatorial It can be performed by high-throughput screening using chemistry, or a combination thereof. In addition, as a means for measuring the bound protein, a biosensor using the surface plasmon resonance phenomenon can also be used.

  Candidate substances selected by the above screening method can be further screened using a non-human animal model of a disease state that has the possibility of developing ATLL. Candidate substances selected in this way may further develop drug efficacy tests using pathological model non-human animals with the possibility of developing ATLL, safety tests using non-human animals or humans, and the possibility of developing ATLL. The present invention may be used for human clinical trials, and by carrying out these tests, it is possible to select and acquire active ingredients of ATLL onset inhibitors that are more practical and highly effective.

  The material thus selected is subjected to structural analysis as necessary, and then, depending on the type of the material, chemical synthesis, biological synthesis (including fermentation), or genetic manipulation, And can be used for the preparation of a pharmaceutical composition for the purpose / effect of delaying or preventing the onset of ATLL.

(II) Reagent kit for screening active ingredients of ATLL onset inhibitor The present invention is also a reagent kit for the above screening.

  The reagent kit includes at least (i) a polypeptide containing at least positions 181 to 200 of the amino acid sequence of Cdc37 (CBD polypeptide), (ii) Tax, and (iii) binding between the CBD polypeptide and Tax. Reagents to be detected are included. The reagent for detecting the binding is a method used to detect the binding between Tax and CBD polypeptide, such as Western blotting, co-immunoprecipitation, immunoprecipitation-immunoblot, column pull-down, mass spectrometry Can be selected as appropriate according to methods using a meter, imaging using fluorescent labels, high-throughput screening using combinatorial chemistry, two-hybrid method, etc., for example, recognizing CBD region of anti-Tax antibody and Csd And various reagents used in the above-described method, and can be used in appropriate combination depending on the detection method.

(III) ATLL Onset Suppressor The present invention also provides an ATLL onset inhibitor comprising as an active ingredient a substance having an action of inhibiting the binding between Tax and the CBD region of Cdc37. The ATLL onset inhibitor is a pharmaceutical composition characterized by delaying the onset of ATLL or preventing the onset of ATLL in HTLV-1-infected patients.

  Examples of the substance having an action of inhibiting the binding between Tax and the CBD region of Cdc37 include an antibody that recognizes and binds at least the CBD region of Cdc37, or an antibody against Tax.

  Since these antibodies inhibit the binding between Tax and CBD polypeptide, the degradation of Tax is promoted, and as a result, the increase of NF-κB is suppressed and the induction of ATLL is prevented.

  An antibody against Tax and an antibody against CBD polypeptide can be prepared by known methods. There is no restriction | limiting in particular in the form of an antibody, A monoclonal antibody is also included besides a polyclonal antibody. Also included are antisera obtained by immunizing rabbits and other immunized animals with an antigen protein, all classes of polyclonal and monoclonal antibodies, human antibodies and humanized antibodies by genetic recombination.

  The protein used as the sensitizing antigen for antibody acquisition is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a human, mouse or rat, and particularly preferably a protein derived from a human.

  A gene encoding a Tax or CBD polypeptide is inserted into a known expression vector system, a host cell is transformed with the vector, and the desired polypeptide is obtained from the inside or outside of the host cell by a known method to sensitize these. What is necessary is just to use as an antigen. Alternatively, cells expressing the polypeptide of interest or a lysate thereof, or chemically synthesized Tax or CBD polypeptide may be used as the sensitizing antigen. In addition, a short peptide can be appropriately combined with a carrier protein such as keyhole limpet hemocyanin, bovine serum albumin or ovalbumin to be used as an antigen.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of the compatibility with the parent cell used for cell fusion in the production of a monoclonal antibody. Birds, rodents, rabbit eyes, and primates are used for this. Chickens are used as avian animals. Examples of rodent animals include mice, rats, and hamsters. For example, a rabbit is used as the animal of the order of rabbit eyes. For example, monkeys are used as the primate animals. As monkeys, monkeys (old world monkeys) with narrow nose, such as cynomolgus monkeys, rhesus monkeys, baboons, chimpanzees and the like are used.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. As a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, a sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if desired. After emulsification, it is administered to mammals. Furthermore, it is preferable to administer a sensitizing antigen mixed with an incomplete Freund's adjuvant several times every 4 to 21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen. Immunization is carried out in this manner, and it is confirmed by a conventional method that the desired antibody level is increased in the serum.

  Here, in order to obtain a polyclonal antibody against a certain protein, after confirming that the desired antibody level in the serum has increased, the blood of the mammal sensitized with the antigen is taken out. Serum is separated from this blood by a known method. As a polyclonal antibody, a serum containing a polyclonal antibody may be used, and if necessary, a fraction containing a polyclonal antibody may be further isolated from this serum and used. For example, by using an affinity column coupled with the protein used for the antigen, a fraction that recognizes only the antigen protein is obtained, and this fraction is further purified using a protein A or protein G column, Immunoglobulin G or M can be prepared. To obtain a monoclonal antibody, after confirming that the desired antibody level rises in the serum of birds and mammals sensitized with the antigen, immune cells are removed from the birds and mammals and subjected to cell fusion. Good. In this case, spleen cells are particularly preferable as immune cells used for cell fusion. The other parent cell to be fused with the immune cell is preferably an avian or mammalian myeloma cell, more preferably a myeloma cell that has acquired the characteristics for selecting fused cells by a drug. The cell fusion between the immune cells and myeloma cells is basically performed according to a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C, Methods Enzymol. (1981) 73, 3-46). Can be done. The hybridoma obtained by cell fusion is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing with the HAT culture solution is carried out for a time sufficient for the cells other than the target hybridoma (non-fused cells) to die, usually several days to several weeks. Subsequently, a normal limiting dilution method is performed to screen and clone hybridomas that produce the target antibody.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized in vitro with proteins, protein-expressing cells or lysates thereof, and sensitized. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain a hybridoma that produces a desired human antibody having a protein binding activity (Japanese Patent Laid-Open No. 63-17688). . Subsequently, the obtained hybridoma was transplanted into the abdominal cavity of the mouse, and ascites was collected from the mouse, and the obtained monoclonal antibody was used as, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, and antigen. It can be prepared by purification using an affinity column coupled with a protein. The antibody targeted in the present invention may be a human antibody or a human antibody. For example, a transgenic animal having a repertoire of human antibody genes is immunized with an antigen protein, a protein-expressing cell or a lysate thereof to obtain an antibody-producing cell, and a hybridoma obtained by fusing this with a myeloma cell is used for the protein. Human antibodies can be obtained (see International Publication Nos. WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735, WO96-34096, etc.).

