WO2002015925A1 - Methof of regulating apoptosis and apoptosis-regulatory polypeptide - Google Patents

Methof of regulating apoptosis and apoptosis-regulatory polypeptide Download PDF

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WO2002015925A1
WO2002015925A1 PCT/JP2001/007179 JP0107179W WO0215925A1 WO 2002015925 A1 WO2002015925 A1 WO 2002015925A1 JP 0107179 W JP0107179 W JP 0107179W WO 0215925 A1 WO0215925 A1 WO 0215925A1
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amino acid
polypeptide
acid sequence
seq
activity
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PCT/JP2001/007179
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French (fr)
Japanese (ja)
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Takashi Tsuruo
Naoya Fujita
Saori Sato
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to AU2001280105A priority Critical patent/AU2001280105A1/en
Priority to JP2002520846A priority patent/JPWO2002015925A1/en
Publication of WO2002015925A1 publication Critical patent/WO2002015925A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to a method for controlling apoptosis, which comprises a step of controlling Akt activity in a cell, in particular, a step of controlling binding between Hsp90 and Akt, a polypeptide having an activity of controlling Akt activity in a cell,
  • the present invention relates to a DNA encoding the polypeptide, an antibody recognizing the polypeptide, a method for producing the polypeptide, a medicament containing the polypeptide or the antibody, and a method for using them.
  • Apoptosis originally means cell death actively caused by cells themselves under physiological conditions, and is deeply involved in morphology, tissue formation, homeostasis maintenance, biological defense during development, and cell division ( Proliferation) plays an important role in maintaining the life of an individual as two sides of the same coin.
  • Apoptosis is triggered by various physiological or pathological signals from outside or inside the cell. Inherited or acquired abnormalities in the process of death controlled by a gene are thought to induce various diseases by over-inducing or suppressing apoptosis.
  • Diseases associated with excessive cell death due to apoptosis include fulminant hepatitis and other viral diseases, neurodegenerative diseases such as Alzheimer's disease, and radiation damage. Conversely, diseases related to the onset of apoptosis suppression include cancer and autoimmune diseases.
  • cancer treatment and immunosuppressive therapy are being carried out, using the fact that radiation and drugs cause apoptosis.
  • protecting cells that play an important role in the living body by suppressing apoptosis from death is the key to suppressing cell death during ischemia in the brain and heart, and from normal cell death during anticancer drugs and radiation therapy. It can be used for medical treatment purposes such as protection of stomach.
  • Akt is phosphoinositide 3-kinase (PI3K) It is known as a serine / threonine kinase that functions in downstream signaling pathways.
  • PI3K phosphoinositide 3-kinase
  • three isoforms Aktl / PKB and Akt2 / PKB? Akt3 / PBa
  • Akt2 and Akt3 are about 82% identical in amino acid to Aktl, and the threonine and serine residues required for phosphorylation and activation are conserved. The division of functions among subtypes has not been disclosed at this time.
  • PI3K is activated in cells by stimulation with growth factors, such as cytokines, and converts PI 4,5 bisphosphonate [PI 4,5 bisphosphonate (PIP2)] to PI 3,4,5 triphosphonic acid [PI 3,4 , 5 triphosphonate (PIP3)].
  • PIP3 acts on Akt as a second messenger, and Akt activity is increased by phosphorylation of Aktl threonine residues 308 and 473 at Aktl.
  • Known substrates of activated Akt include Bad, caspase 9 and IkB kinase, and phosphorylation of these substrates inactivates kt to suppress cell apoptosis.
  • Akt activity may contribute to immortalization of cancer cells and apoptosis resistance.
  • Studies on the expression of Akt in cancer showed that Aktl was colorectal cancer (20% of gastric adenocarcinoma), Akt2 was ovarian cancer (15%) ⁇ knee cancer (12%), breast cancer (3%), and Akt3 was estrogen receptor. It has been reported that such genes are highly expressed in breast cancers lacking Yuichi and androgen-independent prostate cancer [Biochem. J., 1-13 (1998), J. Biol. Chem., 274, 21528]. -21532 (1999)] 0 also, brain, endometrial, prostate cancer, PTEN [Science is a tumor suppressor gene mutations in breast cancer and the like are known, 275.
  • PTEN has been shown to have the activity to dephosphorylate PIP3, which is subsequently generated by PI3K, and to suppress the signal from PI3K to Akt (Cell, 05, 29-39 (1998) ].
  • Loss of PTEN activity in cells is thought to cause Akt homeostatic activation and suppress apoptosis to trigger cancer. Therefore, substances that suppress Akt activity induce apoptosis in cancer cells. It is considered to be useful as a therapeutic drug or a drug that enhances the sensitivity of cancer cells to apoptosis induced by anticancer drugs currently used for cancer treatment
  • Aktl activity is regulated by the balance between phosphorylation and dephosphorylation. It has been shown that protein phosphatase 2A (PP2A) is involved in Aktl dephosphorylation [Proc. Natl. Acad. Sci. USA, 5699-5704 (1996)].
  • P2A protein phosphatase 2A
  • Akt activation regulates glucose metabolism 6-phosphofructose 2-force rice [6-p osphofmctose 2 -kinase (PFK2)] activation caused by insulin stimulation, glucose metabolism enhancement, glucose transporter 4 involved in intracellular glucose uptake [glucose transporter 4] (GLUT4)] and PI3K-mediated suppression of glycogen synthesis due to increased glucose uptake by membrane localization and inactivation of glycogen synthase kinase-3 (GSK3). It has been reported that Akt activation is involved [Biochem. J., 3, 1-13 (1998)]. Therefore, it is considered that Akt-activating substances may be effective for diabetes and the like caused by abnormal glucose metabolism.
  • Heat shock protein is a group of proteins that are expressed in cells when cells are exposed to stress environments such as heat shock, and can be classified into several families according to their molecular weight (Hsp90, Hsp70, Hsp27 etc.). These proteins are It is also called a chaperone, and its main functions are generally considered to be protein folding, membrane permeation, association, and suppression of aggregation.
  • Hsp90 is a heat shock protein belonging to the Hsp90 family composed of a molecular chaperone with a molecular weight of about 90 kDa.
  • Hsp90, Hsp90 ?, Grp94, Hsp75 / TRAP1, etc. have been identified as eukaryotic Hsp90 families, but these Hsp90 family proteins are expressed in cells even in an environment without extracellular stress.
  • Hsp90 specifically forms complexes with various signal transduction factors involved in cell proliferation and canceration and contributes to the cell cycle and cell proliferation signals.
  • Hsp90 Signaling factors that are thought to require binding to the Hsp90 family to maintain intracellular function and stability include steroid hormone receptors (estrogens receptor, progesterone receptors, glucocorticoids). Drecept Yuichi), non-receptor tyrosine kinases (eg, v-src), receptor tyrosine kinases (eg, EGFR, ErbB2), serine threonine kinase (Raf-1, Cdk4, etc.). Therefore, it is thought that by controlling the function of Hsp90, intracellular signal transduction involving an intracellular signaling factor that specifically binds to Hsp90 can be varied.
  • Hsp90 family protein is increased in various cancer cell lines, and the correlation between Hsp90 expression level and prognosis in breast cancer patients has been reported. It is thought that it may play an important role in disease [Pharmacology & Therapeutics, 2, 129-168 (1997), Biochem. Pharmacol., 675-682 (1998), Investigational New Drugs, 11, 361 -373 (1999)].
  • Examples of compounds that bind to Hsp90 and inhibit its function include geldanamycin, herbimycin A and other ansamycin-based compounds and their derivatives, radicicol and its derivatives, and novobiocin. [Investigational New Drugs, 11, 361-373 (1999), J. Natl. Cancer Inst., 92, 242-248 (2000)].
  • Geldanamycin ⁇ ⁇ radicicol has been shown to bind to the ATP / ADP binding region located in the N-terminal region of Hsp90, which is contained in the amino acid sequence from position 221 to position 221). [Cell, m, 239-250 (1997), J. Biol.
  • Hsp90 functions together with co-chaperone molecules such as p50 / Cdc37 and p23 to form a molecular complex with the above-mentioned specific signaling factors, but low-molecular-weight compounds such as geldanamycin ⁇ radicicol
  • co-chaperone molecules such as p50 / Cdc37 and p23
  • low-molecular-weight compounds such as geldanamycin ⁇ radicicol
  • the present invention identifies functional sites on Hsp90 that are involved in the inhibition of apoptosis and the control of Akt activity involved in glucose metabolism, and is used to identify gastric cancer, ovarian cancer, breast cancer, breast cancer, prostate cancer caused by abnormal apoptosis control and glucose metabolism control signals.
  • Therapeutic, prophylactic and diagnostic agents for malignant tumors such as cancer, diabetes, etc., cell death inhibitors during ischemia of the brain, heart, etc., anticancer agents and agents to protect normal cells from cell death during radiation therapy Or to provide agents that enhance the effects of existing therapeutic agents.
  • the present inventors have found for the first time that Hsp90 binds to Akt in a cell, and that the binding of Hsp90 to Akt is important for maintaining Akt activity (phosphorylation). Further, the present invention has been completed by identifying a functional site on Hsp90 involved in the control of Akt activity involved in apoptosis suppression and glucose metabolism, and an Akt site binding to the functional site.
  • the present invention relates to the following inventions (1) to (40).
  • a method for controlling apoptosis which comprises a step of controlling Akt activity in a cell.
  • the control of Akt activity in a cell is carried out by a polypeptide consisting of the 327th to 340th amino acid sequence of SEQ ID NO: 1 or the polypeptide consisting of 335 to 348th amino acid sequence of SEQ ID NO: 3; The method according to any one of 1) to (4).
  • one or more amino acids are deleted or substituted at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3.
  • the method according to any one of (1) to (4) above, wherein the method is performed using a polypeptide comprising an inserted or added amino acid sequence and having an activity of controlling Akt activity in a cell.
  • the control of intracellular Akt activity is at least 60% homologous to the amino acid sequence at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3. Carried out by a polypeptide comprising an amino acid sequence having a property and having an activity of controlling intracellular Akt activity.
  • the control of Akt activity in the cell is from the 229th position to the 309th position of the amino acid sequence of SEQ ID NO: 5, the 23rd position to the 310th position of the amino acid sequence of SEQ ID NO: 7, or The method according to the above (2) or (4), which is carried out using a polypeptide consisting of the amino acids 227 to 306 of the amino acid sequence of SEQ ID NO: 9.
  • the control of Akt activity in the cell is from the 289th to the 309th of the amino acid sequence of SEQ ID NO: 5, from the 290th to the 310th of the amino acid sequence of SEQ ID NO: 7, or The method according to (2) or (4), which is performed using a polypeptide consisting of positions 286 to 306 of the amino acid sequence of SEQ ID NO: 9.
  • the control of Akt activity in the cell is caused by the amino acid sequence of SEQ ID NO: 5 from the 229th position to the 309th position, the amino acid sequence of SEQ ID NO: 7 from the 23rd position to the 310th position, Alternatively, it has an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added at positions 227 to 306 of the amino acid sequence of SEQ ID NO: 9, and has an activity of controlling Akt activity in a cell.
  • the regulation of intracellular Akt activity is from the 229th position to the 309th position of the amino acid sequence of SEQ ID NO: 5, the 23rd position to the 310th position of the amino acid sequence of SEQ ID NO: 7, or A polyamino acid sequence comprising an amino acid sequence having at least 60% homology with the amino acid sequence at positions 227 to 306 of the amino acid sequence set forth in SEQ ID NO: 9, and having an activity of controlling intracellular Akt activity.
  • the control of intracellular Akt activity is carried out by a compound that fluctuates the activity of the polypeptide described in any one of the above (5) to (15), which regulates intracellular Akt activity, The method according to (1) or (2) above.
  • One or more amino acids are deleted at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3.
  • a polypeptide comprising an amino acid sequence substituted, inserted or added, and having an activity of controlling Akt activity in a cell.
  • a polypeptide comprising a sequence and having an activity of controlling Akt activity in a cell.
  • polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added at the 7th to 30th positions, and which has an activity of controlling Akt activity in a cell.
  • polypeptide consisting of an amino acid sequence having 60% or more homology with the amino acid sequence from the 7th to the 36th amino acid and having an activity to control intracellular Akt activity.
  • (27) A DNA that hybridizes with the DNA of (26) under stringent conditions and encodes a polypeptide having an activity of controlling intracellular Akt activity.
  • the transformant according to (29) is cultured in a medium, a polypeptide capable of controlling Akt activity in cells is produced and accumulated in the culture, and the polypeptide is separated from the culture.
  • (19) - (2 5) contains a step of contacting the cell with a test sample which expresses a polypeptide according to any one of the control of Akt activity in cells of said polypeptide A method for screening a compound that changes the activity of the compound.
  • a pharmaceutical composition comprising the antibody or antibody fragment according to 33) and an anticancer agent as active ingredients.
  • Anticancer drugs include psulfan, cyclophosphamide, ifosfamide, melphalan, nitrosoperrea, amsacrine, carboplatin, cisplatin, dacarbazine, azacytidine, cladribine, sieverabine, fludarabine, fluorouracil, hydroxyperia, leuco At least one selected from methotrexate, actinomycin D, bleomycin, daunorubicin, doxorubicin, mitomycin C, mitoxantrone, etoposide, paclitaxel, teniposide, vinblastine, vincristine, vindesine, geldanamycin and peracidicol
  • the pharmaceutical composition according to any one of the above (37) to (39), wherein In cells, when Akt is phosphorylated, Akt becomes stable and apoptosis is suppressed. When Akt is dephosphorylated, Akt becomes unstable and a
  • phosphorylated Akt is stabilized by binding to Hsp90. Furthermore, the fact that phosphorylated Akt binds to Hsp90 and maintains a stable state is due to the involvement of the phosphatase action site on Akt and the binding region between Hsp90 and Akt. was also found. From the above, it is possible to control apoptosis by controlling the intracellular Akt activity, particularly by controlling the binding between Hsp90 and Akt, thereby controlling the intracellular Akt activity.
  • “controlling intracellular Akt activity” includes increasing or decreasing intracellular Akt activity, for example, controlling the binding between Hsp90 and Akt, stabilizing phosphorylated Akt, It is performed by dephosphorylation of Akt.
  • Controlling the binding between Hsp90 and Akt means to stabilize or inhibit (particularly competitive inhibition) the binding between Hsp90 and Akt, and to stabilize or promote the binding between Hsp90 and Akt. Including. “Controlling apoptosis” also includes inducing or suppressing apoptosis.
  • the method for controlling the binding between Hsp90 and Akt is not particularly limited, but preferably includes Hsp90 or a partial polypeptide thereof, a partial polypeptide of Akt, a compound, a peptide, an antibody and an antibody fragment that inhibits the binding of Hsp90 to Akt. It is controlled by adding.
  • Hsp90 is not particularly limited, and any protein may be used as long as it belongs to the Hsp90 family, but Hsp903 or Hsp90 is preferably used.
  • Hsp90 5 and Hsp90 are proteins whose amino acid sequences are represented by SEQ ID NOS: 1 and 3, respectively, and whose base sequences are represented by SEQ ID NOs: 2 and 4, respectively.
  • the partial polypeptide of Hsp90 that controls apoptosis includes a polypeptide consisting of positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or a polypeptide consisting of positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3 (
  • a partial Hsp90 peptide having an activity of controlling Akt activity in cells is abbreviated as “Hsp90 polypeptide of the present invention”.
  • Akt is phosphorylated and apoptosis is suppressed.
  • Hsp90 polypeptide of the present invention binds to Akt but a dephosphorylating enzyme acts on Akt, Akt is dephosphorylated and apoptosis is induced.
  • the binding of Hsp90 to Akt can be controlled by a partial polypeptide of Akt. Is more preferable.
  • Akt is not particularly limited, and may be any protein as long as it belongs to the Akt family.
  • Aktl, Akt2 or Akt3 is used.
  • Aktl, Akt2 and Akt3 are proteins whose amino acid sequences are represented by SEQ ID NOS: 5, 7 and 9, respectively, and whose nucleotide sequences are represented by SEQ ID NOs: 6, 8 and 10, respectively.
  • the partial polypeptide of Akt that controls apoptosis includes amino acids 229 to 309 of the amino acid sequence described in SEQ ID NO: 5, 231 to 310 of the amino acid sequence described in SEQ ID NO: 7, or SEQ ID NO:
  • a polypeptide consisting of the amino acid sequence from position 227 to position 306 of the amino acid sequence 9 is preferred, and in particular, from position 289 to position 309 of the amino acid sequence of sequence number 5, from position 290 to position 310 of the amino acid sequence of sequence number 7, or Amino acid sequence described in SEQ ID NO: 9
  • Polypeptide consisting of positions 286 to 306 hereinafter, a partial Akt polypeptide having an activity of controlling intracellular Akt activity is abbreviated as “Akt polypeptide of the present
  • Akt polypeptide of the present invention As a result of binding of the Akt polypeptide of the present invention to Hsp90, the amount of Akt bound to Hsp90 decreases. Akt not bound to Hsp90 is induced by a phosphatase, and then apoptosis is induced.
  • Hsp90 polypeptide and Akt polypeptide of the present invention are abbreviated as “polypeptide of the present invention”
  • Polypeptides comprising a substituted, inserted or added amino acid sequence and having an activity to control intracellular Akt activity are disclosed in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current 'protocols', 'molecular' biology), Nuc. Acid. Res., 10, 6487 (1982), Proc. Natl. Acad.
  • Deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence constituting the polypeptide of the present invention means that any one or more amino acid sequences in the same sequence Means that there is a deletion, substitution, insertion or addition of one or more amino acid residues at the position, the deletion, substitution, insertion or addition may occur simultaneously, Amino acids can be either natural or non-natural.
  • Natural amino acids include L-alanine, L-asparagine, L-asparaginic acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine , L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
  • amino acid residues that can be substituted for each other.
  • Amino acid residues included in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, norin, norvaline, alanine, 2-aminobutanoic acid, methionine, 0-methylserine, t-butylglycine, t-butylylalanine, cyclohexylalanine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
  • Group D lysine, arginine, ordinine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
  • Group E Proline, 3-hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoserine
  • Group G fenirualanine, tyrosine
  • polypeptide of the present invention In order for the polypeptide of the present invention to have an activity of controlling Akt activity in a cell, When calculated using the amino acid sequence of the polypeptide of the present invention and BLAST [J. Mol. Biol., 2, 403 (1990)] or FASTA [Methods Enzymol., 1M, 63-98 (1990)], etc. It has a homology of at least 60% or more, usually 80% or more, preferably 90% or more, and particularly preferably 95% or more.
  • the present invention also relates to a DNA encoding the polypeptide of the present invention, and a DNA that hybridizes with the DNA under stringent conditions and has an activity of controlling Akt activity in a cell.
  • DNA hereinafter abbreviated as DNAj of the present invention.
  • the DNA encoding the polypeptide of the present invention includes the nucleotides 979 to 1020 of the nucleotide sequence of SEQ ID NO: 2, the nucleotides 1003 to 1044 of the nucleotide sequence of SEQ ID NO: 4, and the nucleotide of SEQ ID NO: 6. 685 to 927, preferably 865 to 927 of the sequence, 691 to 930, preferably 868 to 930 of the salt tomb sequence described in SEQ ID NO: 8, and 679 of the base sequence described in SEQ ID NO: 10 And a DNA having a base sequence from 856th to 918th, preferably from 856th to 918th.
  • a large number of DNAs having a base sequence corresponding to the amino acid sequence of the polypeptide of the present invention other than those described above are all included in the present invention.
  • DNA capable of hybridizing under stringent conditions refers to a DNA having a base sequence corresponding to the amino acid sequence of the polypeptide of the present invention as a probe, a colony hybridization method, a plaque hybridization method. Or DNA obtained by using the Southern blot hybridization method or the like. Specifically, using a filter on which DNA derived from colonies or plaques is immobilized, 0.7 to 1.0 mol / After performing the hybridization at 65 ° C in the presence of sodium chloride in step 1, the SSC solution from 0 :! to 2x concentration (1x concentration of SSC solution is 150 mmol / 1 sodium chloride, (consisting of 1 mmol / sodium citrate) and washing the filter under 65 ° C conditions to obtain DNA that can be identified.
  • DNAs having a homology of preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more can be mentioned.
  • the binding between Hsp90 and Akt is measured by the following method. That is, after adding DNA encoding the tag to DNA encoding the full length or partial fragment of Hsp90 and Akt, the DNA is introduced into cells, and the cells are cultured. After lysing the cells, an immunoprecipitation reaction is performed using an antibody against the tag. Identification of substances in immunoprecipitates by Western-blotting allows Hsp90 and Akt.
  • the state of binding to Akt can be measured.
  • Immunoprecipitation refers to a reaction in which an antigen-antibody complex is formed by reacting a protein with an antibody thereto.
  • Western blotting means that proteins are separated by polyacrylamide gel electrophoresis, and the proteins on the gel are transferred to a nitrocellulose membrane, polyvinylidene 'difluoride membrane, etc., and the proteins are detected using antibodies against the proteins. Refers to the method.
  • the immunoprecipitate of the monoclonal antibody against the tag contains a complex of the Hsp90 partial protein and the Akt protein. Will be obtained. Therefore, after subjecting the complex to polyacrylamide gel electrophoresis, the gel was transferred to a nitrocellulose membrane, and then subjected to an antibody against Hsp90 or a tag attached to Hsp90. By using such an antibody, it can be confirmed that Hsp90 has bound to the Akt protein.
  • the tag may be any tag such as a FLAG tag, V5 tag, HA tag, and the like.
  • a method for measuring Akt activity in cells includes a method for measuring phosphorylation of Akt. That is, after adding DNA encoding a tag to DNA encoding the full length or partial fragment of Hsp90 and Akt, the DNA is introduced into cells, and the cells are cultured. After lysing the cells, an immunoprecipitation reaction is performed using an antibody against the tag. The substance in the immunoprecipitate is identified by Western plotting, and the phosphorylation of Akt can be measured by using an antibody against phosphorylated Akt.
  • Methods for measuring apoptosis include a method of extracting chromosomal DNA in cells and analyzing the chromosomal DNA by electrophoresis, a method of staining cells and analyzing them by morphological observation, and a method of apoptosis. Examples include a method of measuring protease, which is essential for induction.
  • DAPI 6-diamino-2-phenylindole
  • Methods for measuring protease which is essential for apoptosis induction, include a method for measuring caspase [Oncogene, 11, 1295 (1998)]. Specifically, cells are lysed in a lysis solution containing 10 mM HEPES (pH 7.4), 2 mM EDTA, and 0.1% CHAPS. After reacting the cell lysate with acetyl-L-aspartyl-L-glucamyl-L-valyl-L-aswald-7-amino-4-methylcoumarin (DEVD-AMC) in ICE buffer, absorbance Caspase activity can be measured by measuring with a meter.
  • DEVD-AMC acetyl-L-aspartyl-L-glucamyl-L-valyl-L-aswald-7-amino-4-methylcoumarin
  • absorbance Caspase activity can be measured by measuring with a meter.
  • Examples of the method for producing the polypeptide of the present invention include a method using chemical synthesis and a method using a genetic method. This will be described in detail below.
  • polypeptides of the present invention are described, for example, in "Basics and Experiments of Peptide Synthesis” (Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Michinori Waki: Maruzen), “Fourth Edition Experimental Chemistry Course 22—Organic Synthetic IV Acids” -Amino acids and peptides "(Saburo Aimoto, Shoichi Kusumoto, Kuniaki Tatsuta, Yoshihiro Hayakawa, Keizo Yamamoto, Tateaki Wakamiya: Maruzen),” In Yuichi National Journal of Applied and Fudo " Mouth Tin 'Resina (International Journal oi Peptide Protein Research),, 161-214 (1990)', 'Solid-Phase Peptide Synthesis', Method's 'In' Enzymology I Vol.
  • Specific synthesis methods include the azide method, the acid chloride method, the acid anhydride method, the mixed acid anhydride method, the DCC method, the active ester method, the carboimidazole method, and the redox method.
  • both the solid phase synthesis method and the liquid phase synthesis method can be applied. That is, the desired polypeptide is synthesized by condensing the amino acid constituting the polypeptide of the present invention with the remaining portion and, if the product has a protecting group, removing the protecting group.
  • the amino acid residue constituting the polypeptide of the present invention is chemically modified or protected on the side chain and / or the peptide amino terminal and / or the peptide carboxy terminal
  • the peptide is chemically modified after synthesis.
  • a method known in the field of peptide synthesis chemistry such as synthesis of peptides using chemically modified amino acids or appropriate selection of reaction conditions for final deprotection of peptide synthesis (“Peptide synthesis”).
  • the polypeptide of the present invention can also be synthesized using an automatic peptide synthesizer.
  • Peptide synthesis using a peptide synthesizer is performed on a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation, or a peptide synthesizer manufactured by Advanced ChemTech Inc. (USA).
  • a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation, or a peptide synthesizer manufactured by Advanced ChemTech Inc. (USA).
  • N-Fmoc-amino acid or N-Boc-amino acid with appropriately protected side chains the synthesis can be carried out according to the respective synthesis programs.
  • polypeptide of the present invention can be purified by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the polypeptide of the present invention was prepared by the method described in Molecular Biology, Molecular Cloning Second Edition or Current Protocol, Inc. Molecular Biology, etc.
  • a DNA fragment of an appropriate length containing a portion encoding the polypeptide is prepared.
  • a DNA is prepared by substituting the nucleotide sequence of the portion encoding the polypeptide of the present invention so that the nucleotide sequence becomes an optimal codon for expression in a host cell.
  • the DNA is useful for efficient production of the polypeptide of the present invention.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length cDNA downstream of a promoter in an appropriate expression vector.
  • the recombinant vector is introduced into a host cell compatible with the expression vector.
  • any cells that can express the target gene such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
  • the expression vector contains a promoter at a position where it can replicate autonomously in the host cell or can be integrated into the chromosome, and can transcribe DNA encoding the polypeptide of the present invention. Things are used.
  • the recombinant vector containing the DNA encoding the polypeptide of the present invention is capable of autonomous replication in the prokaryote and at the same time, is promoted. It is preferable that the vector be composed of a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence. A gene that controls a promoter may be included.
  • expression vectors include pBrp2, pBTacl, and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega PQE-8 (manufactured by QIAGEN), pKYPIO (JP-A-58-110600), pKYP200 [Agric. Biol. Chem., 669 (1984)], pLSAl [Agric. Biol. Chem., 277 ( 1989)], GELl [Proc. Natl. Acad. Sci.
  • the promoter may be any promoter that functions in the host cell.
  • ⁇ E _ promoter evening one P trp;
  • la promoter P L promoter
  • P R promoter Isseki one T7 promoter Isseki one such, be given promo one evening one derived from Escherichia coli or phage, etc.
  • the promoter evening one obtained by two series of P trp (P trp X 2) , tac promoter Isseki one, LaeT7 promoter Isseki one, k ⁇ I artificially designed modified promoter Isseki one as a promoter Etc. can also be used.
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosomal binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
  • a transcription termination sequence is not always necessary for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
  • Host cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., such as Escherichia coli XLa-Blue, Escherichia coli XL2-Blue Escherichia coli DH1, Escherichia coli MC I 000, Escherichia coli KY3276 N Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli ⁇ ⁇ o.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia Escherichia coli GI6 coli ficaria ⁇ Serratia fonticoia ⁇ Serratia liquefaciens ⁇ Serratia marcescens.Bacillus subtilis, Bacillus am loliquefacines
  • Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, fifi, 2110 (1972)], the protoplast method (Sho 63-248394), or the methods described in Gene, 11, 107 (1982) and Molecular & General Genetics, Hyundai, 111 (1979).
  • examples of expression vectors include YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), pHS19, and pHS15.
  • any promoter can be used as long as it can be expressed in yeast strains.
  • promoters for glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP Promo One Night, ADH Promo One, gal 1 Promo One, Gal 10 Promo One, Heat Shock Polypeptide Motor, MFI Promoter, CUP One Promo One, etc.
  • any method can be used as long as it is a method for introducing DNA into yeast.
  • the method include an elect-portion method [Methods Enzymol., 1Q4, 182 (1990)] and a spheroplast method. [Proc. Natl. Acad. Sci. USA, 15., 1929 (1978)], lithium acetate method [J. BacterioL, 1, 163 (1983)], Proc. Natl. Acad. Sci. USA, IS, 1929 ( 1978)].
  • any promoter can be used as long as it functions in animal cells.
  • the promoter of the cytomegalovirus (CMV) IE (immediate early) gene and the early promoter of SV40 can be used.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • host cells examples include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells, and HBT5637 (63-299).
  • any method for introducing a recombinant vector into animal cells any method can be used as long as DNA can be introduced into animal cells.
  • electroporation method Cytotechnology, 133 (1990)
  • calcium phosphate method Japanese Patent Application Laid-Open No. 2-227075
  • lipofection method Proc. Natl. Acad. Sci. USA, S4, 7413 (1987), Virology, 52, 456 (1973)] and the like.
  • an insect cell When an insect cell is used as a host, for example, the current protocol 'In' Morefu-Noroshi, Bacxuovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology , fi, 47 (1988), etc., to express the polypeptide. That is, a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected into insect cells to express the polypeptide. be able to.
  • Examples of the gene transfer vector used in the method include pVL1392, pVLl393, pBlueBacIII (all manufactured by Invitoi'ogen) and the like.
  • Baculoviruses are, for example, viruses that infect night roth moths Art Grapher's Force Reformi Force ⁇ Nuclea ⁇ Polyhemdosis ⁇ Virus
  • Methods for co-transferring the above-described recombinant gene into insect cells and the above baculovirus to prepare a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [ Proc. Natl. Acad. Sci. USA, M, 7413 (1987)].
  • examples of the expression vector include Ti plasmid and tobacco mosaic virus vector.
  • Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the 35S promoter of cauliflower mosaic virus (CaMV), the inactin 1 promoter, and the like.
  • Examples of the host cell include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfa-alpha, rice, wheat, and wheat.
  • any method for introducing DNA into plant cells can be used.
  • Agrobacterium Agrobacterium ⁇ (Japanese Patent Application No. 59-140885, Japanese Patent Application Laid-Open No. 70080, WO 94/00977), the electoral poration method (JP-A-60-251887), a method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), and the like.
  • a method for expressing a gene in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition.
  • polypeptide with a sugar chain added thereto When expressed by yeast, animal cells, insect cells or plant cells, a polypeptide with a sugar chain added thereto can be obtained.
  • the polypeptide of the present invention is produced by culturing the transformant of the present invention obtained as described above in a medium, producing and accumulating the polypeptide of the present invention in the culture, and collecting from the culture. can do.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • the transformant of the present invention is a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host
  • the transformant is used as a medium for culturing the transformant.
  • Either a natural medium or a synthetic medium may be used as long as the medium contains an assimilable carbon source, a nitrogen source, inorganic salts, and the like, and can efficiently culture the transformant.
  • the carbon source may be any as long as the transformant can be assimilated, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digests thereof can be used.
  • potassium (I) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • the culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH during the culturing is preferably maintained at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia, and the like.
  • an antibiotic such as ambicillin or tetracycline may be added to the medium during the culture.
  • Transform with a recombinant vector using an inducible promoter as a promoter When culturing the isolated microorganism, if necessary, an inducer may be added to the medium. For example, when culturing a microorganism transformed with a recombinant vector using a promoter, isoforms such as isopropyl-/?-D-thiogalactovyranoside are transformed with a recombinant vector using the trp promoter. When culturing the transformed microorganism, indoleacrylic acid or the like may be added to the medium.
  • RPMI1640 medium As a medium for culturing transformants obtained using animal cells as a host, commonly used RPMI1640 medium [The Journal of the American Medical Association, 122., 519 (1967)], Eagle's MEM medium [Science , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, l, 1 (1950)] or fetal bovine serum etc. And the like can be used.
  • the cultivation is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C; and in the presence of 5% CO 2 .
  • antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
  • Culture media for transformants obtained using insect cells as a host include commonly used ⁇ - ⁇ media (Pharmingen), Sf-900 II SFM medium (Life Technologies), Ex Cell 400 And Ex Cell 405 (both manufactured by JRH Biosciences), Grace's Insect Medium [Nature, 1, 788 (1962)] and the like. Cultivation is usually carried out for 1 to 5 days under conditions of pH 6 to 7 and 25 to 30 ° C.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ.
  • a culture medium for culturing the transformant a commonly used Murashige and Skog (MS) medium, a white (White) medium, or a plant formone such as auxin or cytokinin added to these mediums is used. Media or the like can be used.
  • Cultivation is usually performed at pH 5 to 9 and 20 to 40 ° C for 3 to 60 days. If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
  • a transformant derived from a microorganism, an animal cell, or a plant cell having a recombinant vector into which a DNA encoding the polypeptide of the present invention has been incorporated is cultured according to a conventional culture method, and the polypeptide is obtained. Is produced and accumulated, and the polypeptide is collected from the culture, whereby the polypeptide can be produced.
  • a method for expressing the gene, secretory production, fusion polypeptide expression, and the like can be performed according to the method described in Molecular 'Cloning 2nd edition, in addition to direct expression.
  • the polypeptide of the present invention can be produced in a host cell, secreted outside the host cell, or produced on the host cell outer membrane.
  • the host cell to be used or the polypeptide to be produced can be used.
  • the method can be selected by changing the structure of the peptide.
  • polypeptide of the present invention When the polypeptide of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 2 ⁇ 4, 17619 (1989)] and the method of Lowe et al. [Pro Natl. Acad. Sci. USA, M, 8227 (1989), Genes Develop., 4, 1288 (1990) ⁇ , or the method described in JP-A-5-336963, WO 94/23021, etc.
  • the polypeptide can be actively secreted out of the host cell.
  • the polypeptide of the present invention is expressed in a form in which a signal peptide is added in front of the polypeptide containing the active site of the polypeptide of the present invention using a gene recombination technique, whereby the polypeptide of the present invention can be expressed in host cells. It can be actively secreted out. Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the transgenic animal or plant, an animal into which the gene has been introduced (transgenic non-human animal) or a plant (transgenic plant) can be created. The polypeptide of the present invention can also be produced using an individual.
  • the polypeptide can be produced by producing or accumulating the polypeptide, and collecting the polypeptide from the animal or plant individual.
  • a method for producing the polypeptide of the present invention using an animal individual for example, known methods [American Journal of Clinical Nutrition, fiS, 639S (1996), American Journal of Clinical Nutrition, ⁇ 3, 627S (1996), Bio / Technology, S, 830 (1991)] to produce the polypeptide of the present invention in an animal constructed by introducing a gene.
  • a transgenic non-human animal into which DNA encoding the polypeptide of the present invention has been introduced is bred, and the polypeptide is produced and accumulated in the animal.
  • the polypeptide By collecting the polypeptide, the polypeptide can be produced.
  • the place of production and accumulation in the animal include milk (eg, JP-A-63-309192) and eggs of the animal.
  • the promoter used at this time any promoter that can be expressed in an animal can be used.
  • a promoter specific to mammary gland cells, a casein promoter, a casein promoter, A lactoglobulin promoter and a whey acid protein promoter are preferably used.
  • a transgenic plant into which DNA encoding the polypeptide of the present invention has been introduced can be prepared by a known method [tissue culture, 20 (1994)]. Tissue culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)], producing and accumulating the polypeptide in the plant, and collecting the polypeptide from the plant. By doing so, a method for producing the polypeptide can be mentioned.
  • a general enzyme isolation and purification method can be used.
  • the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton.
  • the cells are disrupted using a Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, normal enzyme isolation and purification
  • the cells When the polypeptide is expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to collect the insoluble form of the polypeptide as a precipitate fraction. .
  • the insoluble form of the recovered polypeptide is solubilized with a protein denaturant.
  • the polypeptide is returned to a normal tertiary structure by diluting or dialyzing the lysate and reducing the concentration of the protein denaturant in the lysate. After this operation, a purified preparation of the polypeptide can be obtained by the same isolation and purification method as described above.
  • the polypeptide of the present invention or a derivative such as a polypeptide having a sugar chain added to the polypeptide is secreted extracellularly, the polypeptide or the polypeptide is added to the culture supernatant.
  • Derivatives of the peptide can be recovered. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. You can get it.
  • polypeptide examples include, for example, amino acids 327 to 340 of the amino acid sequence of SEQ ID NO: 1, amino acids 335 to 348 of the amino acid sequence of SEQ ID NO: 3, and amino acids of SEQ ID NO: 5. 229 to 309 and 289 to 309 of the sequence; 231 to 310 and 290 to 310 of the amino acid sequence described in SEQ ID NO: 7, and the amino acid sequence 227 to 306 of SEQ ID NO: 9 It is better to give polypeptides consisting of 286th to 306th. 2.
  • the DNA of the present invention can be prepared by using the method described in Molecular 'Cloning Second Edition or Current Protocols' in Molecular Biology, etc. Alternatively, it can be prepared from cells.
  • the human or non-human animal tissue or cell may be any tissue or cell in which Akt or Hsp90 is expressed, and examples include brain, testis, heart, lung and the like.
  • Total RNA or mRNA is prepared from tissues or cells of a human or non-human animal. Prepare a cDNA library from the prepared total RNA or mRNA.
  • a gene fragment which encodes the polypeptide of the present invention by PCR using the prepared cDNA library as a ⁇ type by preparing a degiene primer based on the amino acid sequence of the polypeptide of the present invention To get.
  • the cDNA library can be screened to obtain the DNA of the present invention encoding the peptide of the present invention.
  • the mRNA of human or non-human animal tissues or cells may be a commercially available one (for example, manufactured by Clontech), or may be prepared from human or non-human animal tissues or cells as described above. .
  • guanidine thiocyanate-cesium trifluoroacetate method As methods for preparing total RNA from tissues or cells of human or non-human animals, guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, l, 3 (1987)], guanidine acid thiocyanate, phenol-clo Oral form (AGPC) method [Analytical Biochemistry, 1Q2, 156 (1987), Experimental Medicine, S, 1937 (1991)].
  • oligo (dT) -immobilized cellulose column method Molecular 'Cloning 2nd edition
  • mRNA can be prepared by using a kit such as the Fast Track mRNA Isolation Kit (Invitrogen) or the Quick Prep mRNA Purification Kit (Pharmacia).
  • a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA. Methods for preparing a cDNA library include methods described in Molecular 'Clothing Second Edition, Current Protocols' In 'Molecular' Biology, A Laboratory Manual, 2nd Ed. (1989), or commercially available. Examples of such kits include a method using the is: Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) and a ZAP-cDNA Synthesis Kit (STRATAGENE).
  • any phage vector, plasmid vector or the like can be used as long as it can replicate autonomously in E. coli K12 strain.
  • ZAP Express [Stratagene, Strategies, 5, 58 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Research, 11, 9494 (1989)]
  • Lambda ZAP II (Stratagene), gtl0, A gtll [DNA cloning, A Practical Approach, 1, 49 (1985)]
  • human riplEx (Clontech), ⁇ ExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (Mol. Cell. Biol) ., S, 280 (1983)]
  • pUC18 Gene,, 103 (1985)].
  • Escherichia coli As the Escherichia coli to which the vector into which the cDNA is incorporated is introduced, any microorganisms belonging to Escherichia coli can be used. Specifically, Escherichia coli XL1-Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, SS, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 ( 1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Kschericliia coli K802 [J. Mol. Biol., 16, 118 (1966)] and Escherichia coli JM105 [Gene,, 275 (1985)].
  • This cDNA library may be used as it is for subsequent analyses, but in order to reduce the proportion of incomplete-length cDNA and obtain full-length cDNA as efficiently as possible, the oligocap method [Gene, Michi'171 (1994) , Gene, Fiber, 149 (1997), Protein Nucleic Acid Enzyme, 603 (1996), Experimental Medicine, 11, 2491 (1993), cDNA Cloning, Yodosha (1996), Gene Ripplery, Yodosha ( 1994)] may be used for the following analyses. Based on the amino acid sequence of the polypeptide of the present invention, a degenerative primer specific to the nucleotide sequence at the 5 'end and 3' end of the nucleotide sequence predicted to encode the amino acid sequence is prepared.
  • a gene fragment encoding the polypeptide of the present invention can be obtained by amplifying DNA using the prepared cDNA library type I as a type II and PG method [PCR Protocols, Academic Press (1990)]. That the obtained gene fragment is a DNA encoding the polypeptide of the present invention can be determined by a commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Pro Natl. Acad. Sci. USA, 24, 5463 ( 1977)] or by using a base sequence analyzer such as ABIPRISM377 DNA Sequencer (manufactured by PE Biosystems).
  • cDNA or cDNA libraries synthesized from mRNAs contained in tissues or cells of human or non-human animals are subjected to colony hybridization / plaque hybridization (Molecular Cloning). Inning (2nd edition), the DNA of the present invention can be obtained.
  • a cDNA or cDNA library synthesized from mRNA contained in tissues or cells of a human or non-human animal can also be obtained by performing screening using the method.
  • the nucleotide sequence of the obtained DNA of the present invention is analyzed from the end by a nucleotide sequence analysis method usually used, for example, the dideoxy method of Sanger et al. [Pro Natl. Acad. Sci. USA, 24, 5463 (1977)] or ABIPRISM377.
  • the base sequence of the DNA is determined by analysis using a base sequence analyzer such as a DNA sequencer (manufactured by PE Biosystems).