  In addition to producing antibodies using hybridomas, cells obtained by immortalizing immune cells such as sensitized lymphocytes producing antibodies with oncogenes may be used. The thus obtained monoclonal antibody can also be obtained as a recombinant antibody produced using a gene recombination technique. A recombinant antibody is produced by cloning DNA encoding it from an immune cell such as a hybridoma or a sensitized lymphocyte producing an antibody, incorporating it into an appropriate vector, and introducing it into a host.

  The antibody may be a known antibody using a chimeric antibody comprising a non-human antibody-derived variable region and a human antibody-derived constant region, or a non-human antibody-derived CDR (complementarity determining region) and a human antibody-derived FR (frame). It can be obtained as a humanized antibody comprising a work region) and a constant region. The antibody obtained as described above can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. However, it is not limited to these. The concentration of the antibody obtained above can be measured by measuring absorbance, enzyme-linked immunosorbent assay, or the like. Examples of columns used for affinity chromatography include a protein A column and a protein G column.

 Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography. These chromatography can be performed using liquid phase chromatography, such as HPLC and FPLC.

 As a method for measuring the antigen binding activity of an antibody, for example, absorbance measurement, ELISA, EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method can be used. When using ELISA, the HK33 protein is added to a plate on which an antibody is immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added. A secondary antibody that recognizes an antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, washed, and then an enzyme substrate such as p-nitrophenyl phosphate is added to measure the absorbance. Binding activity can be evaluated. BIAcore (Pharmacia) can also be used for antibody activity evaluation.

  Substances having an action of inhibiting the binding between Tax and CBD polypeptide, such as the above-mentioned antibody, can be prepared as a pharmaceutical composition formulated by a known pharmaceutical method in addition to directly using the substance itself.

  For example, tablets, capsules, elixirs, microcapsules as required, or aseptic solutions or suspensions with water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

  Examples of additives that can be mixed in tablets and capsules include binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry are used. When the dispensing unit form is a capsule, the above material can further contain a liquid carrier such as fats and oils. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.

  Examples of the aqueous solution for injection include isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethyleneglycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  For example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, percutaneous, or oral administration to patients are performed by methods known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. In addition, if the compound can be encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

The dose of the ATLL onset inhibitor of the present invention varies depending on the symptoms, but in the case of oral administration, in general, for an adult (with a body weight of 60 kg), it is appropriately selected from the range of about 0.1 to 100 mg per day. be able to.
When administered parenterally, the single dose varies depending on the subject of administration, symptoms, and administration method. For example, in the form of an injection, it is usually about 0.01 to 30 mg per day for an adult (with a body weight of 60 kg). It may be convenient to administer the degree by intravenous injection.

  Hereinafter, the contents of the present invention will be specifically described using the following experimental examples. However, the present invention is not limited to these. Unless otherwise stated, “%” means “% by weight” unless otherwise specified.

In addition, main materials used in the following experimental examples, their preparation methods, and experimental procedures are as follows.
(1) Material and preparation method (1-1) Cell preparation Human fetal kidney-derived cell HEK293 (hereinafter referred to as “293 cell”) is 15% FBS, 2 mM L-glutamine, Penicillin-Streptomycin (PC / SM) Culture (37 ° C., 5% CO ₂ ) was maintained in the added Dulbecco's Modified Eagle Medium (DMEM, GIBCO).

Using this, cells for transfection experiments were prepared as follows.
(A) Aspirate the DMEM medium from the subculture flask in which 293 cells have grown almost confluent, and wash once with 5 mL of PBS.
(B) 293 cells adhering to the bottom of the flask were suspended in 3 mL of PBS containing trypsin and 1 mM EDTA, suspended with a pipette, and then DMEM supplemented with 15% FBS, L-glutamine, and PC / SM Add 20 mL and suspend the cells.
(C) Transfer 0.6 mL of cell suspension culture solution to each well of a 12-well plate (5 × 10 ⁴ / well) and culture the plate until it becomes 60-80% confluent (approximately overnight). Used for transfection).

(1-2) Preparation of plasmid
(a) NF-κB-luc
NF-κB-luc (Dr. M. Lienhard Schmitz. Department of Immunochemistry, German Cancer Research Center, Im Neuenheimer Feld, a vector that has an NF-κB binding sequence and induces transcription and translation of firefly luciferase in an NF-κB-dependent manner 280, 69120 (obtained from Heidelberg, Germany)) was used as a reporter plasmid to measure NF-κB activation.

(b) HTLV1-LTR-luc
HTLV-1-LTR activity of the vector HTLV1-LTR-luc (Non-patent Document 7), which has the Long-Terminal-Repeat (LTR) binding sequence of HTLV-1 and induces Tax-dependent transcription and translation of firefly luciferase It was used as a reporter plasmid to measure crystallization.

(c) RSV-βgal
RSV-βgal that expresses β-galactosidase dependently on the Rous sarcoma virus (RSV) promoter (from Dr. Kuan Teh Jeang, Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, the National Institutes of Health, Bethesda, Maryland 20892, USA. Obtained) was used as a transfection monitor.

  In the Tax administration experiment system, the vector pLTR-Tax (Non-patent Document 7) that expresses the Tax protein in a HTLV-1 LTR-dependent manner or the vector pCMV-Tax that expresses the Tax in a cytomegalovirus (CMV) -dependent manner is used. It was.

(2) Experimental procedure (2-1) Luciferase and β-galactosidase assay Luciferase assay substrate (Luciferase Assay Substrate (Promega)) or β-galactosidase assay substrate (Galacto-Star (registered trademark): Reaction Buffer Diluent (Applied Biosystems) )) Was used to analyze with a luminometer (GloMax ™ 96 Microplate Luminometer, Promega) that measures luminescence intensity.

(2-2) Quantification of Tax expression level or Hsp90 expression level by immunoblotting
Quantification of the expression level of Tax protein by introducing the Tax expression vector or Hsp90 protein by introducing the Hsp90 expression vector was performed as follows. First, after quantifying the amount of protein in the cell lysate (Protein Assay Kit, BioRad, 500-0001JA), 10 μg of protein is placed in each lane and separated by SDS-polyacrylamide electrophoresis (SDS-PAGE). . Next, the protein separated by the semi-dry method (Trans-Blot SD, BioRad) was transferred to a PVDF membrane (Immobilon-P, Millipore), which was distributed by Yutaka Tanaka, an anti-Tax rat monoclonal antibody (Immunology Laboratory, University of the Ryukyus) ), Or after binding to each protein using anti-Hsp90 rat monoclonal antibody (Stressgen, SPA-835), using horseradish peroxidase-conjugated secondary antibody and chemiluminescence reagent (ECL Western Blotting Detection Reagents, GE Healthcare) X-ray film is exposed to light, detected by Adobe Photoshop, and quantified.

Experimental Example 1 Tax degradation induction by Cdc37 co-chaperone
Hsp90 is one of the most abundant protein species in cells, and it is known to interact with various protein molecules (Client) group and contribute to correct three-dimensional structure formation and functional expression of these Client groups. (Reference 5). Hsp90 cooperates with various cofactors (called co-chaperones), and functions as a chaperone. Among them, Cdc37 binds to many phosphorylase groups involved in intracellular signal transduction, Since the increase in the expression level is frequently observed in tumorigenic cells, its activity has attracted attention (References 6 and 7). Cdc37 is an amino acid region (residues 40-110) usually called Kinase Biding Domain (KBD) and is considered to interact with a kinase kinase (Kinase) (Reference 6) (FIG. 3A). Recently, in addition to the KBD region, the presence of an amino acid region (181-200 residues) called Client Binding Domain (CBD) has also been pointed out (Reference Document 8).

  In this experiment, intermolecular interaction between Tax and Hsp90 / Cdc37 complex was investigated using Hsp90 / Cdc37 complex and complex of Hsp90 and Cdc37 deletion mutant.

(1) Experimental method (1-1) Construction of Hsp90 and Cdc37 expression vectors PCR amplification using human Hsp90 cDNA (distributed by Professor Keisuke Nagata, University of Tsukuba) or Cdc37 cDNA (Reference 8) as a template, and each amplification obtained The product was cloned into pcDNA3-Flag EcoRI-SalI site (Hsp90) and BamHI-EcoRI site (Cdc37) (pcDNA3-F-Cdc37). For Cdc37, six types of DNA fragments encoding different amino acid regions were prepared [Cdc37 (1-378), Cdc37 (1-278), Cdc37 (1-200), Cdc37 (1-180), Cdc37 (181-378), Cdc37 (201-378)], and cloned into the EcoRI-SalI site (Hsp90) and BamHI-EcoRI site (Cdc37) of pcDNA3-Flag in the same manner as above (pcDNA3-F-Cdc37 ( 1-378), pcDNA3-F-Cdc37 (1-278), pcDNA3-F-Cdc37 (1-200), pcDNA3-F-Cdc37 (1-180), pcDNA3-F-Cdc37 (181-378), pcDNA3 -F-Cdc37 (201-378)).

  Next, 0.1 μg each of NF-κB-Luc and RSV-βGal, pcDNA3 1 μg (FIG. 4A (hereinafter the same), lane 1), pLTR-Tax 0.5 μg + pcDNA3 (lane 2), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (1-378) (lane 3), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (1-200) (lane 4), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (1 -180) (lane 5), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (181-378) (lane 6), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (201-378) (lane 7) Each was transfected according to the method of Experimental Example 3 and the cells were lysed. Then, the luciferase activity and the expression of Tax and Cdc37 were measured using an anti-Tax rat monoclonal antibody and an anti-Flag antibody (Sigma, F1804), respectively. Detected. The results are shown in FIG. 4A.

  In addition, 1 μg of control pcDNA3 (in FIG. 4B (hereinafter the same), see lane 1), pLTR-Tax 0.5 μg (lane 2), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (1-200) 0.125 μg (lane 3) , PLTR-Tax 0.5μg + pcDNA3-F-Cdc37 (1-200) 0.25μg (lane 4), pLTR-Tax 0.5μg + pcDNA3-F-Cdc37 (1-200) 0.5μg (lane 5), pLTR-Tax 0.5μg + pcDNA3-F -Cdc37 (1-180) 0.125μg (lane 6), pLTR-Tax 0.5μg + pcDNA3-F-Cdc37 (1-180) 0.25μg (lane 7), pLTR-Tax 0.5μg + pcDNA3-F-Cdc37 (1-180) 0.5 μg (lane 8), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (181-378) 0.125 μg (lane 9), pLTR-Tax 0.5 μg + pcDNA3-F-Cdc37 (181-378) 0.25 μg (lane 10) and pLTR- Tax 0.5 μg + pcDNA3-F-Cdc37 (181-378) 0.5 μg (lane 11) was transfected, and the expression of Tax, Cdc37 and Tubulin was carried out in the same manner as described above, anti-Tax rat monoclonal antibody, anti-Flag antibody, And anti-α-Tubulin mouse monoclonal antibody It was detected Te. The results are shown in FIG. 4B.