  • the present invention By searching the base sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST based on the determined base sequence of the cDNA, the present invention
  • the gene encoding the polypeptide can be determined.
  • the DNA of the present invention is obtained by chemically synthesizing with a DNA synthesizer such as Perkin 'Elma's DNA synthesizer Model 392 using the phosphoramidite method. You can also.
  • a purified antibody of the polypeptide of the present invention or a partial fragment of the polypeptide, or a peptide having a partial amino acid sequence of the polypeptide of the present invention is used as an antigen to obtain a polyclonal antibody.
  • Antibodies that recognize the polypeptide of the present invention such as monoclonal antibodies (hereinafter, abbreviated as “antibodies of the present invention”) can be produced.
  • the antibody of the present invention also includes those antibody fragments, and may be a recombinant antibody such as a humanized antibody and an antibody fragment thereof.
  • a purified product of the polypeptide of the present invention or a partial polypeptide of the polypeptide, or a peptide having a partial amino acid sequence of the polypeptide of the present invention is used as an antigen and administered to animals.
  • a polyclonal antibody can be prepared.
  • Animals to be administered include egrets, goats, rats, mice, hamsters, and the like.
  • the dose of the antigen is preferably 50 to 100 g per animal.
  • a peptide obtained by covalently binding a peptide to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin.
  • the peptide serving as the antigen can be synthesized using a peptide synthesizer.
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the serum reacts with the antigen used for immunization.
  • Enzyme-linked immunosorbent assay [Enzyme-linked immunosorbent assay (ELISA): published by Medical Shoin (1976 ), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] Confirm.
  • a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum has a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  • Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, force prillic acid precipitation [Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory (1988)], DEAE-Sepharose column, There is a method in which a chromatographic method using an anion exchange column, a protein A or G-column, a gel filtration column, or the like is used alone or in combination.
  • Rats whose serum shows a sufficient antibody titer against the partial polypeptide of the polypeptide of the present invention used for immunization are used as a source of antibody-producing cells.
  • the spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
  • the spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • MEM medium manufactured by Nissui Pharmaceutical
  • the spleen cells in the obtained precipitate fraction are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, washed three times with MEM medium, and the obtained spleen cells are separated from antibody-producing cells. Used as
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine-resistant mouse derived from BALB / c
  • myeloma cell line P3-X63Ag8-Ul (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol., M, 1 (1978), Europ. J. Immunol., Fi, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 27fi, 269 (1978)], P3-X63-Ag8653 (653) [J.
  • HAT medium Hypoxanthin (10 ⁇ 4 inol / l), thymidine (1.5 X 10- 5 mol / l) and amino Noputerin (4 X 10 ⁇ 7 medium mol / 1) was added] are suspended in 100 ml.
  • the suspension is dispensed into 96-well culture plates at 100 ⁇ / well, and cultured at 37 ° C. overnight in a 5% CO 2 incubator for 7 to 14 days.
  • the following methods can be given as specific examples of the enzyme immunoassay.
  • a partial fragment polypeptide of the polypeptide of the present invention used as the antigen was coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below was used as the primary antibody.
  • reacting with an anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like as a second antibody followed by a reaction according to the labeling substance, and the polypeptide of the present invention.
  • Those that specifically react with are selected as hybridomas producing the monoclonal antibody of the present invention.
  • Cloning was repeated twice by the limiting dilution method using the hybridoma [the first time using HT medium (medium obtained by removing aminopterin from HAT medium), and the second time using normal medium].
  • the antibody with a strong antibody titer is selected as a hybridoma strain producing the monoclonal antibody of the present invention.
  • Pristane treatment [2,6,10,14-Tetramethylpenedecane (Pristane) 0.5 ml was intraperitoneally administered and bred for 2 weeks. 8 ⁇ : For 10-week old mice or nude mice, (c) 5 to 20 ⁇ 10 6 cells / animal of the polypeptide monoclonal antibody-producing polypeptide of the present invention obtained in the above, are injected intraperitoneally. In 10 to 21 days, Hypridoma becomes ascites cancer.
  • the ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solid components.
  • a monoclonal antibody can be purified and obtained in the same manner as in the polyclonal method.
  • the subclass of the antibody is determined using a mouse monoclonal antibody evening kit or a rat monoclonal antibody typing kit.
  • the amount of polypeptide is calculated by the Lowry method or from the absorbance at 280 nm.
  • a method for screening a substance using the polypeptide of the present invention Using the polypeptide of the present invention, a compound that changes the activity of the polypeptide that regulates intracellular Akt activity can be screened. Specifically, cells expressing the polypeptide of the present invention are brought into contact with a test sample, and qualitatively or quantitatively examined for the binding of Hsp90 to Akt and for the induction or suppression of apoptosis. A compound that changes the activity of controlling the Akt activity can be selected.
  • the test substance is not particularly limited as long as it can be added to the culture system of the cells expressing the polypeptide of the present invention. Examples of the test substance include a low molecular compound, a polymer compound, an organic compound, an inorganic compound, a protein, Genes, viruses, cells, etc. are listed. The test substance excluding the gene may be added directly to the culture medium.
  • Examples of a method for efficiently introducing a gene into cells include a method in which the gene is placed on a virus vector such as retrovirus, adenovirus, adeno-associated virus, simple virus, lentivirus, etc., and added to a culture system.
  • a virus vector such as retrovirus, adenovirus, adeno-associated virus, simple virus, lentivirus, etc.
  • a method of encapsulating the vesicles in an artificial vesicle structure such as ribosome and adding to a culture system may be used.
  • a report on gene analysis using a recombinant virus vector [Pro Natl. Acad. Sci. USA, 22, 6733 (1995); Nucleic Acids Res., 1 &, 3587, 1990; Nucleic Acids Res., 2 ⁇ , 3816 (1995)].
  • Hsp90 and Akt The binding between Hsp90 and Akt and the induction or suppression of apoptosis can be measured by the methods described above.
  • the polypeptide of the present invention a compound that inhibits the binding between the polypeptide and a protein that binds to the polypeptide, and the antibody of the present invention can be used to activate or activate a protein that binds to the polypeptide of the present invention. It can be used as an active ingredient (hereinafter abbreviated as “active ingredient of the present invention”) of a pharmaceutical preparation such as a therapeutic drug, a preventive drug, or a diagnostic drug for a disease caused by reduction.
  • active ingredient of the present invention a pharmaceutical preparation such as a therapeutic drug, a preventive drug, or a diagnostic drug for a disease caused by reduction.
  • the polypeptide and a protein that binds to the polypeptide are Examples of the compound that inhibits the binding to white matter include a compound obtained by the above screening method.
  • the diseases caused by the activation or reduction of the activity of the protein binding to the polypeptide of the present invention include the following diseases.
  • diseases associated with excessive cell death due to apoptosis include fulminant hepatitis and other viral diseases, neurodegenerative diseases such as Alzheimer's disease, and radiation damage.
  • Diseases associated with suppression of apoptosis include, for example, malignant tumors such as stomach cancer, ovarian cancer, breast cancer, knee cancer, and prostate cancer, and autoimmune diseases.
  • diseases caused by abnormal glucose metabolism control signals include diabetes and the like.
  • the active ingredient of the present invention is effective in inhibiting the effects of existing therapeutic agents such as a cell death inhibitor during ischemia of the brain and heart, an anticancer agent, a protective agent against cell death of normal cells during radiotherapy, and an anticancer agent. Used as a potentiating agent.
  • anticancer drugs include alkylating agents, antimetabolites, antitumor antibiotics, and plant alloids. More specifically, busulfan, cyclophosphamide, cyclophosphamide, and sulfad , Ifosphamiae melphafun (melp alan) N nitrosourea, amsacrine, amsacrine ⁇ carplatin, cisplatin, cisplatin, dacarbazine N azacitidine, cladribine Bottles (cytarabine), fludarabine (fludarabine), funooleurouracil (fluorouracil) N -hydroxyperea (hydroxyurea) lipocoholin (leucovorin) methotrexate (methotrexate ⁇ actinomycin D), bleomycin ), Daunorubicin (danorubicin), doxorubicin (doxorubicin), my toma Shin C (mit
  • a recombinant vector prepared for gene therapy into which a gene encoding a polypeptide as an active ingredient is inserted is introduced into cells taken out of the patient, and then the cells are introduced into a living body.
  • an artificial vehicle structure such as ribosome A peptide fragment that enhances the efficiency of introduction into cells as an active ingredient, such as the TAT peptide described in Examples [Science, 2, 1569-1572 (1999)] Fusion and administration to living organisms.
  • the administration ratio and administration schedule of the anticancer agent and the active ingredient of the present invention are determined in consideration of the age and weight of the patient, and the nature or severity of the condition to be treated.
  • the active ingredients of the present invention can be contained as pharmaceutical preparations, alone or as a mixture with any other active ingredient for treatment.
  • these pharmaceutical preparations are prepared by mixing the active ingredient with one or more pharmacologically acceptable carriers and by any method well-known in the technical field of pharmaceuticals.
  • the most effective route for treatment can be oral or parenteral, for example, intravenous.
  • Dosage forms include tablets, powders, granules, syrups, and injections.
  • Liquid preparations suitable for oral administration include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil It can be produced by using oils such as, for example, preservatives such as P-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. Tablets, powders, granules, etc.
  • excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, and polyvinyl alcohol. , Hydroxypropylcellulose, gelatin and other binders, fatty acid esters And a plasticizer such as glycerin.
  • Formulations suitable for parenteral administration comprise a sterile aqueous preparation containing the active compound which is preferably isotonic with the blood of the recipient.
  • a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of saline and a putose sugar solution.
  • parenteral preparations are also selected from the diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc. exemplified for the oral preparations.
  • auxiliary components can also be added.
  • the dose and frequency of administration of the active ingredient of the present invention will vary depending on the mode of administration, the age and weight of the patient, the nature or severity of the condition to be treated, but usually 0.01 mg to lg per adult for oral administration. Preferably, 0.05 to 50 mg is administered once or several times a day. In the case of parenteral administration such as intravenous administration, 0.001 to 100 mg, preferably 0.01 to: 10 mg per adult is administered once or several times a day. However, the dose and the number of administrations vary depending on the various conditions described above.
  • FIG. 1 shows the results of an experiment in which binding between Hsp90 and Akt in BALB / 3T3 and 293T cells was detected by Western blotting using an anti-Hsp90 antibody after immunoprecipitation with an anti-Akt antibody.
  • FIG. 2 shows the results of an experiment in which a region essential for binding to Hsp90? was analyzed using an Aktl deletion mutant.
  • FIG. 3 shows the results of an experiment in which a region essential for binding to Aktl was analyzed using an Hsp90-deletion mutant.
  • the c Figure 5 shows the results of an experiment analyzing the inhibition and inhibition of phosphorylation of Aktl the binding of Hsp90 ⁇ and Aktl in a cell, Aktl missing
  • the results of an experiment analyzing changes in Akt activity when a mutant gene was introduced into cells to inhibit the binding of Hsp90 to Aktl in cells were shown.
  • FIG. 6 shows that Aktl deletion mutant gene was introduced into cells and Hsp90?
  • FIG. 4 shows the results of an experiment in which the induction of apoptosis of cells when the binding of A was inhibited was analyzed by nuclear staining with DAPI.
  • Figure ⁇ ⁇ shows the results of an experiment in which the induction of cell apoptosis was analyzed by measuring caspase 3-like activity when the Aktl deletion mutant gene was introduced into cells to inhibit the binding of Hsp90 to Aktl in the cells. .
  • FIG. 8 shows that when the Aktl deletion mutant gene was introduced into cells to inhibit the binding of Hsp90 ⁇ to Aktl in the cells, VP-16 increased the sensitivity to apoptosis-inducing stimulus by the caspase substrate PARP.
  • the experimental result analyzed by the intracellular cleavage activity is shown.
  • Fig. 9 shows the results of an analysis of the decrease in sensitivity of cells to apoptosis-inducing stimuli when transfected with a peptide containing an Akt-binding site on Hsp90?
  • a caspase substrate Show.
  • WB is Western 'printing
  • a-Hsp90 is an anti-Hsp90 antibody
  • hi-Akt is an anti-Akt antibody
  • Cont. IgG is control IgG
  • IP is immunoprecipitated
  • pFLAG-akL is FLAG. Is the full-length Aktl gene with pcg, pcDNA3.1-Hsp90?
  • -FLAG is anti-FLAG tag M2 monoclonal antibody
  • -P-Akt Thr 3 ° 8
  • HI-V5 is an anti-V5 tag monoclonal antibody
  • HI-HA is an anti-HA tag monoclonal antibody
  • FLAG-Akt is a full-length Aktl protein with FLAG ligature
  • c-Akt Is the endogenous Aktl full-length protein
  • 1-309 aki is the Aktl partial sequence (1-309) with FLAG fragment.
  • DMEM medium containing 10% fetal bovine serum (FBS), 37 ° C, 5% C0 2 and cultured in the presence, mouse fibroblast BALB / 3T3 (ATCC CCL-163 ) and human embryonic kidney cells 293T cells [Pro Natl. Acad. Sci.
  • the full-length human Aktl gene was obtained from human osteosarcoma Saos-2 cells (ATCC HTB-85) [J. Cell. Physiol., Phoenix 290 (2000)] by reverse transcription PGR (RT-PCR).
  • RT-PCR reverse transcription PGR
  • various fragments of the Aktl gene (1-433 (AC), 120-480 ( ⁇ ), 120-433 (ANC), -175, 1-228, 1-331, 205-480, 340-480, 1-246, 1-267, 1-288, deletion mutants having amino acid sequences corresponding to positions 1-309 Gene fragment).
  • the above full-length Aktl gene and deletion mutant were subcloned into the pFLAG-CMV-2 vector to obtain a vector for cell expression.
  • Hsp90? and Hsp90a genes (manufactured by Invitrogen) subcloned into the pcDNA3.1 / GS vector and the expression vector containing the full-length or deletion mutant of Aktl described above were combined with Superfect transfection reagent (Qiagen).
  • Superfect transfection reagent Qiagen
  • Aktl protein or its deletion mutant protein was immunoprecipitated from the obtained cell lysate using an anti-FLAG tag M2 monoclonal antibody (manufactured by Sigma), and Hsp90? Or Hsp90 in the immunoprecipitate was used.
  • the Hsp90-binding region of Aktl is a region having the amino acid sequence at positions 229 to 309 of the amino acid sequence of Aktl described in SEQ ID NO: 5, and among these, the region having the amino acid sequence at positions 289 to 309 is particularly preferable. It proved to be necessary for strong binding.
  • Example 3
  • Hsp90 gene The full-length human Hsp90 gene (WT) was obtained from Invitrogen. Also,;? Various Hsp90 gene fragment by PCR (SEQ ID NO: 1 1-301 of Hsp90 amino acid sequence set forth 1-530 (Mid), 1-635, 534-724 ( ⁇ ) ⁇ 534-680, 1-315, 1-326, 1-340,
  • Aktl was immunoprecipitated from the obtained cell lysate using an anti- (His) 6 tag antibody, and the presence or absence of Hsp90? Or its deletion mutant in the immunoprecipitate was determined by using an anti-FLAG tag M2 monoclonal antibody (Sigma) ) Was detected by Western blotting.
  • Figure 3 As a result, it was revealed that the Akt-binding region of Hsp90? Is a region having the 327th to 340th amino acid sequences of the amino acid sequence of Hsp90? Described in SEQ ID NO: 1.
  • Aktl deletion mutant gene with HA fragment using pHM6 vector.
  • SEQ ID NO: 5 A gene encoding the amino acid sequence corresponding to positions 1-309, 1-228, and 1-288 of the amino acid sequence described), a full-length Hsp90-tagged gene with a V5 tag (pcDNA3.1-Hsp90-), and The FLAG-tagged Aktl full length gene (pFLAG-Aktl) was introduced into 293T cells. Thereafter, the cells were cultured for 24 hours under serum-free conditions, and a cell lysate was prepared by the method of Example 1. Aktl in the obtained cell lysate was immunoprecipitated with an anti-FLAG tag M2 monoclonal antibody (Sigma).
  • Akt activity of the immunoprecipitate was measured using an Akt kinase assay kit (manufactured by Upstate Biotechnology) using a partial peptide at the phosphorylation site of GSK3, one of the substrates.
  • Fig. 5 shows the results.
  • the partial sequence of Aktl (amino acid sequence 1 to 309) revealed that Akt activity was reduced when the binding between Aktl and Hsp90 was inhibited.
  • caspase-3 like protease activity in the above cells was measured by the method described in Oncogene, 1295-1304 (1998). Specifically, the collected cells were lysed in a lysate containing 10 mM HEPES (pH 7.4), 2 mM EDTA, and 0.1% CHAPS, and the resulting cell lysate was equivalent to 5 g protein and DEVD-AMC ⁇ acetyl -JJ-asp artyl-L-glutamyl-L-valyl-L-aspart-7-amino-4-methylcoumariii) (Peptide Laboratories) in ICE buffer [10 mM HEPES (pH 7.4) / 10% glycerol / 2 ⁇ dithiothreitol] at 37 ° C for 1 hour.
  • ICE buffer 10 mM HEPES (pH 7.4) / 10% glycerol / 2 ⁇ dithiothreitol
  • Akt deletion mutant enhances sensitivity to apoptosis stimulation by VP-16 i
  • Aktl Inhibition of the binding of Aktl to Hsp90 ⁇ by the partial sequence of Aktl (amino acid sequence at position 309 to 309) shown in Examples 1 to 4 and the resulting decrease in Akt activity are associated with apoptosis stimulation by anticancer drugs.
  • VP-16 was used to verify that it increased the sensitivity.
  • the pFLAG-CMV-2 vector is subcloned with an Aktl deletion mutant gene (corresponding to the gene encoding the amino acid sequence of amino acids 1 to 309 of Aktl) or its vector control.
  • HSP327-TAT peptide (LEFRALLFIPRRAP-GYGRKKRRQKRHG) in which the TAT peptide [Science, 2, 1569-1572 (1999)] was conjugated to the C-terminus of Hsp90?
  • HSP287-TAT peptide (PIWTRNPDDITQEE-GYGRKKRRQRRRG), which is a TAT-binding peptide having the amino acid sequence at positions 287-300 of the amino acid sequence shown in SEQ ID NO: 1 that is not the Akt-binding region, and apoptosis.
  • human fibroblast sarcoma cells HT1080 (ATCC CCL-121) were not treated with peptide, and 0.1 mol / L or 1 mol / L of HSP327-TAT or HSP287-TAT peptide was added. And cultured in serum-free DMEM medium for 24 hours.
  • a functional site on Hsp90 involved in apoptosis suppression and regulation of Akt activity involved in glucose metabolism and a functional site on Akt binding to the Hsp90 were identified.
  • Prophylactic and therapeutic drugs against malignant tumors such as gastric cancer, ovarian cancer, breast cancer, breast cancer, prostate cancer, and prostate cancer, and diseases such as diabetes, cell death inhibitors during ischemia in the brain and heart, normal in anticancer drugs and radiation therapy Drugs that protect cells from cell death or enhance the effects of existing therapies Agent is provided,

Abstract

There have been needed remedies, preventives and diagnostics for diseases caused by apoptosis regulatory or sugar metabolism regulatory signal errors including malignant tumors such as gastric cancer, ovarian cancer, breast cancer, pancreatic cancer and prostatic cancer, cell death inhibitors in ischemic brain, heart, etc., agents protecting normal cells from cell death in treatments with anticancer agents or radiation, or drugs potentiating the effects of existing remedies. A method of regulating apoptosis which involves the step of regulating the Akt activity in cells, in particular, the step of regulating the step of regulating the binding of Hsp90 to Akt; and a means useful in treating or diagnosing diseases which are caused by apoptosis regulatory or sugar metabolisms regulatory signal errors by using the regulation of apoptosis.

Description

明 細 アポト一シスの制御方法およびアポトーシス制御ポリべプチド <技術分野 >  Akira Apoptosis control method and apoptosis control polypeptide <Technical field>
本発明は、 細胞内の Akt活性を制御する工程、 特に Hsp90と Aktとの結合を 制御する工程を含有する、 アポトーシスを制御する方法、 細胞内の Akt活性を制 御する活性を有するポリペプチド、 該ポリペプチドをコードする DNA、 該ポリ ペプチドを認識する抗体、 該ポリペプチドの製造方法、 該ポリペプチドまたは該 抗体を含有する医薬、 およびそれらの利用方法に関する。  The present invention relates to a method for controlling apoptosis, which comprises a step of controlling Akt activity in a cell, in particular, a step of controlling binding between Hsp90 and Akt, a polypeptide having an activity of controlling Akt activity in a cell, The present invention relates to a DNA encoding the polypeptide, an antibody recognizing the polypeptide, a method for producing the polypeptide, a medicament containing the polypeptide or the antibody, and a method for using them.