(2) Experimental results FIG. 4A verified the suppression of Tax-induced NF-κB activity by the Cdc37 deletion mutant from the luciferase activity (FIG. 4A, upper panel) and the expression level of Tax (FIG. 7A, middle panel). Results are shown.

  As shown in FIG. 4A, when full-length Cdc37 (Cdc37 (1-378)) is co-expressed with Tax (see lane 3), luciferase activity is 17 compared to when only Tax is expressed (lane 2). %Rose. However, mutants with deletion of each functional region (Cdc37 deletion mutant: Cdc37 (1-200), Cdc37 (1-180), Cdc37 (181-378), Cdc37 (201- In all cases 378)), the luciferase activity was reduced as compared with the case where only Tax was expressed (lane 2). Mutants Cdc37 (1-200) (lane 4) and Cdc37 (181-378) (lane 6) both had a significant decrease in luciferase activity (33%, 16%) (upper panel) and significant degradation of Tax ( Middle panel) was observed.

  As shown in FIG. 4B, the Tax-degrading activity of the Cdc37-deficient mutant was expressed when only Tax was expressed (lane 2); Tax, 1/4, 1/2, or an equivalent amount of Cdc37 (1-200) Co-expressed (lanes 3-5); Tax co-expressed with 1/4, 1/2, or equal amount of Cdc37 (1-180) (lanes 6-8); Tax and 1 / 4, 1/2, or equal amounts of Cdc37 (181-378) co-expressed (lanes 9-11), Cdc37 (1-200), Cdc37 (181-378) and Tax Only when co-expressed (lanes 3-5, 9-11), degradation of Tax was observed in a dose-dependent manner for Cdc37-deficient mutants (upper panel). In addition, it was confirmed that the amount of each Cdc37-deficient mutant also increased in a dose-dependent manner (middle panel) and that there was no change in the expression level of tubulin (lower panel).

  From the above experiments, it was found that Cdc37-deficient mutants, particularly Cdc37-deficient mutants that retain the 181-200 amino acid region (CBD region) of Cdc37, promoted the degradation of Tax and also suppressed NF-κB activity. . This means that a Cdc37 deletion mutant with a CBD region competitively inhibits the binding of the endogenous Cdc37 / Hsp90 complex to Tax, making it unable to maintain a stable structure in the cell. To do.

Experimental Example 2 Binding of Tax and Cdc37-CBD (1) Experimental method
To which region of Cdc37 Tax was bound was verified by co-immunoprecipitation.

  From the results of Experimental Example 1, it was clarified that the degradation of Tax was induced by co-expression with the Cdc37-deficient mutant, so that Tax and Cdc37-deficient mutants were expressed independently, and the cell lysate was equivalent. Each was mixed and immunoprecipitation was performed (upper panel of FIG. 5).

  The cell lysates expressing the Tax and Cdc37-deficient mutants were each electrophoresed by SDS-PAGE at 10 μg, and then the Tax was treated with an anti-Tax rat monoclonal antibody (middle panel of FIG. 5), and Hsp90 and Cdc37. The deletion mutant was detected using an anti-Flag antibody (FIG. 5: lower panel).

  Specifically, the immunoprecipitation method includes 0.5 μg of pcDNA3 (FIG. 5 (hereinafter the same): middle panel lane 1, lower panel lanes 1 and 2), pLTR-Tax 0.5 μg (middle panel lanes 2 to 9), pcDAN3- F-Hsp90 0.5 μg (panel lower lane 3), pcDAN3-F-Cdc37 (1-378) 0.5 μg (panel lower lane 4), pcDAN3-F-Cdc37 (1-278) 0.5 μg (panel lower lane 5), pcDAN3-F-Cdc37 (1-200) 0.5μg (panel lower lane 6), pcDAN3-F-Cdc37 (1-180) 0.5μg (panel lower lane 7), pcDAN3-F-Cdc37 (181-378) 0.5μg (Panel lower lane 8), pcDAN3-F-Cdc37 (201-378) 0.5 μg (panel lower lane 9) were separately transfected into 293 cells, and 40 hours later, the cells were lysed with Co-IP buffer. Mix 250 µg of lysate solution in the same lane, and add 2 µg of anti-Flag rabbit polyclonal antibody (Sigma, F7425) and 30 µl of ProteinG-agarose using the same procedure as in Experimental Example 4 described later. It was carried out by. The immunoprecipitation complex was washed 6 times with Co-IP buffer and then electrophoresed by SDS-PAGE, and Tax was detected with an anti-Tax rat monoclonal antibody (upper panel of FIG. 5).

(2) Experimental Results As shown in FIG. 5, Tax bound to Hsp90 (lane 3) and full-length Cdc37 (lane 4). Among the Cdc37-deficient mutants, those that did not bind to Tax were Cdc37-deficient mutants lacking the 181-200th amino acid region (CBD region) of Cdc37, ie, Cdc37 (1-180) (lane 7) and Cdc37. (201-378) (lane 9) (upper panel in FIG. 5). These facts revealed that 20 amino acid residues in the CBD region of Cdc37 (residues 181-1200) are essential for the binding between Tax and Cdc37.

  This means that in order for Tax to bind to and stabilize the Hsp90 / Cdc37 complex, the binding to the CBD region of Cdc37 is important. This means that it is important for stabilization of (see FIG. 3B). That is, by inhibiting the binding between Tax and the CBD region of Cdc37, it is considered that the degradation of Tax is induced and the activation of NF-κB is suppressed, and thus the onset of ATLL can be suppressed.