<背景技術 > <Background technology>
アポトーシスは、 本来生理的条件下で細胞自らが積極的に引き起こす細胞死を 意味しており、 発生過程での形態、 組織の形成、 ホメォス夕シスの維持、 生体防 御に深く関わり、 細胞*** （増殖） と表裏一体となって個体の生命維持に重要な 役割を持つ。 アポトーシスは、 細胞外あるいは細胞内からの生理的または病的な 様々なシグナルによって誘発される。 遺伝子により制御された死の過程で先天的 あるいは後天的に異常が生じると、 アポトーシスが過剰に誘発されたり抑制され たりして様々な疾患を引き起こすと考えられる。 アポトーシスによる過剰な細胞 死が発症に関わる疾患としては、 劇症肝炎やその他のウィルス疾患、 アルヅハイ マー病などの神経変性疾患、 放射線障害などがあげられる。 逆にアポトーシス抑 制が発症に関わる疾患としては、 癌ゃ自己免疫疾患などがあげられる。  Apoptosis originally means cell death actively caused by cells themselves under physiological conditions, and is deeply involved in morphology, tissue formation, homeostasis maintenance, biological defense during development, and cell division ( Proliferation) plays an important role in maintaining the life of an individual as two sides of the same coin. Apoptosis is triggered by various physiological or pathological signals from outside or inside the cell. Inherited or acquired abnormalities in the process of death controlled by a gene are thought to induce various diseases by over-inducing or suppressing apoptosis. Diseases associated with excessive cell death due to apoptosis include fulminant hepatitis and other viral diseases, neurodegenerative diseases such as Alzheimer's disease, and radiation damage. Conversely, diseases related to the onset of apoptosis suppression include cancer and autoimmune diseases.
また、 放射線や薬物がアポトーシスを引き起こすことを利用した、 癌治療や免 疫抑制療法が行われている。 逆に、 アポトーシスを抑制することにより生体で重 要な役割を果たす細胞を死から守ることは、 脳、 心臓などの虚血時における細胞 死抑制、 抗癌剤や放射線治療時における正常細胞の細胞死からの保護などの医学 的な治療目的に利用することができる。  In addition, cancer treatment and immunosuppressive therapy are being carried out, using the fact that radiation and drugs cause apoptosis. Conversely, protecting cells that play an important role in the living body by suppressing apoptosis from death is the key to suppressing cell death during ischemia in the brain and heart, and from normal cell death during anticancer drugs and radiation therapy. It can be used for medical treatment purposes such as protection of stomach.
Aktは、 ホスホイノシチド 3-カイネース [phosphoinositide 3-kinase (PI3K)] 下流のシグナル伝達経路で機能するセリン ·スレオニンカイネースとして知られ ている。 Aktの哺乳動物ァイソフォーム （mammalian isoform) としては、 これ までに 3つのアイソフォーム （Aktl/PKBひ、 Akt2/PKB ? Akt3/P Bァ） が知 られている [Biochem. J.， , 1- 13 (1998)] 0Akt2と Akt3ともに Aktlと約 82 % のアミノ酸が同一であり、リン酸化されて活性化する時に必要なスレオニン残基、 セリン残基も保存されている。 サブタイプ間の機能分担等は現時点では明らかに されていない。 Akt is phosphoinositide 3-kinase (PI3K) It is known as a serine / threonine kinase that functions in downstream signaling pathways. As the mammalian isoform of Akt, three isoforms (Aktl / PKB and Akt2 / PKB? Akt3 / PBa) have been known so far [Biochem. J.,, 1-. 13 (1998)] 0 Both Akt2 and Akt3 are about 82% identical in amino acid to Aktl, and the threonine and serine residues required for phosphorylation and activation are conserved. The division of functions among subtypes has not been disclosed at this time.
PI3Kは、 細胞内で増殖因子 'サイ トカイン等による刺激によって活性化され、 PI 4,5 ビスホスホン酸 [PI 4,5 bisphosphonate (PIP2)] を PI 3,4,5 トリホスホ ン酸 [PI 3,4,5 triphosphonate (PIP3)] に変換する。 PIP3がセカンドメッセンジ ャ一として Aktに働き、 Aktlの 308番スレオニン残基と 473番セリン残基のリ ン酸化によって Akt活性は亢進する。 活性化した Aktの基質としては、 Bad、 キ ヤスパ一ゼ 9 (caspase 9)、 IkBカイネースなどが知られており、 これら基質をリ ン酸化することにより ktが不活性化して細胞のアポトーシスを抑制することか ら、 Akt活性の亢進は癌細胞の不死化やアポトーシス耐性に閧与する可能性が考 えられている。 癌における Akt の発現を調べた報告としては、 Aktl が大腸癌 (gastric adenocarcinomaの 20%)、 Akt2が卵巣癌 (15 %)ヽ膝癌 (12%)、乳癌 （3 %)、 Akt3がエストロゲンレセプ夕一を欠失した乳癌、アンドロゲン非依存性前立腺癌 などでそれそれ高発現しているという報告がある [Biochem. J., 1- 13 (1998)、 J. Biol. Chem., 274, 21528-21532 (1999)] 0 また、 脳腫瘍、 子宮内膜癌、 前立腺 癌、 乳癌等での遺伝子変異が知られている癌抑制遺伝子である PTEN [Science, 275. 1943- 1947 (1997) Nature Genetics, , 356-362 (1997)]は、 発見された当 初から、 ホスファタ一ゼとホモロジ一が高いことが知られていた。 PTENは、 そ の後 PI3Kによって生成される PIP3の脱リン酸化活性を持ち、 PI3K→Aktへの シグナルを抑制する機能を持つことが明らかにされている [Cell, 05, 29-39 (1998)]。細胞における PTEN活性の消失は、 Aktの恒常的な活性化を引き起こし、 アポトーシスが抑制されることにより癌化の引き金となると考えられる。 これら のことから、 Akt活性を抑制する物質は癌細胞に対してアポト一シスを誘導する 治療薬、 あるいは現在癌治療に用いられている抗癌剤によって誘導される癌細胞 のアポト一シスの感受性を増強させる薬剤として有用であると考えられるPI3K is activated in cells by stimulation with growth factors, such as cytokines, and converts PI 4,5 bisphosphonate [PI 4,5 bisphosphonate (PIP2)] to PI 3,4,5 triphosphonic acid [PI 3,4 , 5 triphosphonate (PIP3)]. PIP3 acts on Akt as a second messenger, and Akt activity is increased by phosphorylation of Aktl threonine residues 308 and 473 at Aktl. Known substrates of activated Akt include Bad, caspase 9 and IkB kinase, and phosphorylation of these substrates inactivates kt to suppress cell apoptosis. Therefore, it is considered that enhanced Akt activity may contribute to immortalization of cancer cells and apoptosis resistance. Studies on the expression of Akt in cancer showed that Aktl was colorectal cancer (20% of gastric adenocarcinoma), Akt2 was ovarian cancer (15%) ヽ knee cancer (12%), breast cancer (3%), and Akt3 was estrogen receptor. It has been reported that such genes are highly expressed in breast cancers lacking Yuichi and androgen-independent prostate cancer [Biochem. J., 1-13 (1998), J. Biol. Chem., 274, 21528]. -21532 (1999)] 0 also, brain, endometrial, prostate cancer, PTEN [Science is a tumor suppressor gene mutations in breast cancer and the like are known, 275. 1943- 1947 (1997) Nature Genetics, , 356-362 (1997)] was known to be high in phosphatases and homologues from the very beginning. PTEN has been shown to have the activity to dephosphorylate PIP3, which is subsequently generated by PI3K, and to suppress the signal from PI3K to Akt (Cell, 05, 29-39 (1998) ]. Loss of PTEN activity in cells is thought to cause Akt homeostatic activation and suppress apoptosis to trigger cancer. Therefore, substances that suppress Akt activity induce apoptosis in cancer cells. It is considered to be useful as a therapeutic drug or a drug that enhances the sensitivity of cancer cells to apoptosis induced by anticancer drugs currently used for cancer treatment
[Biochem. J., 335, 1-13 (1998)、 Genes Develop., IS, 2905-2927 (1999) J. Natl. Cancer Inst., ai, 1922-1932 (1999)、 Proc. Natl. Acad. Sci. USA, 2£, 4240-4245 (1999)、 Biochem. Biophys. Acta, 1470. M21-M35 (2000)]。 [Biochem. J., 335, 1-13 (1998), Genes Develop., IS, 2905-2927 (1999) J. Natl. Cancer Inst., Ai, 1922-1932 (1999), Proc. Natl. Acad. Sci. USA, 2 £, 4240-4245 (1999), Biochem. Biophys. Acta, 1470. M21-M35 (2000)].
Aktlの活性はリン酸化、脱リン酸化のバランスによって制御されている。 Aktl の脱リン酸化にはプロテインホスファターゼ 2A [protein phosphatase 2 A (PP2A)] が関与することが明らかにされている [Proc. Natl. Acad. Sci. USA, 5699-5704 (1996)]。  Aktl activity is regulated by the balance between phosphorylation and dephosphorylation. It has been shown that protein phosphatase 2A (PP2A) is involved in Aktl dephosphorylation [Proc. Natl. Acad. Sci. USA, 5699-5704 (1996)].
最近、 ゲルダナマイシン （Geldanamycin) で細胞を処理することにより、 Akt/PKBセリン ■スレオニン力イネ一スが細胞内で不安定化することから、 Akt が以下に述べる Hsp90 に結合して活性制御を受けている可能性が示唆されてい る [Proceedings American Association for Cancer Research, 41, 1987 (2000)]。 しかしながら、 Aktと Hsp90が直接結合するか否か、 互いの結合領域、 結合によ る Aktの活性制御などに関しては、 何ら明らかにされていない。 \  Recently, treatment of cells with geldanamycin destabilizes Akt / PKB serine ■ threonine-potential rice in cells, and Akt binds to Hsp90 described below to regulate the activity. Has been suggested [Proceedings American Association for Cancer Research, 41, 1987 (2000)]. However, whether Akt and Hsp90 bind directly, the binding region of each other, the control of Akt activity by binding, etc., have not been clarified. \
また、 Aktの活性化がグルコース代謝を制御していることも明らかにされてい る。 インシュリン剌激によって起こる、 6-ホスホフルクトース 2-力イネ一ス [6- p osphofmctose 2 -kinase (PFK2)]活性化による糖代謝亢進、 細胞内グルコース 取り込みに関与するグルコース トランスポーター 4 [glucose transporter 4 (GLUT4)] の膜局在化によるグルコース取り込みの増加、 およびグリコーゲンシ ン夕一ゼカイネース -3 [glycogen synthase kinase-3 (GSK3)] の不活化によるグ リコーゲン合成抑制などにはすべて PI3Kを介した Aktの活性化が関与している ことが報告されている [Biochem. J., 3 , 1-13 (1998)]。 従って、 糖代謝異常に よって生じる糖尿病等に対しては、 Aktを活性化する物質が有効である可能性が 考えられる。  It has also been shown that Akt activation regulates glucose metabolism. 6-phosphofructose 2-force rice [6-p osphofmctose 2 -kinase (PFK2)] activation caused by insulin stimulation, glucose metabolism enhancement, glucose transporter 4 involved in intracellular glucose uptake [glucose transporter 4] (GLUT4)] and PI3K-mediated suppression of glycogen synthesis due to increased glucose uptake by membrane localization and inactivation of glycogen synthase kinase-3 (GSK3). It has been reported that Akt activation is involved [Biochem. J., 3, 1-13 (1998)]. Therefore, it is considered that Akt-activating substances may be effective for diabetes and the like caused by abnormal glucose metabolism.
ヒートショックプロテイン （Hsp) は細胞が熱ショック等のストレス環境にさ らされた時に細胞内で発現する一連の蛋白質群であり、 その分子量によって幾つ かのフアミリーに分類ざれる （Hsp90、 Hsp 70, Hsp27など)。 これら蛋白質は分 子シャペロン （chaperone) とも呼ばれ、一般的には蛋白質のフォールデイング、 膜透過、 会合、 凝集の抑制等がその主な機能と考えられている。 Heat shock protein (Hsp) is a group of proteins that are expressed in cells when cells are exposed to stress environments such as heat shock, and can be classified into several families according to their molecular weight (Hsp90, Hsp70, Hsp27 etc.). These proteins are It is also called a chaperone, and its main functions are generally considered to be protein folding, membrane permeation, association, and suppression of aggregation.
Hsp90は分子量約 90kDaの分子シャペロンによって構成される Hsp90フアミ リーに属するヒートショック蛋白質である。現在のところ真核生物の Hsp90ファ ミリ一としては、 Hsp90 、 Hsp90 ?、 Grp94, Hsp75/TRAP1 などが同定され ているが、これらの Hsp90フアミリー蛋白質は細胞外ストレスがかからない環境 下でも細胞内で発現しており、近年 Hsp 90が細胞増殖や癌化に関わる種々のシグ ナル伝達因子と特異的に複合体を形成し、 細胞周期や細胞増殖シグナルに闋与し ているこどが明らかになつてきた。細胞内での機能や安定性の保持に Hsp90ファ ミリーとの結合が必要であると考えられているシグナル伝達因子としては、 ステ ロイ ドホルモン受容体 （エス トロゲンレセプ夕一、 プロゲステロンレセプ夕一、 グルココルチコィ ドレセプ夕一など)、非受容体型チロシンキナーゼ （v-srcなど)、 受容体型チロシンキナーゼ （EGFR、 ErbB2 など)、 セリンスレオニンキナ一ゼ (Raf-1、 Cdk4など） などがあげられる。従って、 Hsp90の機能を制御することに よって Hsp90 と特異的に結合する細胞内シグナル伝達因子の関わる細胞内情報 伝達を変動させることができると考えられる。例えば、 種々の癌細胞株で Hsp90 フアミリ一蛋白質の発現上昇が報告されていることや、乳癌患者における Hsp90 発現量と予後との相関が報告されていることからも、 Hsp90の機能が癌などの疾 患において重要な働きをしている可能性が考えられている [Pharmacology & Therapeutics, 2 , 129-168 (1997)、 Biochem. Pharmacol., 675-682 (1998)、 Investigational New Drugs, 11, 361-373 (1999)]。  Hsp90 is a heat shock protein belonging to the Hsp90 family composed of a molecular chaperone with a molecular weight of about 90 kDa. At present, Hsp90, Hsp90 ?, Grp94, Hsp75 / TRAP1, etc. have been identified as eukaryotic Hsp90 families, but these Hsp90 family proteins are expressed in cells even in an environment without extracellular stress. In recent years, it has become clear that Hsp90 specifically forms complexes with various signal transduction factors involved in cell proliferation and canceration and contributes to the cell cycle and cell proliferation signals. Was. Signaling factors that are thought to require binding to the Hsp90 family to maintain intracellular function and stability include steroid hormone receptors (estrogens receptor, progesterone receptors, glucocorticoids). Drecept Yuichi), non-receptor tyrosine kinases (eg, v-src), receptor tyrosine kinases (eg, EGFR, ErbB2), serine threonine kinase (Raf-1, Cdk4, etc.). Therefore, it is thought that by controlling the function of Hsp90, intracellular signal transduction involving an intracellular signaling factor that specifically binds to Hsp90 can be varied. For example, it has been reported that the expression of Hsp90 family protein is increased in various cancer cell lines, and the correlation between Hsp90 expression level and prognosis in breast cancer patients has been reported. It is thought that it may play an important role in disease [Pharmacology & Therapeutics, 2, 129-168 (1997), Biochem. Pharmacol., 675-682 (1998), Investigational New Drugs, 11, 361 -373 (1999)].
Hsp90に結合してその機能を阻害する化合物としては、 ゲルダナマイシン、 ハ ービマイシン A (Herbimycin A) などのアンサマイシン系化合物とその誘導体、 ラデイシコ一ル （Radicicol) とその誘導体、 ノボビォシン （Novobiocin) などの クマリン系化合物があげられる [Investigational New Drugs, 11, 361-373 (1999)、 J. Natl. Cancer Inst., 92, 242-248 (2000)]。 ゲルダナマイシンゃラディ シコールに関しては、 Hsp90の N末端領域に位置する ATP/ADP結合領域 ひ〜 221 番目のアミノ酸配列に含まれる） に結合することが明らかにされている [Cell, m, 239-250 (1997)、 J. Biol. Chem., 212, 23843-23850 (1997)、 Cell Stres s and Chaperones, a, 100-108 (1998)、 J. Med. Chem., 42, 260-266 (1999)]。 Hsp90は、 p50/Cdc37、 p23などの共シャペロン （co-chaperone)分子とともに、 前述の特異的シグナル伝達因子と分子複合体を形成して機能するが、 ゲルダナマ イシンゃラデイシコールなどの低分子化合物が Hsp90 に結合することにより、 Hsp90を含む分子複合体の構成が変化し、 Hsp90の機能が失われることにより、 結果的に分子複合体に結合していたシグナル伝達因子の機能や、 細胞内局在、 細 胞内安定性などを変化させることになり、 癌細胞の増殖抑制をはじめとする種々 の生物活性を示すと考えられている [Investigational New Drugs, H, 361-373 (1999)]。一方、 ノボビォシンは Hsp90の C末端領域 （380〜 728番目のアミノ酸 配列に含まれる） に結合することにより、同様の効果を示すと報告されている [J. Natl. Cancer Inst., S2, 242-248 (2000)]。 Examples of compounds that bind to Hsp90 and inhibit its function include geldanamycin, herbimycin A and other ansamycin-based compounds and their derivatives, radicicol and its derivatives, and novobiocin. [Investigational New Drugs, 11, 361-373 (1999), J. Natl. Cancer Inst., 92, 242-248 (2000)]. Geldanamycin デ ィ radicicol has been shown to bind to the ATP / ADP binding region located in the N-terminal region of Hsp90, which is contained in the amino acid sequence from position 221 to position 221). [Cell, m, 239-250 (1997), J. Biol. Chem., 212, 23843-23850 (1997), Cell Stres s and Chaperones, a, 100-108 (1998), J. Med. Chem., 42, 260-266 (1999)]. Hsp90 functions together with co-chaperone molecules such as p50 / Cdc37 and p23 to form a molecular complex with the above-mentioned specific signaling factors, but low-molecular-weight compounds such as geldanamycin ゃ radicicol By binding to Hsp90, the composition of the molecular complex containing Hsp90 changes, and by losing the function of Hsp90, the function of the signal transduction factor bound to the molecular complex, intracellular localization, It is thought to cause changes in intracellular stability, etc., and to exhibit various biological activities including suppression of the growth of cancer cells [Investigational New Drugs, H, 361-373 (1999)]. On the other hand, novobiocin has been reported to show the same effect by binding to the C-terminal region of Hsp90 (included in the amino acid sequence at positions 380 to 728) [J. Natl. Cancer Inst., S2, 242- 248 (2000)].
<発明の開示 > <Disclosure of Invention>
本発明は、 アポトーシス抑制や糖代謝に関与する Akt の活性制御に関わる Hsp90上の機能部位を同定し、 アポトーシス制御や糖代謝制御シグナルの異常に よって生じる胃癌、 卵巣癌、 乳癌、 脬癌、 前立腺癌等の悪性腫瘍、 糖尿病等の疾 患に対する治療薬、 予防薬および診断薬、 脳、 心臓などの虚血時における細胞死 抑制剤、 抗癌剤や放射線治療時における正常細胞の細胞死からの保護剤、 または 既存の治療薬の効果を増強する薬剤を提供することを目的とする。  The present invention identifies functional sites on Hsp90 that are involved in the inhibition of apoptosis and the control of Akt activity involved in glucose metabolism, and is used to identify gastric cancer, ovarian cancer, breast cancer, breast cancer, prostate cancer caused by abnormal apoptosis control and glucose metabolism control signals. Therapeutic, prophylactic and diagnostic agents for malignant tumors such as cancer, diabetes, etc., cell death inhibitors during ischemia of the brain, heart, etc., anticancer agents and agents to protect normal cells from cell death during radiation therapy Or to provide agents that enhance the effects of existing therapeutic agents.
本発明者らは、 Hsp90が Aktと細胞内で結合すること、 さらにこの Hsp90と Aktの結合が Aktの活性 （リン酸化）維持に重要であることを初めて見出した。 さらにアポトーシス抑制や糖代謝に関与する Aktの活性制御に関わる Hsp90上 の機能部位、 および該機能部位に結合する Aktの部位を同定することにより、 本 発明を完成するに至った。  The present inventors have found for the first time that Hsp90 binds to Akt in a cell, and that the binding of Hsp90 to Akt is important for maintaining Akt activity (phosphorylation). Further, the present invention has been completed by identifying a functional site on Hsp90 involved in the control of Akt activity involved in apoptosis suppression and glucose metabolism, and an Akt site binding to the functional site.
即ち、 本発明は以下の (1)〜(40)の発明に関する。  That is, the present invention relates to the following inventions (1) to (40).
( 1 ) 細胞内の Akt活性を制御する工程を含有する、 アポトーシスの制御方 法。 (2) アポト一シスの制御がアポトーシスの誘導である上記 （1) 記載の方 法。 (1) A method for controlling apoptosis, which comprises a step of controlling Akt activity in a cell. (2) The method according to the above (1), wherein the control of apoptosis is induction of apoptosis.
(3) アポトーシスの制御がアポト一シスの抑制である上記 （ 1) 記載の方 法。  (3) The method according to the above (1), wherein the control of apoptosis is suppression of apoptosis.
(4) 細胞内の Akt活性の制御が、 Hsp90と Aktとの結合の制御である、 上記 （ 1) に記載の方法。  (4) The method according to (1), wherein the control of Akt activity in the cell is a control of binding between Hsp90 and Akt.
( 5 ) 細胞内の Akt活性の制御が、 Hsp90フアミリーに属する少なくとも一 つの蛋白質またはその部分ポリペプチドにより行われる、 上記 （ 1) 〜 （4) の いずれか 1項に記載の方法。  (5) The method according to any one of (1) to (4) above, wherein the control of Akt activity in the cell is performed by at least one protein belonging to Hsp90 family or a partial polypeptide thereof.
(6) Hsp90フアミリーに属する蛋白質が、 HspSO^または Hsp90ひである、 上記 （ 5 ) 記載の方法。  (6) The method according to (5) above, wherein the protein belonging to Hsp90 family is HspSO ^ or Hsp90.
( 7 ) 細胞内の Akt活性の制御が、 配列番号 1記載のアミノ酸配列の 327 番目から 340番目または配列番号 3記載のアミノ酸配列の 335番目から 34 8番目からなるポリペプチドにより行われる、 上記 （1) 〜 （4) のいずれか 1 項に記載の方法。  (7) The control of Akt activity in a cell is carried out by a polypeptide consisting of the 327th to 340th amino acid sequence of SEQ ID NO: 1 or the polypeptide consisting of 335 to 348th amino acid sequence of SEQ ID NO: 3; The method according to any one of 1) to (4).
(8) 細胞内の Akt活性の制御が、 配列番号 1記載のアミノ酸配列の 327 番目から 340番目または配列番号 3記載のアミノ酸配列の 335番目から 34 8番目において 1以上のアミノ酸が欠失、 置換、 挿入または付加されたアミノ酸 配列からなり、 かつ細胞内の Akt活性を制御する活性を有するポリペプチドによ り行われる、 上記 （ 1) 〜 （4) のいずれか 1項に記載の方法。  (8) In the control of Akt activity in cells, one or more amino acids are deleted or substituted at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3. The method according to any one of (1) to (4) above, wherein the method is performed using a polypeptide comprising an inserted or added amino acid sequence and having an activity of controlling Akt activity in a cell.
(9) 細胞内の Akt活性の制御が、 配列番号 1記載のアミノ酸配列の 327 番目から 340番目または配列番号 3記載のアミノ酸配列の 335番目から 34 8番目のァミノ酸配列と 60 %以上の相同性を有するァミノ酸配列からなり、 か つ細胞内の Akt活性を制御する活性を有するポリべプチドにより行われる、上記 (9) The control of intracellular Akt activity is at least 60% homologous to the amino acid sequence at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3. Carried out by a polypeptide comprising an amino acid sequence having a property and having an activity of controlling intracellular Akt activity.
( 1) 〜 （4) のいずれか 1項に記載の方法。 The method according to any one of (1) to (4).
(10) 細胞内の Akt活性の制御が、 Aktフアミリーに属する少なくとも一つ の蛋白質の部分ペプチドにより行われる、 上記 （2) または （4) 記載の方法。 (10) The method according to (2) or (4), wherein the control of Akt activity in the cell is performed by a partial peptide of at least one protein belonging to Akt family.
• (11) Aktファミリ一に属する蛋白質が、 Alctl、 Akt2または Akt3である、 上記 （10) に記載の方法。 • (11) the protein belonging to the Akt family is Alctl, Akt2 or Akt3; The method according to (10) above.
(12) 細胞内の Akt活性の制御が、配列番号 5記載のアミノ酸配列の 2 2 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 2 7番目から 3 0 6番目からなる ポリペプチドにより行われる、 上記 （2 ) または （4 ) に記載の方法。  (12) The control of Akt activity in the cell is from the 229th position to the 309th position of the amino acid sequence of SEQ ID NO: 5, the 23rd position to the 310th position of the amino acid sequence of SEQ ID NO: 7, or The method according to the above (2) or (4), which is carried out using a polypeptide consisting of the amino acids 227 to 306 of the amino acid sequence of SEQ ID NO: 9.
( 13) 細胞内の Akt活性の制御が、配列番号 5記載のアミノ酸配列の 2 8 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 9 0番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 8 6番目から 3 0 6番目からなる ポリペプチドにより行われる、 上記 （2 ) または （4 ) に記載の方法。  (13) The control of Akt activity in the cell is from the 289th to the 309th of the amino acid sequence of SEQ ID NO: 5, from the 290th to the 310th of the amino acid sequence of SEQ ID NO: 7, or The method according to (2) or (4), which is performed using a polypeptide consisting of positions 286 to 306 of the amino acid sequence of SEQ ID NO: 9.
( 14) 細胞内の Akt活性の制御が、配列番号 5記載のァミノ酸配列の 2 2 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 2 7番目から 3 0 6番目において 1以上のアミノ酸が欠失、 置換、 挿入または付加されたアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有するポリぺプチドにより行われる、上 記 （2 ) または （4 ) に記載の方法。  (14) The control of Akt activity in the cell is caused by the amino acid sequence of SEQ ID NO: 5 from the 229th position to the 309th position, the amino acid sequence of SEQ ID NO: 7 from the 23rd position to the 310th position, Alternatively, it has an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added at positions 227 to 306 of the amino acid sequence of SEQ ID NO: 9, and has an activity of controlling Akt activity in a cell. The method according to the above (2) or (4), which is carried out using a polypeptide having the same.
( 15) 細胞内の Akt活性の制御が、配列番号 5記載のアミノ酸配列の 2 2 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 2 7番目から 3 0 6番目のァミノ 酸配列と 6 0 %以上の相同性を有するァミノ酸配列からなり、 かつ細胞内の Akt 活性を制御する活性を有するポリぺプチドにより行われる、上記（2 )または（4 ) に記載の方法。  (15) The regulation of intracellular Akt activity is from the 229th position to the 309th position of the amino acid sequence of SEQ ID NO: 5, the 23rd position to the 310th position of the amino acid sequence of SEQ ID NO: 7, or A polyamino acid sequence comprising an amino acid sequence having at least 60% homology with the amino acid sequence at positions 227 to 306 of the amino acid sequence set forth in SEQ ID NO: 9, and having an activity of controlling intracellular Akt activity. The method according to the above (2) or (4), which is carried out with a peptide.
( 16) 細胞内の Akt活性の制御が、 上記 （5 ) 〜 （15) のいずれか 1項に記 載のポリべプチドの細胞内の Akt活性を制御する活性を変動させる化合物により 行われる、 上記 （ 1 ) または （2 ) に記載の方法。  (16) The control of intracellular Akt activity is carried out by a compound that fluctuates the activity of the polypeptide described in any one of the above (5) to (15), which regulates intracellular Akt activity, The method according to (1) or (2) above.
( 17) 該化合物が、 該ポリペプチドと該ポリペプチドに結合する蛋白質との 結合を阻害するポリペプチドである、 （16) に記載の方法。  (17) The method according to (16), wherein the compound is a polypeptide that inhibits binding between the polypeptide and a protein that binds to the polypeptide.
( 18) 細胞内の Akt活性の制御が、 上記 （5 ) 〜 （15) のいずれか 1項に記 載のポリべプチドを認識する抗体またはその抗体断片により行われる、上記（ 2 ) または （4 ) に記載の方法。 (18) The control of intracellular Akt activity is performed by the antibody or antibody fragment thereof that recognizes the polypeptide described in any one of (5) to (15) above. Or the method described in (4).
( 19) 配列番号 1記載のァミノ酸配列の 3 2 7番目から 3 4 0番目または配 列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8番目からなるポリぺプチド。  (19) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 from 327 to 340th or the amino acid sequence of SEQ ID NO: 3 from 335 to 348.
(20) 配列番号 1記載のァミノ酸配列の 3 2 7番目から 3 4 0番目または配 列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8番目において 1以上のアミ ノ酸が欠失、 置換、 挿入または付加されたアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有するポリべプチド。 - (20) One or more amino acids are deleted at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3. A polypeptide comprising an amino acid sequence substituted, inserted or added, and having an activity of controlling Akt activity in a cell. -
(21) 配列番号 1記載のアミノ酸配列の 3 2 7番目から 3 4 0番目または配 列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8番目と 6 0 %以上の相同性 を有するアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有する ポリべプチド。 (21) amino acids having a homology of 60% or more with amino acids 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or amino acids 335 to 348 of the amino acid sequence of SEQ ID NO: 3 A polypeptide comprising a sequence and having an activity of controlling Akt activity in a cell.
(22) 配列番号 5記載のアミノ酸配列の 2 2 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列 2 2 7番目から 3 0 6番目からなるポリべプチド。  (22) 229 to 309 of the amino acid sequence of SEQ ID NO: 5, 231 to 310 of the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence 22 of SEQ ID NO: 9 Polypeptides consisting of 7th to 30th.
(23) 配列番号 5記載のアミノ酸配列の 2 8 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 9 0番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列 2 8 6番目から 3 0 6番目からなるポリぺプチド。  (23) 289 to 309 of the amino acid sequence of SEQ ID NO: 5, 290 to 310 of the amino acid sequence of SEQ ID NO: 7, or 28 of the amino acid sequence of SEQ ID NO: 9 6th to 30th polypeptides.
(24) 配列番号 5記載のアミノ酸配列の 2 2 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列 2 2 7番目から 3 0 6番目において 1以上のアミノ酸が欠失、置換、 挿入または付加されたァミノ酸配列からなり、 かつ細胞内の Akt活性を制御する 活性を有するポリべプチド。  (24) 229 to 309 in the amino acid sequence described in SEQ ID NO: 5, 231 to 310 in the amino acid sequence described in SEQ ID NO: 7, or the amino acid sequence 22 in SEQ ID NO: 9 A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added at the 7th to 30th positions, and which has an activity of controlling Akt activity in a cell.
(25) 配列番号 5記載のアミノ酸配列の 2 2 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列の 2 2 7番目から 3 0 6番目のアミノ酸配列と 6 0 %以上の相同性 を有するアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有する ポリべプチド。  (25) 229 to 309 of the amino acid sequence of SEQ ID NO: 5, 231 to 310 of the amino acid sequence of SEQ ID NO: 7, or 2 of the amino acid sequence of SEQ ID NO: 9 27 A polypeptide consisting of an amino acid sequence having 60% or more homology with the amino acid sequence from the 7th to the 36th amino acid and having an activity to control intracellular Akt activity.
(26) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチドをコードす る DNA。 (26) The polypeptide encoding the polypeptide according to any one of (19) to (25) above. DNA.
(27) 上記 （26) に記載の DNAとストリンジェントな条件下でハイブリダ ィズし、 かつ細胞内の Akt活性を制御する活性を有するポリペプチドをコードす る DNA。  (27) A DNA that hybridizes with the DNA of (26) under stringent conditions and encodes a polypeptide having an activity of controlling intracellular Akt activity.
(28) 上記 （26) または （27) 記載の DNAをベクターに組み込んで得られ る組換え体ベクター。  (28) A recombinant vector obtained by incorporating the DNA according to (26) or (27) into a vector.
(29) 上記 （28) に記載の組換えベクターを宿主細胞に導入して得られる形 質転換体。  (29) A transformant obtained by introducing the recombinant vector according to (28) into a host cell.
(30) 上記 （29) に記載の形質転換体を培地に培養し、 培養物中に細胞内の Akt活性を制御可能なポリべプチドを生成蓄積させ、 該培養物から該ポリぺプチ ドを採取することを特徴とする細胞内の Akt活性を制御する活性を有するポリべ プチドの製造方法。  (30) The transformant according to (29) is cultured in a medium, a polypeptide capable of controlling Akt activity in cells is produced and accumulated in the culture, and the polypeptide is separated from the culture. A method for producing a polypeptide having an activity of controlling intracellular Akt activity, which is characterized by being collected.
(31) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチドを発現する 細胞と被験試料とを接触させる工程を含有する、 該ポリぺプチドの細胞内の Akt 活性を制御する活性を変動させる化合物のスクリーニング方法。 (31) In (19) - (2 5) contains a step of contacting the cell with a test sample which expresses a polypeptide according to any one of the control of Akt activity in cells of said polypeptide A method for screening a compound that changes the activity of the compound.
(32) 該化合物が、 該ポリペプチドと該ポリペプチドに結合する蛋白質との 結合を阻害する化合物である、 上記 （31) に記載の方法。  (32) The method according to (31), wherein the compound is a compound that inhibits the binding between the polypeptide and a protein that binds to the polypeptide.
(33) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチドを認識する 抗体またはその抗体断片。  (33) An antibody or an antibody fragment thereof that recognizes the polypeptide according to any one of (19) to (25).
(34) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチド、 該ポリべ プチドと該ポリべプチドに結合する蛋白質との結合を阻害するポリべプチド、 ま たは上記 （33) に記載の抗体または抗体断片を有効成分として含有する医薬。 (34) In (19) to the polypeptide of any one of (2 5), Poribe peptide that inhibits binding to proteins that bind to the Poribe peptide and said Poribe peptide, or above A medicament comprising the antibody or antibody fragment according to (33) as an active ingredient.
(35) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチドに結合する 蛋白質の活性化または活性低下により引き起こされる疾患の治療薬または予防薬 である、 上記 （34) 記載の医薬。  (35) The drug according to the above (34), which is a therapeutic or prophylactic drug for a disease caused by activation or decreased activity of a protein binding to the polypeptide according to any one of the above (19) to (25). Medicine.
(36) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチドに結合する 蛋白質の活性化または活性低下により引き起こされる疾患の診断薬である、 上記 (36) The diagnostic agent for a disease caused by activation or decreased activity of a protein binding to the polypeptide according to any one of the above (19) to (25).
(34) 記載の医薬。 (37) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチド、 該ポリべ プチドと該ポリぺプチドに結合する蛋白質との結合を阻害するポリぺプチド、 ま たは上記 （33) に記載の抗体あるいは抗体断片、 および抗癌剤を組み合わせてな る医薬組成物。 (34) The medicament according to the above. (37) The polypeptide according to any one of the above (19) to (25), a polypeptide that inhibits binding between the polypeptide and a protein that binds to the polypeptide, or the above ( 33. A pharmaceutical composition comprising a combination of the antibody or antibody fragment described in 33) and an anticancer agent.
(38) 上記 （19) 〜 （25) のいずれか 1項に記載のポリペプチド、 該ポリべ プチドと該ポリぺプチドに結合する蛋白質との結合を阻害するポリぺプチド、 ま たは上記 （33) に記載の抗体あるいは抗体断片、 および抗癌剤を有効成分として 含有する医薬組成物。  (38) The polypeptide according to any one of the above (19) to (25), a polypeptide that inhibits binding between the polypeptide and a protein that binds to the polypeptide, or the above ( 33. A pharmaceutical composition comprising the antibody or antibody fragment according to 33) and an anticancer agent as active ingredients.
(39) 抗癌剤が、 アルキル化剤、 代謝拮抗剤、 抗腫瘍性抗生物質、 植物アル カノィ ドから選ばれる少なくとも一つの抗癌剤である上記 （37) または （38) に 記載の医薬組成物。  (39) The pharmaceutical composition according to the above (37) or (38), wherein the anticancer agent is at least one anticancer agent selected from an alkylating agent, an antimetabolite, an antitumor antibiotic, and a plant alkanoid.
(40) 抗癌剤が、 プスルファン、 シクロフォスフアミ ド、 ィホスフアミ ド、 メルファラン、 ニトロソゥレア、 アムサクリン、 カルボプラチン、 シスプラチン、 ダカルバジン、 ァザシチジン、 クラ ドリビン、 シ夕ラビン、 フルダラビン、 フル ォロウラシル、 ヒドロキシゥレア、 ロイコボリン、 メ ト トレキサ一ト、 ァクチノ マイシン D、 ブレオマイシン、 ダウノルビシン、 ドキソルビシン、 マイ トマイシ ン C、 ミ トキサントロン、 エトポシド、 パクリタキセル、 テニポシド、 ビンブラ スチン、 ビンクリスチン、 ビンデシン、 ゲルダナマイシンおよぴラデイシコール から選ばれる少なくとも一つである上記 （37) 〜 （39) のいずれか 1項に記載の 医薬組成物。 細胞内で、 Aktがリン酸化されると Aktは安定な状態となり、 アポトーシスは 抑制されている。 Aktが脱リン酸化を受けると Aktが不安定な状態となりアポト —シスは誘導される。  (40) Anticancer drugs include psulfan, cyclophosphamide, ifosfamide, melphalan, nitrosoperrea, amsacrine, carboplatin, cisplatin, dacarbazine, azacytidine, cladribine, sieverabine, fludarabine, fluorouracil, hydroxyperia, leuco At least one selected from methotrexate, actinomycin D, bleomycin, daunorubicin, doxorubicin, mitomycin C, mitoxantrone, etoposide, paclitaxel, teniposide, vinblastine, vincristine, vindesine, geldanamycin and peracidicol The pharmaceutical composition according to any one of the above (37) to (39), wherein In cells, when Akt is phosphorylated, Akt becomes stable and apoptosis is suppressed. When Akt is dephosphorylated, Akt becomes unstable and apoptosis is induced.
本発明では、 リン酸化された Aktが Hsp90 と結合して安定化されていること が見出された。 さらに、 リン酸化された Aktが Hsp90 と結合して安定な状態を 保っているのは、 脱リン酸酵素の Aktへの作用部位と、 Hsp90 と Aktとの結合 領域とが関与していることをも見出された。 以上のことから、細胞内の Akt活性を制御することにより、特に Hsp90と Akt との結合を制御することで細胞内 Akt活性を制御することにより、 アポトーシス を制御することが可能である。 ここで、 「細胞内の Akt活性を制御する」 とは、 細胞内の Akt活性を亢進または低下させることを含み、 例えば、 Hsp90 と Akt との結合の制御、 リン酸化された Aktの安定化、 Aktの脱リン酸化などにより行 われる。 「Hsp90と Aktとの結合を制御する」 とは、 Hsp90と Aktとの結合を不 安定化または阻害 （特に競争阻害） すること、 および Hsp90と Aktとの結合を安 定化または促進することを含む。 また、 「アポトーシスを制御する」 とは、 アポト 一シスを誘導または抑制することを含む。 In the present invention, it has been found that phosphorylated Akt is stabilized by binding to Hsp90. Furthermore, the fact that phosphorylated Akt binds to Hsp90 and maintains a stable state is due to the involvement of the phosphatase action site on Akt and the binding region between Hsp90 and Akt. Was also found. From the above, it is possible to control apoptosis by controlling the intracellular Akt activity, particularly by controlling the binding between Hsp90 and Akt, thereby controlling the intracellular Akt activity. Here, “controlling intracellular Akt activity” includes increasing or decreasing intracellular Akt activity, for example, controlling the binding between Hsp90 and Akt, stabilizing phosphorylated Akt, It is performed by dephosphorylation of Akt. “Controlling the binding between Hsp90 and Akt” means to stabilize or inhibit (particularly competitive inhibition) the binding between Hsp90 and Akt, and to stabilize or promote the binding between Hsp90 and Akt. Including. “Controlling apoptosis” also includes inducing or suppressing apoptosis.
Hsp90 と Akt との結合を制御する方法は特に限定されないが、 好ましくは Hsp90 またはその部分ポリペプチド、 Aktの部分ポリペプチド、 Hsp90 と Akt との結合を阻害する化合物、 ペプチド、 抗体および抗体断片等を加えることによ り制御される。  The method for controlling the binding between Hsp90 and Akt is not particularly limited, but preferably includes Hsp90 or a partial polypeptide thereof, a partial polypeptide of Akt, a compound, a peptide, an antibody and an antibody fragment that inhibits the binding of Hsp90 to Akt. It is controlled by adding.