Experimental Example 3 Inhibition of Tax-induced transcriptional activation (NF-κB, HTLV-1-LTR) by 17-DMAG (1) Experimental method (1-1) Transfection
In the Tax administration experiment system, NF-κB-luc (0.1 μg), pRSV-βgal (0.1 μg), vector pLTR-Tax or pCMV-Tax (0.5 μg), and TNF-α In the experimental system, NF-κB-luc (0.1 μg), pRSV-βgal (0.1 μg), and pcDNA3 (Invitrogen) (0.5 μg) were respectively introduced (transfected) into cells for transfection experiments.

  Specifically, first, Fugene-HD (registered trademark, Roche) 2.1 μL (plasmid: Fugene-HD = 1: 3) and DMEM 100 μL were mixed and allowed to stand at room temperature for 15 minutes. This was mixed with each plasmid DNA solution and allowed to stand for 15 minutes at room temperature, and finally mixed with 500 μL of 5% FBS-containing DMEM and allowed to stand for 15 minutes at room temperature. Carefully pour the prepared DNA transfection mixture onto the cells for transfection experiments (60-80% confluent) on a 12-well cell culture plate with the medium removed, taking care not to detach the cells. The operation was performed.

  The next morning, 0.5 mL of 5% FBS-containing DMEM was added thereto, and 17-DMAG (0.1, 0.5, 2.5, 5 μM) or PBS for control was added 24 hours after the transfection operation.

  In the Tax administration experiment system, the cells were washed twice with 1 mL of PBS 24 hours later (48 hours after the transfection operation), and the cells were released from the bottom of the plate with 0.8 mL of PBS containing 1 mM EDTA, and suspended with a pipette. It collect | recovered to a 1.5 mL microtube.

  In addition, in the TNF-α administration experiment system, 16 hours after adding 17-DMAG, TNF-α (ProSpec) dissolved in 100 μL of DMEM was added so that the final concentration was 10 ng / mL, and then 8 hours later. At 48 hours after the transfection, the cells were washed twice with 1 mL of PBS, and the cells were released from the bottom of the plate with 0.8 mL of 1 mM EDTA-containing PBS, suspended with a pipette, and collected in a 1.5 mL microtube.

  Next, the microtube containing the cell suspension was centrifuged (6000 rpm, 2 min, 20 ° C.) with a cooling centrifuge (Centrifuge 5415R, eppendorf), and then 0.8 mL of 1 mM EDTA-containing PBS was aspirated and removed on ice. The microtube was cooled. The cells in the tube are lysed with 100 μl of Lysis Buffer (Applied Biosystems), stirred (vortex, 15 seconds), and centrifuged (13200 rpm, 5 min, 4 ° C) to separate the insoluble precipitate on the bottom of the tube. Kiyoshi (cell lysate) was obtained.

(1-2) Luciferase and β-galactosidase assay 5 μL of the cell lysate was dispensed into each well of a 96-well plate and immediately subjected to luciferase assay and β-galactosidase assay.

(1-3) Quantitative Analysis of NF-κB and HTLV-1-LTR Transcriptional Activity NF-κB and HTLV-1LTR transcriptional activity (%) was determined by measuring the luciferase assay measured from the same experiment at least three times. After dividing by the galactosidase assay measurement value and normalizing, the mean value ± SD was determined. 24 hours after the transfection operation, the luciferase value and β-galactosidase value of the cells to which PBS was added for control were always set as the reference values (100%), and the NF-κB activity of each cell was calculated from the relative values.

(1-4) Quantitative analysis of Tax expression level by immunoblotting
Quantification of the expression level of Tax protein by introducing the Tax expression vector is based on the amount of protein in the cell lysate (Protein Assay Kit, BioRad, 500-0001JA). After separation by electrophoresis (SDS-PAGE), the protein is transferred to a PVDF membrane (Immobilon-P, Millipore) by the semi-dry method (Trans-Blot SD, BioRad), and anti-Tax rat monoclonal antibody (Immunology Course, University of the Ryukyus) After binding to each protein using anti-Hsp90 rat monoclonal antibody (Stressgen, SPA-835) and anti-α-Tubulin mouse monoclonal antibody (SIGMA, T-5168), secondary binding with horseradish peroxidase After exposure to an X-ray film using an antibody and a chemiluminescent reagent (ECL Western Blotting Detection Reagents, GE Healthcare), it was quantified with Adobe Photoshop.

(2) Experimental results The results are shown in FIG. Specifically, FIG. 6A shows the inhibitory effect of 17-DMAG on NF-κB transcriptional enhancement induced by pLTR-Tax (lane 2 in FIG. 6A) in 293 cells (see lanes 3 to 6 in FIG. 6A). ), FIG. 6C shows the inhibitory effect of 17-DMAG on HTLV1-LTR transcriptional enhancement induced by pLTR-Tax (see lane 2 in FIG. 6C) in 293 cells (see lanes 4-6 in FIG. 6C), and FIG. 6D shows the inhibitory effect of 17-DMAG (see lanes 4 to 6 in FIG. 6D) on the HTLV1-LTR transcriptional enhancement (lane 2 in FIG. 6D) induced by pCMV-Tax in 293 cells.

  As can be seen from FIG. 6A, NF-κB overactivated by Tax was suppressed to about 27% by administration of 2.5 μM 17-DMAG (FIG. 6A, lane 5). Therefore, 17-DMAG has an action of suppressing NF-κB activity excessively enhanced by Tax, and it is considered that malignant tumor formation is suppressed based on such action. Moreover, HTLV1-LTR transcriptional activity enhanced by Tax was suppressed by administration of 17-DMAG regardless of the type of promoter inducing the expression of Tax (FIGS. C and D). Further, as can be seen from FIG. 6B, the protein expression level of Tax was attenuated to 1/10 or less by administration of 2.5 μM 17-DMAG.