Hsp90 と Akt との結合は、 Hsp90 またはその部分ポリべプチドにより制御す ることがさらに好ましい。 Hsp90は特に限定されず、 Hsp90フアミリーに属する ものであれば、 いずれの蛋白質でもよいが、 好ましくは Hsp90 3または Hsp90 ひが用いられる。 Hsp90 5および Hsp90ひはアミノ酸配列がそれそれ配列番号 1 および 3、 塩基配列がそれそれ配列番号 2および 4で示される蛋白質である。 ァ ポト一シスを制御する Hsp90の部分ポリぺプチドとしては、配列番号 1記載のァ ミノ酸配列の 327番目から 340番目または配列番号 3記載のアミノ酸配列の 335 番目から 348番目からなるポリペプチド （以下、 細胞内の Akt活性を制御する活 性を有する Hsp90の部分ぺプチドを 「本発明の Hsp90ポリぺプチド」 と略記す る） が好ましい。  More preferably, the binding between Hsp90 and Akt is controlled by Hsp90 or a partial polypeptide thereof. Hsp90 is not particularly limited, and any protein may be used as long as it belongs to the Hsp90 family, but Hsp903 or Hsp90 is preferably used. Hsp90 5 and Hsp90 are proteins whose amino acid sequences are represented by SEQ ID NOS: 1 and 3, respectively, and whose base sequences are represented by SEQ ID NOs: 2 and 4, respectively. The partial polypeptide of Hsp90 that controls apoptosis includes a polypeptide consisting of positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or a polypeptide consisting of positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3 ( Hereinafter, a partial Hsp90 peptide having an activity of controlling Akt activity in cells is abbreviated as “Hsp90 polypeptide of the present invention”.
本発明の Hsp90ポリぺプチドが Aktと結合し Aktが脱リン酸化酵素による作 用を受けない場合には、 Aktがリン酸化された状態が保たれてアポトーシスが抑 制される。 本発明の Hsp90ポリぺプチドが Aktと結合するが Aktに脱リン酸化 酵素が作用する場合には、 Aktは脱リン酸化されてアポト一シスが誘導される。 同様に、 Hsp90と Aktとの結合は、 Aktの部分ポリぺプチドにより制御するこ とがさらに好ましい。 Aktは特に限定されず、 Aktファミリーに属するものであ れば、 いずれの蛋白質でもよいが、 好ましくは Aktl、 Akt2または Akt3が用い られる。 Aktl、 Akt2および Akt3はアミノ酸配列がそれそれ配列番号 5、 7およ ぴ 9、 塩基配列がそれそれ配列番号 6、 8および 10で示される蛋白質である。 ァ ポトーシスを制御する Aktの部分ポリぺプチドとしては、配列番号 5記載のァミ ノ酸配列の 229番目から 309番目、配列番号 7記載のァミノ酸配列の 231番目か ら 310番目、または配列番号 9記載のアミノ酸配列 227番目から 306番目からな るポリペプチドが好ましく、 特に、 配列番号 5記載のアミノ酸配列の 289番目か ら 309番目、配列番号 7記載のアミノ酸配列の 290番目から 310番目、 または配 列番号 9記載のァミノ酸配列 286番目から 306番目からなるポリぺプチド （以下、 細胞内の Akt活性を制御する活性を有する Aktの部分べプチドを「本発明の Akt ポリペプチド」 と略記する） が好ましい。 When the Hsp90 polypeptide of the present invention binds to Akt and Akt is not affected by phosphatase, Akt is phosphorylated and apoptosis is suppressed. When the Hsp90 polypeptide of the present invention binds to Akt but a dephosphorylating enzyme acts on Akt, Akt is dephosphorylated and apoptosis is induced. Similarly, the binding of Hsp90 to Akt can be controlled by a partial polypeptide of Akt. Is more preferable. Akt is not particularly limited, and may be any protein as long as it belongs to the Akt family. Preferably, Aktl, Akt2 or Akt3 is used. Aktl, Akt2 and Akt3 are proteins whose amino acid sequences are represented by SEQ ID NOS: 5, 7 and 9, respectively, and whose nucleotide sequences are represented by SEQ ID NOs: 6, 8 and 10, respectively. The partial polypeptide of Akt that controls apoptosis includes amino acids 229 to 309 of the amino acid sequence described in SEQ ID NO: 5, 231 to 310 of the amino acid sequence described in SEQ ID NO: 7, or SEQ ID NO: A polypeptide consisting of the amino acid sequence from position 227 to position 306 of the amino acid sequence 9 is preferred, and in particular, from position 289 to position 309 of the amino acid sequence of sequence number 5, from position 290 to position 310 of the amino acid sequence of sequence number 7, or Amino acid sequence described in SEQ ID NO: 9 Polypeptide consisting of positions 286 to 306 (hereinafter, a partial Akt polypeptide having an activity of controlling intracellular Akt activity is abbreviated as “Akt polypeptide of the present invention”) ) Is preferred.
本発明の Aktポリぺプチドが Hsp90と結合した結果、 Hsp90と結合した Akt 量が減少する。 Hsp90と結合していない Aktは脱リン酸化酵素による作用を受け た後、 アポト一シスが誘導される。  As a result of binding of the Akt polypeptide of the present invention to Hsp90, the amount of Akt bound to Hsp90 decreases. Akt not bound to Hsp90 is induced by a phosphatase, and then apoptosis is induced.
本発明の Hsp90ポリぺプチドおよび Aktポリぺプチド （以下、 Hsp90ポリぺ プチドおよび Aktポリへプチドを 「本発明のポリペプチド」 と略記する） のアミ ノ酸配列において 1以上のアミノ酸が欠失、 置換、 挿入または付加されたァミノ 酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有するポリべプチドは、 Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (以下、 モレキュラー .クロ一ニング第 2版と略す)、 Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (以下、 カレント 'プロトコ一ルズ 'ィン 'モレキュラー 'バイオロジーと略す)、 Nuc. Acid. Res., 10, 6487 (1982)、 Proc. Natl. Acad. Sci. USA, IS, 6409 (1982)、 Gene, , 315 (1985)、 N c. Acid. Res" 13, 4431 (1985)、 Proc. Natl. Acad. Sci. USA, ， 488 (1985)等に記載の部位特異的変異導入法を用いて、本発明のポリべプチドの ァミノ酸配列を有するポリペプチドをコードする DNAに部位特異的変異を導入 することにより、 取得することができる。 欠失、 置換、 挿入または付加されるアミノ酸の数は特に限定されないが、 上記 の部位特異的変異法等の周知の方法により欠失、 置換もしくは付加できる程度の 数であり、 1個から数十個、 好ましくは 1〜20個、 より好ましくは 1〜10個、 さ らに好ましくは 1~5個である。 One or more amino acids are deleted in the amino acid sequence of the Hsp90 polypeptide and Akt polypeptide of the present invention (hereinafter, Hsp90 polypeptide and Akt polypeptide are abbreviated as “polypeptide of the present invention”); Polypeptides comprising a substituted, inserted or added amino acid sequence and having an activity to control intracellular Akt activity are disclosed in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current 'protocols', 'molecular' biology), Nuc. Acid. Res., 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, IS, 6409 (1982), Gene,, 315 (1985), Nc. Acid. Res "13, 4431 (1985). Natl. Acad. Sci. USA, 488 (1985) and the like. Using, by introducing a site-specific mutation into DNA encoding a polypeptide having the Amino acid sequence of Poribe peptide of the present invention can be obtained. Although the number of amino acids to be deleted, substituted, inserted or added is not particularly limited, it is a number that can be deleted, substituted or added by a well-known method such as the above-described site-directed mutagenesis, and is one to several tens. , Preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.
本発明のポリぺプチドを構成するァミノ酸配列中、 1残基以上のアミノ酸残基 が欠失、 置換、 挿入または付加されたとは、 同一配列中の任意かつ一もしくは複 数のアミノ酸配列中の位置において、一または複数のアミノ酸残基の欠失、置換、 挿入または付加があることを意味し、 欠失、 置換、 挿入または付加が同時に生じ てもよく、 欠失、 置換、 挿入または付加されるアミノ酸は天然型と非天然型とを 問わない。 天然型アミノ酸としては、 L-ァラニン、 L-ァスパラギン、 L-ァスパラ ギン酸、 L-アルギニン、 L-グルタミン、 L-グルタミン酸、 グリシン、 L-ヒスチジ ン、 L-イソロイシン、 L-ロイシン、 L-リジン、 L-メチォニン、 L-フエ二ルァラ二 ン、 L-プロリン、 L-セリン、 L-スレオニン、 L-トリプトファン、 L-チロシン、 L- バリン、 L-システィンなどがあげられる。  Deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence constituting the polypeptide of the present invention means that any one or more amino acid sequences in the same sequence Means that there is a deletion, substitution, insertion or addition of one or more amino acid residues at the position, the deletion, substitution, insertion or addition may occur simultaneously, Amino acids can be either natural or non-natural. Natural amino acids include L-alanine, L-asparagine, L-asparaginic acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine , L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
以下に、 相互に置換可能なアミノ酸残基の例を示す。 同一群に含まれるァミノ 酸残基は相互に置換可能である。  The following are examples of amino acid residues that can be substituted for each other. Amino acid residues included in the same group can be substituted for each other.
A群： ロイシン、 イソロイシン、 ノルロイシン、 ノ リン、 ノルバリン、 ァラニ ン、 2-アミノブタン酸、 メチォニン、 0 -メチルセリン、 t-プチルグリシン、 t-ブ チルァラニン、 シクロへキシルァラニン  Group A: leucine, isoleucine, norleucine, norin, norvaline, alanine, 2-aminobutanoic acid, methionine, 0-methylserine, t-butylglycine, t-butylylalanine, cyclohexylalanine
B群：ァスパラギン酸、 グルタミン酸、 イソァスパラギン酸、 イソグルタミン 酸、 2-アミノアジピン酸、 2-アミノスべリン酸  Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
C群：ァスパラギン、 グルタミン  Group C: Asparagine, Glutamine
D群： リジン、 アルギニン、 オル二チン、 2，4-ジアミノブタン酸、 2,3-ジァミノ プロピオン酸  Group D: lysine, arginine, ordinine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
E群： プロリン、 3-ヒドロキシプロリン、 4-ヒドロキシプロリン  Group E: Proline, 3-hydroxyproline, 4-hydroxyproline
F群：セリン、 スレオニン、 ホモセリン  Group F: serine, threonine, homoserine
G群： フエ二ルァラニン、 チロシン  Group G: fenirualanine, tyrosine
本発明のポリペプチドが細胞内の Akt活性を制御する活性を有するためには、 本発明のポリペプチドのアミノ酸配列と、 BLAST [J. Mol. Biol., 2 , 403 (1990)] や FASTA [Methods Enzymol., 1M, 63-98 (1990)] 等を用いて計算したときに、 少なくとも 60%以上、 通常は 80%以上、 好ましくは 90%以上、 特に好ましくは 95%以上の相同性を有する。 In order for the polypeptide of the present invention to have an activity of controlling Akt activity in a cell, When calculated using the amino acid sequence of the polypeptide of the present invention and BLAST [J. Mol. Biol., 2, 403 (1990)] or FASTA [Methods Enzymol., 1M, 63-98 (1990)], etc. It has a homology of at least 60% or more, usually 80% or more, preferably 90% or more, and particularly preferably 95% or more.
また、 本発明は、 本発明のポリペプチドをコードする DNA、 およぴ該 DNAと ストリンジェントな条件下でハイプリダイズする DNAであり、かつ細胞内の Akt 活性を制御する活性を有するポリペプチドをコードする DNA (以下、 「本発明の DNAj と略記する） を包含する。  The present invention also relates to a DNA encoding the polypeptide of the present invention, and a DNA that hybridizes with the DNA under stringent conditions and has an activity of controlling Akt activity in a cell. DNA (hereinafter abbreviated as DNAj of the present invention).
本発明のポリぺプチドをコ一ドする DNAとしては、 配列番号 2記載の塩基配 列の 979番目から 1020番目、 配列番号 4記載の塩基配列の 1003番目から 1044 番目、配列番号 6記載の塩基配列の 685番目から 927番目、好ましくは 865番目 から 927番目、配列番号 8記載の塩墓配列の 691番目から 930番目、好ましくは 868番目から 930番目、 および配列番号 10記載の塩基配列の 679番目から 918 番目、 好ましくは 856番目から 918番目の塩基配列を有する DNAがあげられる。 なお、 本発明のポリペプチドのアミノ酸配列に対応する塩基配列を有する DNA は、 上記に記載したもの以外に多数存在するが、 それらはすべて本発明に包含さ れる。  The DNA encoding the polypeptide of the present invention includes the nucleotides 979 to 1020 of the nucleotide sequence of SEQ ID NO: 2, the nucleotides 1003 to 1044 of the nucleotide sequence of SEQ ID NO: 4, and the nucleotide of SEQ ID NO: 6. 685 to 927, preferably 865 to 927 of the sequence, 691 to 930, preferably 868 to 930 of the salt tomb sequence described in SEQ ID NO: 8, and 679 of the base sequence described in SEQ ID NO: 10 And a DNA having a base sequence from 856th to 918th, preferably from 856th to 918th. In addition, a large number of DNAs having a base sequence corresponding to the amino acid sequence of the polypeptide of the present invention other than those described above are all included in the present invention.
ストリンジェントな条件下でハイブリダイズ可能な DNAとは、 本発明のポリ ぺプチドのアミノ酸配列に対応する塩基配列を有する DNAをプローブとして、 コロニー ·ハイブリダィゼ一シヨン法、 プラーク 'ハイブリダィゼーシヨン法あ るいはサザンプロッ トハイブリダィゼーシヨン法等を用いることにより得られる DNAを意味し、 具体的には、 コロニーあるいはプラーク由来の DNAを固定化し たフィルタ一を用いて、 0.7〜： 1.0 mol/1の塩化ナトリウム存在下、 65°Cでハイプ リダィゼ一シヨンを行った後、 0.：!〜 2倍濃度の SSC溶液 （1倍濃度の SSC溶液 の組成は、 150 mmol/1塩化ナトリウム、 15 mmol/1クェン酸ナトリウムよりなる） を用い、 65°C条件下でフィルターを洗浄することにより同定できる DNAをあげ ることができる。 ハイブリダィゼ一シヨンは、 モレキュラー ·クロ一ニング第 2 版、カレント 'プロ トコールズ 'ィン 'モレキュラー'バイオロジー、 DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995)等に記載されている方法に準じて行うことができる。ハイブリダィズ可能 な DNAとして具体的には、 BLAST [J. Mol. Biol., 215, 403 (1990)] や FASTA [Methods Enzymol., 1 , 63-98 (1990)] 等を用いて計算したときに、 配列番号 2 記載の塩基配列の 979番目から 1020番目、 配列番号 4記載の塩基配列の 1003 番目から 1044番目、 配列番号 6記載の塩基配列の 685番目から 927番目または 865番目から 927番目、配列番号 8記載の塩基配列の 691番目から 930番目また は 868番目から 930番目、および配列番号 10記載の塩基配列の 679番目から 918 番目または 856番目から 918番目の塩基配列を有する DNAと少なくとも 60%以 上、 好ましくは 80%以上、 さらに好ましくは 90%以上、 特に好ましくは 95%以 上の相同性を有する DN Aをあげることができる。 DNA capable of hybridizing under stringent conditions refers to a DNA having a base sequence corresponding to the amino acid sequence of the polypeptide of the present invention as a probe, a colony hybridization method, a plaque hybridization method. Or DNA obtained by using the Southern blot hybridization method or the like. Specifically, using a filter on which DNA derived from colonies or plaques is immobilized, 0.7 to 1.0 mol / After performing the hybridization at 65 ° C in the presence of sodium chloride in step 1, the SSC solution from 0 :! to 2x concentration (1x concentration of SSC solution is 150 mmol / 1 sodium chloride, (consisting of 1 mmol / sodium citrate) and washing the filter under 65 ° C conditions to obtain DNA that can be identified. Hybridization, Molecular Cloning 2nd Edition, Current 'Protocols', 'Molecular' Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995), etc. Specifically, the DNA that can be hybridized is calculated using BLAST [J. Mol. Biol., 215, 403 (1990)] or FASTA [Methods Enzymol., 1, 63-98 (1990)]. 979 to 1020 of the nucleotide sequence of SEQ ID NO: 2, 1003 to 1044 of the nucleotide sequence of SEQ ID NO: 4, 685 to 927 or 865 to 927 of the nucleotide sequence of SEQ ID NO: 6, sequence At least 60% of the DNA having the nucleotide sequence of positions 691 to 930 or 868 to 930 of the nucleotide sequence of No. 8 and the nucleotide sequence of 679 to 918 or 856 to 918 of the nucleotide sequence of SEQ ID NO: 10 As described above, DNAs having a homology of preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more can be mentioned.
Hsp90 と Akt との結合の測定は、 以下の方法により行う。 すなわち、 Hsp90 および Aktの全長あるいは部分断片をコードする DNAにそれそれタグをコード する DNAを付加したのちに、 該 DNAを細胞内に導入し、 細胞を培養する。 該 細胞を溶解後に、 タグに対する抗体を用いて免疫沈降反応を行う。 ウェスタン - ブロッテイングにより免疫沈降物中の物質の特定を行うことにより、 Hsp90 と The binding between Hsp90 and Akt is measured by the following method. That is, after adding DNA encoding the tag to DNA encoding the full length or partial fragment of Hsp90 and Akt, the DNA is introduced into cells, and the cells are cultured. After lysing the cells, an immunoprecipitation reaction is performed using an antibody against the tag. Identification of substances in immunoprecipitates by Western-blotting allows Hsp90 and
Aktとの結合状態を測定することができる。 The state of binding to Akt can be measured.
免疫沈降法とは、 蛋白質とそれに対する抗体を反応させることにより、 抗原抗 体複合体を生じる反応をいう。  Immunoprecipitation refers to a reaction in which an antigen-antibody complex is formed by reacting a protein with an antibody thereto.
ウエスタン · ブロヅティングとは、 ポリアクリルアミ ドゲル電気泳動によって 蛋白質を分離後、 該ゲルの蛋白質をニトロセルロース膜、 ポリビニリデン ' ジフ ルォロイ ド膜などに転移し、 該蛋白質に対する抗体を用いて蛋白質を検出する方 法をいう。  Western blotting means that proteins are separated by polyacrylamide gel electrophoresis, and the proteins on the gel are transferred to a nitrocellulose membrane, polyvinylidene 'difluoride membrane, etc., and the proteins are detected using antibodies against the proteins. Refers to the method.
具体的には、 Hsp90部分蛋白質と、 タグを付した Akt蛋白質とが細胞内におい て結合した状態である場合、 タグに対するモノクローナル抗体による免疫沈降物 は、 Hsp90部分蛋白質と Akt蛋白質との複合体が得られることになる。 したがつ て、 該複合体をポリアクリルアミ ドゲル電気泳動に供したのちに該ゲルをニトロ セルロース膜に転移後、 Hsp90 に対する抗体または Hsp90 に付したタグに対す る抗体を用いることにより、 Hsp90が Akt蛋白質に結合していることを確認する ことができる。 Specifically, when the Hsp90 partial protein and the tagged Akt protein are bound in a cell, the immunoprecipitate of the monoclonal antibody against the tag contains a complex of the Hsp90 partial protein and the Akt protein. Will be obtained. Therefore, after subjecting the complex to polyacrylamide gel electrophoresis, the gel was transferred to a nitrocellulose membrane, and then subjected to an antibody against Hsp90 or a tag attached to Hsp90. By using such an antibody, it can be confirmed that Hsp90 has bound to the Akt protein.
タグとしては、 FLAGタグ、 V5タグ、 HAタグなどいかなるものでもよい。 細胞内における Akt活性を測定する方法としては、 Aktのリン酸化を測定する 方法が挙げられる。 すなわち、 Hsp90および Aktの全長あるいは部分断片をコー ドする DNAにそれそれタグをコ一ドする DNAを付加したのちに、該 DNAを細 胞内に導入し、 細胞を培養する。 該細胞を溶解後に、 タグに対する抗体を用いて 免疫沈降反応を行う。 ウエスタンプロッティングにより免疫沈降物中の物質の特 定を行い、 リン酸化された Aktに対する抗体を用いることにより、 Aktのリン酸 化を測定することができる。  The tag may be any tag such as a FLAG tag, V5 tag, HA tag, and the like. A method for measuring Akt activity in cells includes a method for measuring phosphorylation of Akt. That is, after adding DNA encoding a tag to DNA encoding the full length or partial fragment of Hsp90 and Akt, the DNA is introduced into cells, and the cells are cultured. After lysing the cells, an immunoprecipitation reaction is performed using an antibody against the tag. The substance in the immunoprecipitate is identified by Western plotting, and the phosphorylation of Akt can be measured by using an antibody against phosphorylated Akt.