  By the way, HTLV1-LTR transcriptional enhancement does not involve NF-κB activity at all, and in the place where Tax functions, NF-κB activation is in the cytoplasm (binding to NEMO component protein NEMO, Non-Patent Document 7). On the other hand, HTLV1-LTR transcriptional activation is different from each other, as is in the nucleus (binding to transcription factor CBP / p300, Non-Patent Documents 5 and 6), and the functionally interacting proteins are also completely different. doing. Thus, the suppression of Tax-induced NF-κB enhancement by 17-DMAG may occur by reducing the abundance of the Tax molecule itself, unlike the known inhibition of IKK complex formation (Reference 4). found. This means that the intracellular stability of the Tax molecule depends on the Hsp90 / Cdc37 complex.

Experimental Example 4 Induction of Tax degradation in C8166 cells from ATL patients by 17-DMAG (1) Experimental method
ATL patient culture C8166 maintained in RPMI 1640 medium + 15% fetal bovine serum is suspended in 2 ml of the same medium at 4x10 ^{6 and} transferred to ⁶ wells. For control, PBS or 17-DMAG (0.1, 0.5, 2.5 , 5 μM). After 24 hours, the cells were collected and washed twice with PBS, then Co-IP buffer (0.5% NP40, 50 mM HEPES (pH 7.3), 150 mM NaCL, 1 mM EDTA, 10 mM β-glycerophosphate, 10 mM NaF, 10% glycerol, Roche protease inhibitor tablet) was dissolved in 500 μl. After sonicating the cell lysate and destroying the nuclei and other compartments, 250 μg of protein was collected, the Co-IP buffer was added to adjust the final volume to 500 μl, and anti-Hsp90 mouse monoclonal antibody (Stressgen, SPA -830) was added with 2 μg and protein G-agarose (Calbiochem) 30 μl, and immunoprecipitation was performed at 15 rpm for 2 hours in the refrigerator (4 degrees) using ATTO Mini Desk Rotor BC710. The resulting immunoprecipitation complex was washed 6 times with 500 μl of Co-IP buffer, separated by SDS-PAGE, transferred to a PVDF membrane, anti-Hsp90 rat monoclonal antibody (Stressgen, SPA-835) or anti-IKKβ Detection was performed with a rabbit polyclonal antibody (Cell Signaling Technology, 2684). Tax was detected with anti-Tax rat monoclonal antibody for each 10 μg of cell lysate. The result is shown in FIG. 7A.

  In addition, PBS or 2.5 μM 17-DMAG was added to the C8166 cell suspension prepared in the same manner as described above, and the cells were collected after 12, 24, 36 and 48 hours, and the anti-Tax rat monoclonal antibody described above was recovered. The expression level was detected with anti-Hsp90 mouse monoclonal antibody and anti-α-Tubulin mouse monoclonal antibody (SIGMA, T-5168). The result is shown in FIG. 7B.

(2) Experimental Results As shown in the leftmost lane in the middle panel of FIG. 7A, the IKK complex (see FIG. 2) and Hsp90 form a complex with each other. However, when 17-DMAG was added to this, the degradation of Tax progressed in a concentration-dependent manner (see the upper panel of FIG. 7A), and the amount of complex formed between IKK complex and Hsp90 was proportionally reduced accordingly. (See the middle panel of FIG. 7A). On the other hand, as shown in the lower panel of FIG. 7A, the expression level of Hsp90 itself is not affected by the addition of 17-DMAG.

  As shown in FIGS. 6 and 7A, 17-DMAG has a sufficient inhibitory effect on Tax at a concentration of 2.5 μM. Therefore, FIG. 7B is a diagram showing the results of measuring the effect of 17-DMAG at the same concentration on the stability of Tax over time. As shown in FIG. 7B, the degradation of Tax already started 12 hours after the addition of 17-DMAG, and disappeared completely after 48 hours.

  The results of Experimental Example 4 described above strongly suggest that the suppression mechanism of the Tax function by 17-DMAG is due to the promotion of decomposition of Tax as discussed in Experimental Example 3.

Experimental Example 5 Inhibition of ATL Cell Growth and Apoptosis Induction by 17-DMAG (1) Experimental Method Two blood samples collected from patients by the inventor Ogata (hereinafter referred to as “ATL01” and “ATL02”), Blood samples collected from 2 healthy adult males (hereinafter referred to as “PBL1” and “PCL2”) and C8166 cell line, using a method similar to that used in Experimental Example 2, RPMI1640 medium + It was maintained in 15% fetal bovine serum, suspended in 2 ml of the same medium at ⁴ × 10 ⁶ aliquots, and transferred to a 6-well dish. C8166 is a cell line obtained by co-culturing umbilical cord blood with the cell line HuT102 established from an ATL patient (Salahuddin, SZ, Markham, PD, Wong-Staal, F., Franchini, G., Kalyanaraman, VS, Gallo, RC (1983) Restricted expression of human T-cell leukemia-lymphoma virus (HTLV) in transformed human umbilical cord blood lymphocytes. Virology 129: 51-64.).

PBS (control) or 2.5 μM 17-DMAG was added thereto, and 1 × 10 ⁵ cells were collected three times in a 96-well dish every 24 hours. To this, 10 μl of CCK-8 (Dojindo) was added and cultured at 37 ° C. for 1 hour, and then the absorbance at 475 nm was measured with an E-max microplate reader (Molecular Devices). The value (control value) of the PBS addition group (control group) was set to 100, and the relative ratio of the value of the 2.5 μM 17-DMAG addition group to this was determined, and this was expressed as the cell viability (%) ( (See FIG. 8A).

  In addition, 25 μl of Apo-ONE (registered trademark) Homogeneous Caspase-3 / 7 Assay solution was added to the cells collected in the 96 well dish, and the luminescence intensity was measured using a luminometer (GloMax 96 Microplate Luminometer, Promega). . The value (control value) of the PBS addition group (control group) was set to 1, and the relative ratio of the value of the 2.5 μM 17-DMAG addition group to this was determined, and this was shown as the relative caspase value (fold) (FIG. 8B).