アポトーシスを測定する方法としては、 細胞内の染色体 DNAを抽出し、 該染 色体 DNAを電気泳動して解析する方法、 細胞を染色したのちに、 形態学的な観 察により解析する方法、 アポトーシス誘導に必須であるプロテア一ゼを測定する 方法などがあげられる。  Methods for measuring apoptosis include a method of extracting chromosomal DNA in cells and analyzing the chromosomal DNA by electrophoresis, a method of staining cells and analyzing them by morphological observation, and a method of apoptosis. Examples include a method of measuring protease, which is essential for induction.
細胞内の染色体 DNAを抽出し、 該染色体 DNAを電気泳動法して解析する方 法を行うことにより、 アポトーシスを誘導した細胞の DNAに特徴的なラダ一を 観察することができる。  By extracting chromosomal DNA from the cells and analyzing the chromosomal DNA by electrophoresis, a ladder characteristic of the DNA of cells that have induced apoptosis can be observed.
細胞を染色したのちに、形態学的な観察により解析する方法を行うことにより、 アポトーシスを誘導した細胞の細胞核の染色体凝集、 細胞核の断片化、 細胞表面 微絨毛の消失、 細胞質の凝集を観察することができる。 細胞を染色する溶液とし ては、 4'，6-ジァミノ- 2-フエニルインドール （DAPI) などがあげられる。  After staining the cells, morphological observation is performed to observe the chromosome aggregation of the cell nucleus, fragmentation of the cell nucleus, loss of cell surface microvilli, and cytoplasmic aggregation of the cells that have induced apoptosis. be able to. Solutions for staining cells include 4 ', 6-diamino-2-phenylindole (DAPI).
アポトーシス誘導に必須であるプロテア一ゼを測定する方法としては、 キャス パ一ゼを測定する方法 [Oncogene, 11， 1295 (1998)]などがあげられる。 具体的に は、 細胞を 10mM HEPES(pH7.4)、 2mM EDTA、 0.1% CHAPSを含む溶解液中 で溶解する。 該細胞溶解液とァセチル -L-ァスパルチル -L-グル夕ミル- L-バリル- L-ァスバルト- 7-ァミノ- 4-メチルクマリン（DEVD-AMC)を ICE緩衝液で反応し た後に、 吸光光度計で測定することにより、 キャスパーゼ活性を測定することが できる。 以下、 本発明のポリペプチドの製造方法を例をあげて説明するが、 本発明はこ れらに限定されるものではない。 Methods for measuring protease, which is essential for apoptosis induction, include a method for measuring caspase [Oncogene, 11, 1295 (1998)]. Specifically, cells are lysed in a lysis solution containing 10 mM HEPES (pH 7.4), 2 mM EDTA, and 0.1% CHAPS. After reacting the cell lysate with acetyl-L-aspartyl-L-glucamyl-L-valyl-L-aswald-7-amino-4-methylcoumarin (DEVD-AMC) in ICE buffer, absorbance Caspase activity can be measured by measuring with a meter. Hereinafter, the method for producing the polypeptide of the present invention will be described by way of examples, but the present invention is not limited thereto.
1. 本発明のポリペプチドの製造  1. Production of the polypeptide of the present invention
本発明のポリペプチドの製造方法としては、 化学的合成による方法、 遺伝子ェ 学的な手法を用いる方法などがあげられる。 以下に詳細に説明する。  Examples of the method for producing the polypeptide of the present invention include a method using chemical synthesis and a method using a genetic method. This will be described in detail below.
(1) 化学的合成によるポリペプチドの製造方法 (1) Method for producing polypeptide by chemical synthesis
本発明のポリべプチドは、 例えば「ぺプチド合成の基礎と実験」（泉屋信夫、 加 藤哲夫、青柳東彦、脇道典著：丸善)、「第 4版実験化学講座 22—有機合成 IV酸 - アミノ酸，ペプチド」 （相本三郎、 楠本正一、 竜田邦明、 早川芳宏、 山本景祚、 若 宮建昭著：丸善)、 「イン夕一ナショナル · ジャーナル ·ォブ ·ぺプタイ ド ·アン ド ·フ。口ティン 'リサ一ナ (International Journal oi Peptide Protein Research), , 161-214 (1990)」、 「ソリ ヅ ドーフェーズ 'ぺプタイ ド · シンセシス （Solid- Phase Peptide Synthesis) , メソッズ ' イ ン ' ェンザィモロジ一 第 289 卷 (Methods in Enzymology, vol. 289)、 グレツグ · B ·フィ一ルズ （Gregg B. Fields) 編、 ァカデミヅク ·プレス （Academic Press)、 （1997)」、 「ぺプタイ ド ·シンセシ ス · プロトコール （Peptide Synthesis Protocols), メソヅズ .イン .モレキユラ —'バイオロジー 第 35卷 （Methods in Molecular Biology, vol. 35)、 マイケル ' W ·ぺニントン （Michael W. Pennington)s ベン · M ·ダン （Ben M. Dunn) 編、 ヒュ一マナ ·プレス （Humana Press)、 （1994)」等に記載の通常のぺプチド合成法 により合成後、 精製することによって得ることができる。 具体的な合成法として は、 アジド法、 酸クロライ ド法、 酸無水物法、 混合酸無水物法、 DCC法、 活性ェ ステル法、 カルボイミダゾ一ル法、 酸化還元法等が挙げられる。 また、 その合成 は、 固相合成法及び液相合成法のいずれをも適用することができる。 すなわち、 本発明のポリぺプチドを構成するァミノ酸と残余部分とを縮合させ、 生成物が保 護基を有する場合は保護基を脱離することにより目的とするポリペプチドを合成 する。 さらに、 本発明のポリぺプチドを構成するァミノ酸残基の側鎖および/または ぺプチドアミノ末端および/またはぺプチドカルボキシ末端が化学修飾や保護が なされている場合には、 ペプチド合成後に化学修飾するか、 あるいは化学修飾さ れたアミノ酸を用いてぺプチド合成するか、 あるいはぺプチド合成の最終脱保護 の反応条件を適当に選ぶ等、 ぺプチド合成化学の分野において従来公知の方法 (「ぺプチド合成の基礎と実験」 （泉屋信夫、 加藤哲夫、 青柳東彦、 脇道典著：丸 善)、 「続医薬品の開発一第 14卷ーペプチド合成」矢島治明監修：広川書店)、 「生 化学実験講座一 1—夕ンパク質の化学 IV—化学修飾とペプチド合成」 [日本生化学 会編、 東京化学同人、 「蛋白質の化学修飾」 <上> <下> (大野素徳、 金岡祐一、 崎山文夫、 前田浩)] によって製造することができる。 The polypeptides of the present invention are described, for example, in "Basics and Experiments of Peptide Synthesis" (Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Michinori Waki: Maruzen), "Fourth Edition Experimental Chemistry Course 22—Organic Synthetic IV Acids" -Amino acids and peptides "(Saburo Aimoto, Shoichi Kusumoto, Kuniaki Tatsuta, Yoshihiro Hayakawa, Keizo Yamamoto, Tateaki Wakamiya: Maruzen)," In Yuichi National Journal of Applied and Fudo " Mouth Tin 'Resina (International Journal oi Peptide Protein Research),, 161-214 (1990)', 'Solid-Phase Peptide Synthesis', Method's 'In' Enzymology I Vol. 289 (Methods in Enzymology, vol. 289), edited by Gregg B. Fields, Academic Press, (1997), "Applied Synthesis. Protocol (Peptide Synthesis Protocols), .. Uz's Inn Morekiyura - 'Biology 35 Certificates (. Methods in Molecular Biology, vol 35), Michael' W · Bae Ninton (Michael W. Pennington) s Ben · M · Dan (Ben M. Dunn), ed., It can be obtained by synthesizing by a general peptide synthesis method described in, for example, "Humana Press, (1994)" and then purifying. Specific synthesis methods include the azide method, the acid chloride method, the acid anhydride method, the mixed acid anhydride method, the DCC method, the active ester method, the carboimidazole method, and the redox method. For the synthesis, both the solid phase synthesis method and the liquid phase synthesis method can be applied. That is, the desired polypeptide is synthesized by condensing the amino acid constituting the polypeptide of the present invention with the remaining portion and, if the product has a protecting group, removing the protecting group. Furthermore, when the amino acid residue constituting the polypeptide of the present invention is chemically modified or protected on the side chain and / or the peptide amino terminal and / or the peptide carboxy terminal, the peptide is chemically modified after synthesis. Or a method known in the field of peptide synthesis chemistry, such as synthesis of peptides using chemically modified amino acids or appropriate selection of reaction conditions for final deprotection of peptide synthesis (“Peptide synthesis”). "Basics and Experiments of Synthesis" (Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Michinori Waki: Zen Maru), "Development of Continuing Pharmaceuticals, Volume 14, Peptide Synthesis", supervised by Haruaki Yajima: Hirokawa Shoten), "Biochemical Experiments" Lecture 1 1—Chemistry of Protein IV—Chemical Modification and Peptide Synthesis ”[edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin,“ Chemical Modification of Proteins ”<top><bottom> (Sononori Ohno, Yuichi Kanaoka, Fumio Sakiyama, Hiroshi Maeda)].
本発明ポリべプチドは、 自動べプチド合成機を用いて合成することもできる。 ペプチド合成機によるペプチドの合成は、 島津製作所製ペプチド合成機、 アドバ ンスト -ケムテック社 （Advanced ChemTech Inc., USA、 以後 「ACT社」 と略称 する） 製ペプチド合成機等の市販のペプチド合成機上で、 適当に側鎖を保護した Nひ- Fmoc-アミノ酸あるいは Nひ- Boc-アミノ酸等を用い、 それそれの合成プロ グラムに従って実施することができる。 原料となる保護アミノ酸および担体樹脂 は、 ABI社、 島津製作所、 国産化学 （株)、 ノバビオケム社 （NoVaBiochem)、 渡 辺化学 （株)、 ACT社、 アナスペック社 （AaaSpec In )、 またはペプチド研究所 (株） 等から入手することができる。 The polypeptide of the present invention can also be synthesized using an automatic peptide synthesizer. Peptide synthesis using a peptide synthesizer is performed on a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation, or a peptide synthesizer manufactured by Advanced ChemTech Inc. (USA). Using N-Fmoc-amino acid or N-Boc-amino acid with appropriately protected side chains, the synthesis can be carried out according to the respective synthesis programs. Protected amino acids and carrier resins as a raw material, ABI Inc., Shimadzu, Kokusan Chemical Co., Novabiochem (No V aBiochem), Watanabe Chemical Co., ACT, Inc., Ana Espec Corp. (AaaSpec an In), or peptide It can be obtained from Research Laboratories.
本発明のポリペプチドの精製は、 通常の精製法、 例えば溶媒抽出、 蒸留、 カラ ムクロマトグラフィー、 液体クロマトグラフィー、 再結晶などを組み合わせて行 なうことができる。  The polypeptide of the present invention can be purified by a combination of ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
(2) 遺伝子工学的手法によるポリペプチドの製造方法 (2) Method for producing polypeptide by genetic engineering technique
本発明のポリぺプチドは、 モレキュラー 'クローニング第 2版やカレント ·プ 口トコールズ'ィン 'モレキュラー .バイオロジー等に記載された方法等を用い、 例えば以下の方法により、 後述する方法で作製した本発明の DNAを宿主細胞中 で発現させて、 製造することができる。 The polypeptide of the present invention was prepared by the method described in Molecular Biology, Molecular Cloning Second Edition or Current Protocol, Inc. Molecular Biology, etc. The DNA of the present invention in a host cell And can be produced.
全長 cDNAをもとにして、 必要に応じて、 該ポリペプチドをコードする部分を 含む適当な長さの DNA断片を調製する。  Based on the full-length cDNA, if necessary, a DNA fragment of an appropriate length containing a portion encoding the polypeptide is prepared.
また、 必要に応じて、 本発明のポリペプチドをコードする部分の塩基配列を、 宿主細胞の発現に最適なコドンとなるように塩基を置換した DNAを調製する。 該 DNAは本発明のポリぺプチドの効率的製造に有用である。  If necessary, a DNA is prepared by substituting the nucleotide sequence of the portion encoding the polypeptide of the present invention so that the nucleotide sequence becomes an optimal codon for expression in a host cell. The DNA is useful for efficient production of the polypeptide of the present invention.
該 DNA断片、 または全長 cDNAを適当な発現べクタ一のプロモーターの下流 に挿入することにより、 組換えベクターを作製する。  A recombinant vector is prepared by inserting the DNA fragment or full-length cDNA downstream of a promoter in an appropriate expression vector.
該組換えベクターを、 該発現ベクターに適合した宿主細胞に導入する。  The recombinant vector is introduced into a host cell compatible with the expression vector.
宿主細胞としては、 細菌、 酵母、 動物細胞、 昆虫細胞、 植物細胞等、 目的とす る遺伝子を発現できるものであればいずれも用いることができる。  As the host cell, any cells that can express the target gene, such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
発現べクタ一としては、 上記宿主細胞において自立複製可能ないしは染色体中 への組込が可能で、 本発明のポリぺプチドをコ一ドする DNAを転写できる位置 にプロモー夕一を含有しているものが用いられる。  The expression vector contains a promoter at a position where it can replicate autonomously in the host cell or can be integrated into the chromosome, and can transcribe DNA encoding the polypeptide of the present invention. Things are used.
細菌等の原核生物を宿主細胞として用いる場合は、 本発明のポリぺプチドをコ 一ドする DNAを含有してなる組換えべクタ一は原核生物中で自立複製可能であ ると同時に、 プロモー夕一、 リボソーム結合配列、 本発明の DNA、 転写終結配 列より構成されたベクターであることが好ましい。 プロモーターを制御する遺伝 子が含まれていてもよい。  When a prokaryote such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the polypeptide of the present invention is capable of autonomous replication in the prokaryote and at the same time, is promoted. It is preferable that the vector be composed of a ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence. A gene that controls a promoter may be included.
発現べクタ一としては、 例えば、 pB rp2、 pBTacl、 pBTac2 (いずれもベーリ ンガ一マンハイ ム社よ り市販）、 pKK233-2 (Pharmacia 社製）、 pSE280 (Invitrogen社製)、 pGEMEX-1 (Promega社製)、 pQE-8 (QIAGEN社製)、 pKYPIO (特開昭 58-110600)、 pKYP200 [Agric. Biol. Chem., 669 (1984)]、 pLSAl [Agric. Biol. Chem., ， 277 (1989)]、 GELl [Proc. Natl. Acad. Sci. USA, £2, 4306 (1985)]ヽ pBluescript II SK (-) (Stratagene社製)、 Trs30 [Escherichia coli J l09/pTrS30 (FERM BP-5407) よ り 調製〗、 p¾s32 [Escherichia coli JMl09/pTrS32 (FERM BP-5408) より調製]、 pGHA2 [Escherichia coli IGHA2 (FERM B-400) より調製、 特閧昭 60-221091]、 pGKA2 [Escherichia coli IGKA2 (FERM BP-6798) よ り調製、 特開昭 60-221091]、 pTerm2 (US4686191、 US4939094, US5160735)、 pSupex, pUB110、 pTP5、 pCl94、 pEG400 [J. Bacteriol., 112, 2392 (1990)]、 pGEX (Pharmacia社製)、 pETシステム （Novagen 社製）等をあげることができる。 Examples of expression vectors include pBrp2, pBTacl, and pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega PQE-8 (manufactured by QIAGEN), pKYPIO (JP-A-58-110600), pKYP200 [Agric. Biol. Chem., 669 (1984)], pLSAl [Agric. Biol. Chem.,, 277 ( 1989)], GELl [Proc. Natl. Acad. Sci. USA, £ 2, 4306 (1985)] ヽ pBluescript II SK (-) (Stratagene), Trs30 (Escherichia coli Jl09 / pTrS30 (FERM BP-5407) ), P¾s32 [prepared from Escherichia coli JMl09 / pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM B-400), p. 60-221091], pGKA2 [Escherichia coli IGKA2 (FERM BP-6798), JP-A-60-221091], pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pCl94, pEG400 [J. Bacteriol., 112, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen) and the like can be mentioned.
プロモー夕一としては、 宿主細胞中で機能するものであればいかなるものでも よい。 例えば、 ±E _プロモー夕一 （Ptrp；)、 la プロモーター、 PLプロモーター、 PR プロモ一夕一、 T7プロモ一夕一等の、 大腸菌やファージ等に由来するプロモ一 夕一をあげることができる。 また Ptrpを 2つ直列させたプロモー夕一 （Ptrp X 2)、 tacプロモ一夕一、 laeT7プロモ一夕一、 k± Iプロモーターのように人為的に設 計改変されたプロモ一夕一等も用いることができる。 The promoter may be any promoter that functions in the host cell. For example, ± E _ promoter evening one (P trp;), la promoter, P L promoter, P R promoter Isseki one, T7 promoter Isseki one such, be given promo one evening one derived from Escherichia coli or phage, etc. Can be. The promoter evening one obtained by two series of P trp (P trp X 2) , tac promoter Isseki one, LaeT7 promoter Isseki one, k ± I artificially designed modified promoter Isseki one as a promoter Etc. can also be used.
リボソ一ム結合配列であるシャイン-ダルガノ （Shine-Dalgarno) 配列と開始 コドンとの間を適当な距離 （例えば 6〜： 18塩基） に調節したプラスミ ドを用いる ことが好ましい。  It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosomal binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
本発明の組換えベクターにおいては、 本発明の DNAの発現には転写終結配列 は必ずしも必要ではないが、 構造遺伝子の直下に転写終結配列を配置することが 好ましい。  In the recombinant vector of the present invention, a transcription termination sequence is not always necessary for expression of the DNA of the present invention, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.
宿主細胞としては、 ェシヱリヒア属、 セラチア属、 バチルス属、 ブレビバクテ リウム属、 コリネバクテリウム属、 ミクロバクテリウム属、 シユードモナス属等 に属する微生物、例えば、 Escherichia coli XL Ί -Blue, Escherichia coli XL2-Blue Escherichia coli DH1、 Escherichia coli MC I 000, Escherichia coli KY3276N Escherichia coli W1485、 Escherichia coli JM109、 Escherichia coli HB101、 Escherichia coli 丄ヾ o.49、 Escherichia coli W3110、 Escherichia coli NY49、 Escherichia coli GI698、 Escherichia coli TBIヽ Serratia ficaria^ Serratia fonticoia^ Serratia liquefaciens^ Serratia marcescens. Bacillus subtilis、 Bacillus am loliquefacines. Brevibacterium ammoniagenes^ Brevibacterium immariophilum ATCC 14068、 Brevibacterium saccharolyticum ATCC 14066、 Brevibacterium flavum ATCC 14067、 Brevibacterium lar. ofermentum ATCC 13869 ヽ Corynebacterium glutamicum ATCC 13032 ヽ CorynebacteriumHost cells include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., such as Escherichia coli XLa-Blue, Escherichia coli XL2-Blue Escherichia coli DH1, Escherichia coli MC I 000, Escherichia coli KY3276 N Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli 丄 ヾ o.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia Escherichia coli GI6 coli ficaria ^ Serratia fonticoia ^ Serratia liquefaciens ^ Serratia marcescens.Bacillus subtilis, Bacillus am loliquefacines.
O glu amicum ATCC 13869N Corvnebar erium acetoacidophilum ATCC 13870、 Microb acterium ammoniaphilum ATCC 15354ヽ Pseudomonas putida ヽ Ps udomonas sp. D-0110等をあげることができる。 O glu amicum ATCC 13869 N Corvnebar erium acetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354, Pseudomonas putida, Ps udomonas sp. D-0110.
組換えべクタ一の導入方法としては、 上記宿主細胞へ DNAを導入する方法で あればいずれも用いることができ、例えば、カルシウムイオンを用いる方法 [Proc. Natl. Acad. Sci. USA, fifi, 2110 (1972)]、 プロトプラスト法 （特閧昭 63-248394)、 または Gene, 11, 107 (1982) や Molecular & General Genetics,遍, 111 (1979) に記載の方法等をあげることができる。  Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into the above host cells. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, fifi, 2110 (1972)], the protoplast method (Sho 63-248394), or the methods described in Gene, 11, 107 (1982) and Molecular & General Genetics, Hyundai, 111 (1979).
酵母を宿主細胞として用いる場合には、 発現ベクターとして、 例えば、 YEP13 (ATCC 37115)、 YEp24 (ATCC 37051), YCp50 (ATCC 37419), pHS19、 pHS15 等をあげることができる。  When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419), pHS19, and pHS15.
プロモ一夕一としては、 酵母菌株中で発現できるものであればいずれのものを 用いてもよく、例えば、へキソースキナーゼ等の解糖系の遺伝子のプロモー夕一、 PH05プロモーター、 PGKプロモーター、 GAPプロモ一夕一、 ADHプロモー夕 一、 gal 1プロモー夕一、 gal 10プロモ一夕一、 ヒートショヅクポリぺプチドプ 口モーター、 MF l プロモーター、 CUP 1プロモー夕一等をあげることができ る。  As the promoter, any promoter can be used as long as it can be expressed in yeast strains. For example, promoters for glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP Promo One Night, ADH Promo One, gal 1 Promo One, Gal 10 Promo One, Heat Shock Polypeptide Motor, MFI Promoter, CUP One Promo One, etc.