(2) Experimental results FIG. 8A is a graph quantifying the activity of the intracellular mitochondrial electron transport system by increasing the absorbance using CCK-8, and shows the cell survival rate (%). Therefore, a decrease in the relative value with respect to the control value means a decrease in viable cells. As shown in FIG. 8A, the number of viable cells derived from healthy subjects (PBL1, PCL2) did not change after 4 days (96 hours) after addition of 2.5 μM 17-DMAG, whereas ATL patients The derived cells (ATL01, ATL02) decreased to 30% or less after 2 days.

  FIG. 8B shows the activity of Caspase 3/7 in which apoptosis of cells is irreversibly induced, quantified by the amount of chemiluminescence, and an increase in the relative caspase value (fold) means that the apoptotic reaction is enhanced. As shown in FIG. 5B, as in FIG. 5A, ATL patient-derived cells (ATL01, ATL02) are more active than healthy human-derived cells (PBL1, PCL2), and C8166, ATL01, and ATL04 have the fastest growth rates. Induction of activity occurs sequentially.

  From the above two types of experimental results, it was shown that 17-DMAG significantly induces growth inhibition or apoptosis in ATL cells at a concentration that does not affect the survival of healthy cells.

Experimental Example 6 17-DMAG suppresses multiorgan invasion of ATL cells From the above experiment, Tax and Cdc37 CBD region (residues 181 to 200) bind directly through molecular interaction. Tax is important for binding to the Hsp90 / Cdc37 complex and stabilizing in the cell, 17-DMAG induces Tax degradation, suppresses NF-κB enhancement, and 17-DMAG It was revealed that the growth of ATL cells was suppressed and apoptosis was induced on ATL cells.

Then, the inhibitory effect with respect to the multi-organ invasion of ATL cell by 17-DMAG oral administration was verified using the ATLL onset model mouse (reference document 4) which one of the present inventors developed by Hasegawa.

(1) Experimental method The outline of the experimental method is shown in FIG. Lymphocytes that express Tax in a Lck-proxymal promoter-dependent manner were collected from the spleen of an ATL model mouse (SCID mouse: Reference 4) and suspended in RPMI1640 medium. The cell suspension 10 ⁶ cells were transplanted into the abdominal cavity of SCID mice, reared for 28 days, the spleen was removed, and lymphocytes were prepared by lymphoprep (Axis-Shield, Oslo, Norway). 2 × 10 ⁶ lymphoma cells were transplanted into the peritoneal cavity of SCID mice, and 17-DMAG was orally administered (100 μg / animal or 300 μg / animal) and given for 5 consecutive days in the first and second weeks. A control group of mice (control group) was given water. 21 days after transplantation, each organ was verified by pathological dissection. Tissue fixation was performed with 4% formalin-PBS, and staining was performed using the hemotoxylin / eosin (HE) method. PBL was stained by Giemsa method.

(2) Experimental results The results are shown in FIG. As can be seen from this, 350,000 ATL cells were present in 1 ml of peripheral blood leukocytes (PBL) of mice 21 days after transplantation of ATL cells in the water control group to which physiological saline was administered (the upper panel of FIG. 10). , Left column), up to 90,000 in the 17-DMAG 100 μg-treated mice (upper panel in FIG. 10, middle column) and 60,000 in the 17-DMAG 300 μg-treated mice (upper panel in FIG. 10). They decreased in the right column).

  Infiltration of ATL cells is indicated by dark purple portions in the spleen (second row from the top in FIG. 10), liver (third row from the top), and lung (bottom row). Since the spleen was originally an organ that accumulates lymphocytes, there was no significant difference in purple, but the size of the spleen itself was clearly different between the 17-DMAG administration group and the control group. On the other hand, infiltration of ATL cells into the liver and lung decreased depending on the dose of 17-DMAG. Although not shown here, even when 600 μg of 17-DMAG was orally administered to SCID mice in the same manner, no side effects such as abnormal behavior or weight loss were observed.

References cited in the above specification are as follows.
Reference 1: DeBoer, C., Meulman, PA, Wnuk, RJ, Peterson, DH (1970) “Geldanamycin, a new antibiotic.” J. Antibiot. 23 (9): 442-447.
2: Supko, JG, Hickman, RL, Grever, MR, Malspeis, L. (1995) “14 Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent.” Cancer Chemother. Pharmacol. 36 (4): 305-315.
3: Egorin, MJ, Lagattuta, TF, Hamburger, DR, Covey, JM, White, KD, Musser, SM, Eiseman, JL (2002) “Pharmacokinetics, tissue distribution, and metabolism of 17- (dimethylaminoethylamino) -17-demethoxygeldanamycin (NSC 707545) in CD2F1 mice and Fischer 344 rats. “Cancer Chemother Pharmacol. 49 (1): 7-19
4: Hasegawa, H., Sawa, H., Lewis, MJ, Orba, Y., Sheehy, N., Yamamoto, Y., Ichinohe, T., Tsunetsugu-Yokota, Y., Katano, H., Takahashi, H., Matsuda, J., Sata, T., Kurata, T., Nagashima, K., Hall, WW (2006) “Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I. “Nat. Med. 12 (4): 466-472.
5: Caplan, AJ, Mandal, AK, Theodoraki, MA (2007) “Molecular chaperones and protein kinase quality control.” Trends Cell Biol. 17 (2): 87-92.
6: Roe, SM, Ali, MM, Meyer, P., Vaughan, CK, Panaretou, B., Piper, PW, Prodromou, C., Pearl, LH (2004) “The Mechanism of Hsp90 regulation by the protein kinase- specific cochaperone p50 (cdc37). “Cell 116 (1): 87-98.
7: Calderwood, SK, Khaleque, MA, Sawyer, DB, Ciocca, DR (2006) “Heat shock proteins in cancer: chaperones of tumorigenesis.” Trends Biochem. Sci. 31 (3): 164-172.
8: Terasawa, K., Minami, Y. (2006) “A client-binding site of Cdc37.” FEBS J. 272 (18): 4684-4690.

It is a conceptual diagram which shows the onset mechanism of ATLL. It is a conceptual diagram which shows the signal transduction mechanism which brings about NF-κB activation. Fig. A is a conceptual diagram showing that the Hsp90 / Cdc37 complex and the kinase kinase (Kinase) bind through an amino acid region (40-110 residues) called Kinase Biding Domain (KBD). is there. FIG. B is a conceptual diagram showing the binding mode of Hsp90 / Cdc37 complex and Tax, which is assumed from the results of Experimental Examples 3 and 4. FIG. A shows the results of Experimental Example 1 in which the interaction between Tax and the Hsp90 / Cdc37 complex or Hsp90 / Cdc37-deficient mutant / complex was examined. Lane 1 is control, lane 2 is expressing only Tax, lane 3 is when full length Cdc37 (Cdc37 (1-378)) is coexpressed with Tax, lanes 4-7 are missing each functional region Luciferase activity when co-expressed with mutant mutants (Cdc37 (1-200), Cdc37 (1-180), Cdc37 (181-378), Cdc37 (201-378)) in order from Lane 4 (Panel upper panel), Tax expression level (Panel middle panel), and Cdc37-deficient mutant expression level (Panel lower panel). Figure B shows the expression level of Tax by the Cdc37-deficient mutant (upper), the expression level of the Cdc37-deficient mutant (middle), and the expression level of Tubulin (lower) when only Tax is expressed (lane 2); Co-expressed with 1/4, 1/2, or equal amount of Cdc37 (1-200) (lanes 3-5); Tax and 1/4, 1/2, or equal amount of Cdc37 (1 -180) (lanes 6-8); Tax and 1/4, 1/2, or equivalent amount of Cdc37 (181-378) (lanes 9-11) (Experimental example 1) is shown. In Experimental Example 2, Tax, Hsp90 (lane 3), full-length Cdc37 (lane 4), and various Cdc37-deficient mutants (lane 5: Cdc37 (1-278), lane 6: Cdc37 (1-200), lane 7 : Cdc37 (1-180), lane 8: Cdc37 (181-378), lane 9: Cdc37 (201-378)) are expressed separately (middle panel, lower panel), and the cell lysates are mixed in equal amounts. The results of immunoprecipitation (upper panel) are shown. The result of Experimental example 3 is shown. Fig. A shows the inhibitory effect of 17-DMAG (see lanes 3-6) on NF-κB transcription enhancement (lane 2) induced by pLTR-Tax in 293 cells. Fig. C shows pLTR in 293 cells. The inhibitory effect of 17-DMAG on HTLV1-LTR transcriptional enhancement induced by -Tax (lane 2) (see lanes 4-6), Figure D shows the enhanced HTLV1-LTR transcription induced by pCMV-Tax in 293 cells ( The inhibitory effect of 17-DMAG on lane 2) is shown (see lanes 4-6). FIG. B shows the protein expression levels of Tax, Hsp90 and Tubulin. The result of Experimental example 4 which investigated the tax degradation induction | guidance | derivation in C8166 cell derived from ATL patient by 17-DMAG is shown. Fig. A shows the amount of expression of Tax (upper), the amount of IKK complex-Hsp90 complex formed (middle), and the amount of Hsp90 expression (lower) when various amounts of 17-DMAG (0-5 μM) are administered. Show. FIG. B is a graph showing the results of measuring the influence of 2.5 μM 17-DMAG on the stability of Tax over time. From the top, the expression level of Tax, the expression level of Hsp90, and the expression level of Tubulin are shown. Figure A shows the cell viability (%) when ATL patient blood (ATL01, ATL02), healthy adult blood (PBL1, PCL2), and C8166 were added with 2.5 μM 17-DMAG and cultured over time. (Experimental example 5). Moreover, FIG. B shows the result of having measured the relative caspase value (fold) which shows the enhancement of an apoptotic reaction about each blood sample. FIG. 10 is a conceptual diagram showing an experimental scheme of Experimental Example 6. The result of Experimental example 6 is shown. The result which verified the inhibitory effect with respect to the multiorgan invasion of ATL cell by 17-DMAG oral administration from the ATL cell number contained in PBL, a spleen, a liver, and a lung using an ATL onset model mouse is shown.

Claims (3)

A method for screening an active ingredient of an adult T cell leukemia / lymphoma onset inhibitor, characterized by using binding inhibition of Tax and Cdc37 as a CBD region as an index. A method for screening an active ingredient of an adult T cell leukemia / lymphoma onset inhibitor comprising the following steps (a) to (d):
(A) contacting a polypeptide containing at least 181 to 200 of the amino acid sequence of Cdc37 with Tax in the presence of a test substance;
(B) measuring the amount of binding between the polypeptide and Tax,
(C) The amount of binding obtained above is obtained by contacting a polypeptide containing at least positions 181 to 200 of the amino acid sequence of Cdc37 with Tax in the absence of the test substance (control binding). Based on the results of steps (d) and (d) and (c), a substance that decreases the binding level compared to the control binding level is selected from the test substances as the active ingredient of the ATLL development inhibitor. Process. Reagent kit for screening an active ingredient of an adult T cell leukemia / lymphoma onset inhibitor containing the following (i) to (iii):
(I) a polypeptide comprising at least positions 181 to 200 of the amino acid sequence of Cdc37,
(Ii) Tax,
(Iii) A reagent for detecting the binding between the polypeptide and Tax.

Images