ί百王細胞 と し て ί 、 Saccharomyces ヽ Schizosaccharomyces fe ヽ Kluyveromyces feゝ Trichosporon i 、 Schwanniomyces fe、 j ichia ¾、 Candida 属等に禹 3—る微生物、 ί列兄ば、 Saccharomyces cerevisiae^ S chiz o s acch aromyces pombeゝ Kluyveromyces lactis、 Trichosporon puilulans、 Schwanniomyces alluvius. Gandida iiiili ^等をあげることができる。  Saccharomyces ί Schicharomyces iz Schizosaccharomyces fe ヽ Kluyveromyces fe ゝpombe ゝ Kluyveromyces lactis, Trichosporon puilulans, Schwanniomyces alluvius. Gandida iiiili ^ and the like.
組換えベクターの導入方法としては、 酵母に DNAを導入する方法であればい ずれも用いることができ、 例えば、 エレク ト口ポレーシヨン法 [Methods Enzymol., 1Q4, 182 (1990)]、 スフエロプラスト法 [Proc. Natl. Acad. Sci. USA, 15., 1929 (1978)]、 酢酸リチウム法 [J. BacterioL, 1 , 163 (1983)]、 Proc. Natl. Acad. Sci. USA, IS, 1929 (1978)] 記載の方法等をあげることができる。  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. Examples of the method include an elect-portion method [Methods Enzymol., 1Q4, 182 (1990)] and a spheroplast method. [Proc. Natl. Acad. Sci. USA, 15., 1929 (1978)], lithium acetate method [J. BacterioL, 1, 163 (1983)], Proc. Natl. Acad. Sci. USA, IS, 1929 ( 1978)].
動物細胞を宿主として用いる場合には、発現べクタ一として、例えば、 pcDNAI、 pcDM8 (フナコシ社製)、 AGEl07 [特開平 3-22979、 Cytotechnology, a, 133 (1990)]、 pAS3-3 (特開平 2-227075)、 pCDM8 [Nature, , 840 (1987)]、 pcDNAI/Amp (Invitrogen 社製）、 pREP4 (Invitrogen 社製）、 p AGE 103 [J. Biochem., 1Q1, 1307 (1987)]、 pAGE210等をあげることができる。 When an animal cell is used as a host, pcDNAI, pcDM8 (manufactured by Funakoshi), AGEl07 [JP-A-3-22979, Cytotechnology, a, 133 (1990)], pAS3-3 (JP-A-2-227075), pCDM8 [Nature, 840 (1987)], pcDNAI / Amp (Manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 1Q1, 1307 (1987)], pAGE210 and the like.
プロモー夕一としては、 動物細胞中で機能するものであればいずれも用いるこ とができ、 例えば、 サイ トメガロウィルス （CMV)の IE (immediate early)遺伝 子のプロモー夕一、 SV40の初期プロモーター、 レトロウイルスのプロモーター、 メタロチォネィンプロモー夕一、 ヒ一トショックプロモー夕一、 SRひプロモ一夕 —等をあげることができる。また、 ヒト CMVの IE遺伝子のェンハンサ一をプロ モー夕一と共に用いてもよい。  Any promoter can be used as long as it functions in animal cells. For example, the promoter of the cytomegalovirus (CMV) IE (immediate early) gene and the early promoter of SV40 can be used. , Retrovirus promoters, metallotion promoters, heat shock promoters, SR promoters, etc. The enhancer of the IE gene of human CMV may be used together with the promoter.
宿主細胞としては、 ヒトの細胞であるナマルバ （Namalwa)細胞、 サルの細胞 である COS細胞、チャイニーズ'ハムスターの細胞である CHO細胞、 HBT5637 (特閧昭 63-299) 等をあげることができる。  Examples of host cells include Namalwa cells, which are human cells, COS cells, which are monkey cells, CHO cells, which are Chinese hamster cells, and HBT5637 (63-299).
動物細胞への組換えベクターの導入方法としては、 動物細胞に DNAを導入す る方法であればいずれも用いることができ、 例えば、 エレクトロボレ一シヨン法 [Cytotechnology, 133 (1990)]、 リン酸カルシウム法 （特開平 2-227075)、 リポ フエクション法 [Proc. Natl. Acad. Sci. USA, S4, 7413 (1987)、 Virology, 52, 456 (1973)] 等をあげることができる。  As a method for introducing a recombinant vector into animal cells, any method can be used as long as DNA can be introduced into animal cells. For example, electroporation method [Cytotechnology, 133 (1990)], calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA, S4, 7413 (1987), Virology, 52, 456 (1973)] and the like.
昆虫細胞を宿主として用いる場合には、 例えばカレント ·プロトコ一ルズ 'ィ ン'モレ ュフ—ノ ィォロシ一、 Bacxuovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)、 Bio/Technology, fi, 47 (1988) 等に記載された方法によって、 ポリペプチドを発現することができる。 即ち、 組換え遺伝子導入ベクターおよびバキュロウィルスを昆虫細胞に共導入 して昆虫細胞培養上清中に組換えウィルスを得た後、 さらに組換えウィルスを昆 虫細胞に感染させ、 ポリペプチドを発現させることができる。  When an insect cell is used as a host, for example, the current protocol 'In' Morefu-Noroshi, Bacxuovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology , fi, 47 (1988), etc., to express the polypeptide. That is, a recombinant gene transfer vector and a baculovirus are co-transfected into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is infected into insect cells to express the polypeptide. be able to.
該方法において用いられる遺伝子導入べクタ一としては、 例えば、 pVL1392、 pVLl393、 pBlueBacIII (ともに Invitoi'ogen社製） 等をあげることができる。 バキュロウィルスとしては、 例えば、 夜盗蛾科昆虫に感染するウィルスである ァゥトグラファ ' 力リフォルニ力 · ヌクレア一 ■ ポリへドロシス · ウィルスExamples of the gene transfer vector used in the method include pVL1392, pVLl393, pBlueBacIII (all manufactured by Invitoi'ogen) and the like. Baculoviruses are, for example, viruses that infect night roth moths Art Grapher's Force Reformi Force · Nuclea ■ Polyhemdosis · Virus
(Autographa caliiornica nuclear polyhedrosis virus)等 ¾用いることがでさる。 昆虫細胞としては、 Spodoptera frugiperda の ¾巣細胞である Sf9、 Sf21 [Baculo virus Expression Vectors, A Laboratory Manual, W. H. Freeman ana Company, New York (1992)]、 Trichoplusia の卵巣細胞である High 5 (Invitrogen社製） 等を用いることができる。 (Autographa caliiornica nuclear polyhedrosis virus) etc. ¾Can be used. As insect cells, Spodoptera frugiperda nest cells Sf9, Sf21 [Baculo virus Expression Vectors, A Laboratory Manual, WH Freemanana Company, New York (1992)], Trichoplusia ovary cells High 5 (manufactured by Invitrogen) ) Can be used.
組換えウィルスを調製するための、 昆虫細胞への上記組換え遺伝子導入べク夕 —と上記バキュロウィルスの共導入方法としては、 例えば、 リン酸カルシウム法 (特開平 2-227075)、 リポフエクション法 [Proc. Natl. Acad. Sci. USA, M, 7413 (1987)] 等をあげることができる。  Methods for co-transferring the above-described recombinant gene into insect cells and the above baculovirus to prepare a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [ Proc. Natl. Acad. Sci. USA, M, 7413 (1987)].
植物細胞を宿主細胞として用いる場合には、 発現べクタ一として、 例えば、 Ti プラスミ ド、 タバコモザイクウィルスベクタ一等をあげることができる。  When a plant cell is used as a host cell, examples of the expression vector include Ti plasmid and tobacco mosaic virus vector.
プロモーターとしては、 植物細胞中で発現できるものであればいずれのものを 用いてもよく、 例えば、 カリフラワーモザイクウィルス （CaMV) の 35S プロモ 一夕一、 ィネアクチン 1プロモーター等をあげることができる。  Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the 35S promoter of cauliflower mosaic virus (CaMV), the inactin 1 promoter, and the like.
宿主細胞としては、 タバコ、 ジャガイモ、 トマト、 ニンジン、 ダイズ、 ァブラ ナ、 アルフアルファ、 イネ、 コムギ、 ォォムギ等の植物細胞等をあげることがで ぎる。  Examples of the host cell include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfa-alpha, rice, wheat, and wheat.
組換えベクターの導入方法としては、 植物細胞に DNAを導入する方法であれ ばいずれも用いることができ、例えば、ァグロパクテリゥム (Agrobacterium^ (特 閧昭 59-140885、 特開昭 60-70080、 WO 94/00977), エレクト口ポレーシヨン法 (特開昭 60-251887)、 パーティクルガン （遺伝子銃） を用いる方法 （日本特許第 2606856、 日本特許第 2517813) 等をあげることができる。  As a method for introducing a recombinant vector, any method for introducing DNA into plant cells can be used. For example, Agrobacterium (Agrobacterium ^ (Japanese Patent Application No. 59-140885, Japanese Patent Application Laid-Open No. 70080, WO 94/00977), the electoral poration method (JP-A-60-251887), a method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), and the like.
遺伝子の発現方法としては、 直接発現以外に、 モレキュラー · クロ一ニング第 2版に記載されている方法等に準じて、 分泌生産、 融合タンパク質発現等を行う ことができる。  As a method for expressing a gene, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, 2nd edition.
酵母、 動物細胞、 昆虫細胞または植物細胞により発現させた場合には、 糖ある I、は糖鎖が付加されたポリペプチドを得ることができる。 以上のようにして得られる本発明の形質転換体を培地に培養し、 培養物中に本 発明のポリペプチドを生成蓄積させ、 該培養物から採取することにより、 本発明 のポリべプチドを製造することができる。 When expressed by yeast, animal cells, insect cells or plant cells, a polypeptide with a sugar chain added thereto can be obtained. The polypeptide of the present invention is produced by culturing the transformant of the present invention obtained as described above in a medium, producing and accumulating the polypeptide of the present invention in the culture, and collecting from the culture. can do.
本発明の形質転換体を培地に培養する方法は、 宿主の培養に用いられる通常の 方法に従って行うことができる。  The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
本発明の形質転換体が大腸菌等の原核生物あるいは酵母等の真核生物を宿主と して得られた形質転換体である場合、 該形質転換体を培養する培地として、 該形 質転換体が資化し得る炭素源、 窒素源、 無機塩類等を含有し、 該形質転換体の培 養を効率的に行える培地であれば天然培地、 合成培地のいずれを用いてもよい。 炭素源としては、 該形質転換体が資化し得るものであればよく、 グルコース、 フラクトース、 スクロース、 これらを含有する糖蜜、 デンプンあるいはデンプン 加水分解物等の炭水化物、 酢酸、 プロピオン酸等の有機酸、 エタノール、 プロパ ノ一ルなどのアルコール類等を用いることができる。  When the transformant of the present invention is a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host, the transformant is used as a medium for culturing the transformant. Either a natural medium or a synthetic medium may be used as long as the medium contains an assimilable carbon source, a nitrogen source, inorganic salts, and the like, and can efficiently culture the transformant. The carbon source may be any as long as the transformant can be assimilated, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, Alcohols such as ethanol and propanol can be used.
窒素源としては、 アンモニア、 塩化アンモニゥム、 硫酸アンモニゥム、 酢酸ァ ンモニゥム、 リン酸アンモニゥム等の無機酸もしくは有機酸のアンモニゥム塩、 その他の含窒素化合物、 ならびに、 ペプトン、 肉エキス、 酵母エキス、 コーンス チープリカー、 カゼイン加水分解物、 大豆粕および大豆粕加水分解物、 各種発酵 菌体およびその消化物等を用いることができる。  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells and digests thereof can be used.
無機塩としては、 リン酸第一カリウム、 リン酸第二カリウム、 リン酸マグネシ ゥム、 硫酸マグネシウム、 塩化ナトリウム、 硫酸第一鉄、 硫酸マンガン、 硫酸銅、 炭酸カルシウム等を用いることができる。  As the inorganic salt, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
培養は、 振盪培養または深部通気攪拌培養などの好気的条件下で行う。 培養温 度は 15~40°Cがよく、 培養時間は、 通常 16時間〜 7 日間である。 培養中の pH は 3.0〜9.0に保持することが好ましい。 pHの調整は、 無機または有機の酸、 ァ ルカリ溶液、 尿素、 炭酸カルシウム、 アンモニアなどを用いて行う。  The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days. The pH during the culturing is preferably maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, alkali solution, urea, calcium carbonate, ammonia, and the like.
また、 培養中必要に応じて、 アンビシリンやテトラサイクリン等の抗生物質を 培地に添加してもよい。  If necessary, an antibiotic such as ambicillin or tetracycline may be added to the medium during the culture.
プロモー夕一として誘導性のプロモーターを用いた組換えべクタ一で形質転換 した微生物を培養するときには、 必要に応じてィンデューサ一を培地に添加して もよい。 例えば、 プロモー夕一を用いた組換えベクターで形質転換した微生 物を培養するときにはィソプロピル-/? -D-チォガラクトビラノシド等を、 trpプロ モーターを用いた組換えべクタ一で形質転換した微生物を培養するときにはィ ンドールァクリル酸等を培地に添加してもよい。 Transform with a recombinant vector using an inducible promoter as a promoter When culturing the isolated microorganism, if necessary, an inducer may be added to the medium. For example, when culturing a microorganism transformed with a recombinant vector using a promoter, isoforms such as isopropyl-/?-D-thiogalactovyranoside are transformed with a recombinant vector using the trp promoter. When culturing the transformed microorganism, indoleacrylic acid or the like may be added to the medium.
動物細胞を宿主として得られた形質転換体を培養する培地としては、 一般に使 用されている RPMI1640 培地 [The Journal of the American Medical Association, 122., 519 (1967)]、 Eagleの MEM培地 [Science, 122, 501 (1952)]、 ダルベッコ改変 MEM培地 [Virology, 396 (1959)]、 199培地 [Proceeding of the Society for the Biological Medicine, l , 1 (1950)] またはこれら培地に牛胎 児血清等を添加した培地等を用いることができる。  As a medium for culturing transformants obtained using animal cells as a host, commonly used RPMI1640 medium [The Journal of the American Medical Association, 122., 519 (1967)], Eagle's MEM medium [Science , 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine, l, 1 (1950)] or fetal bovine serum etc. And the like can be used.
培養は、 通常 pH 6〜8、 30〜40°C；、 5% CO2存在下等の条件下で 1〜7日間行 Ό。 The cultivation is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C; and in the presence of 5% CO 2 .
また、 培養中必要に応じて、 カナマイシン、 ペニシリン等の抗生物質を培地に 添加してもよい。  If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
昆虫細胞を宿主として得られた形質転換体を培養する培地としては、 一般に使 用されている ΤΝΜ-ΪΉ培地 （Pharmingen社製)、 Sf-900 II SFM培地 （Life Technologies社製)、 Ex Cell 400、 Ex Cell 405 (いずれも JRH Biosciences社製)、 Grace's Insect Medium [Nature, 1 , 788 (1962)] 等を用いることができる。 培養は、 通常 pH 6〜7、 25〜30°C等の条件下で、 1〜5日間行う。  Culture media for transformants obtained using insect cells as a host include commonly used 宿主 -ΪΉ media (Pharmingen), Sf-900 II SFM medium (Life Technologies), Ex Cell 400 And Ex Cell 405 (both manufactured by JRH Biosciences), Grace's Insect Medium [Nature, 1, 788 (1962)] and the like. Cultivation is usually carried out for 1 to 5 days under conditions of pH 6 to 7 and 25 to 30 ° C.
また、 培養中必要に応じて、 ゲン夕マイシン等の抗生物質を培地に添加しても よい。  If necessary, an antibiotic such as genyumycin may be added to the medium during the culture.
植物細胞を宿主として得られた形質転換体は、 細胞として、 または植物の細胞 や器官に分化させて培養することができる。 該形質転換体を培養する培地として は、 一般に使用されているムラシゲ · アンド ·スク一グ （MS) 培地、 ホワイ ト (White) 培地、 またはこれら培地にオーキシン、 サイ トカイニン等、 植物ホルモ ンを添加した培地等を用いることができる。  A transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ. As a culture medium for culturing the transformant, a commonly used Murashige and Skog (MS) medium, a white (White) medium, or a plant formone such as auxin or cytokinin added to these mediums is used. Media or the like can be used.
培養は、 通常 pH 5〜9、 20〜40°Cの条件下で 3〜60日間行う。 また、 培養中必要に応じて、 カナマイシン、 ハイグロマイシン等の抗生物質を 培地に添加してもよい。 Cultivation is usually performed at pH 5 to 9 and 20 to 40 ° C for 3 to 60 days. If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
上記のとおり、 本発明のポリぺプチドをコードする DNAを組み込んだ組換え 体ベクターを保有する微生物、動物細胞、あるいは植物細胞由来の形質転換体を、 通常の培養方法に従って培養し、 該ポリペプチドを生成蓄積させ、 該培養物より 該ポリべプチドを採取することにより、該ポリぺプチドを製造することができる。 遺伝子の発現方法としては、 直接発現以外に、 モレキュラー 'クローニング第 2版に記載されている方法等に準じて、 分泌生産、 融合ポリペプチド発現等を行 うことができる。  As described above, a transformant derived from a microorganism, an animal cell, or a plant cell having a recombinant vector into which a DNA encoding the polypeptide of the present invention has been incorporated is cultured according to a conventional culture method, and the polypeptide is obtained. Is produced and accumulated, and the polypeptide is collected from the culture, whereby the polypeptide can be produced. As a method for expressing the gene, secretory production, fusion polypeptide expression, and the like can be performed according to the method described in Molecular 'Cloning 2nd edition, in addition to direct expression.
本発明のポリペプチドの生産方法としては、 宿主細胞内に生産させる方法、 宿 主細胞外に分泌させる方法、 あるいは宿主細胞外膜上に生産させる方法があり、 使用する宿主細胞や、 生産させるポリペプチドの構造を変えることにより、 該方 法を選択することができる。  The polypeptide of the present invention can be produced in a host cell, secreted outside the host cell, or produced on the host cell outer membrane. The host cell to be used or the polypeptide to be produced can be used. The method can be selected by changing the structure of the peptide.
本発明のポリべプチドが宿主細胞内あるいは宿主細胞外膜上に生産される場合、 ポールソンらの方法 [J. Biol. Chem., 2β4, 17619 (1989)]、 ロウらの方法 [Pro Natl. Acad. Sci. USA, M, 8227 (1989)、 Genes Develop., 4, 1288 (1990)〗、 また は特閧平 5-336963、 WO 94/23021等に記載の方法を準用することにより、 該ポ リぺプチドを宿主細胞外に積極的に分泌させることができる。  When the polypeptide of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 2β4, 17619 (1989)] and the method of Lowe et al. [Pro Natl. Acad. Sci. USA, M, 8227 (1989), Genes Develop., 4, 1288 (1990)〗, or the method described in JP-A-5-336963, WO 94/23021, etc. The polypeptide can be actively secreted out of the host cell.
すなわち、 遺伝子組換えの手法を用いて、 本発明のポリペプチドの活性部位を 含むポリぺプチドの手前にシグナルぺプチドを付加した形で発現させることによ り、 本発明のポリペプチドを宿主細胞外に積極的に分泌させることができる。 また、 特開平 2-227075 に記載されている方法に準じて、 ジヒドロ葉酸還元酵 素遺伝子等を用いた遺伝子増幅系を利用して生産量を上昇させることもできる。 さらに、 遺伝子導入した動物または植物の細胞を再分化させることにより、 遺 伝子が導入された動物個体 （トランスジエニック非ヒ ト動物） または植物個体 (トランスジエニック植物） を造成し、 これらの個体を用いて本発明のポリべプチ ドを製造することもできる。  That is, the polypeptide of the present invention is expressed in a form in which a signal peptide is added in front of the polypeptide containing the active site of the polypeptide of the present invention using a gene recombination technique, whereby the polypeptide of the present invention can be expressed in host cells. It can be actively secreted out. Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the transgenic animal or plant, an animal into which the gene has been introduced (transgenic non-human animal) or a plant (transgenic plant) can be created. The polypeptide of the present invention can also be produced using an individual.
形質転換体が動物個体または植物個体の場合は、 通常の方法に従って、 飼育ま たは栽培し、 該ポリペプチドを生成蓄積させ、 該動物個体または植物個体より該 ポリぺプチドを採取することにより、 該ポリべプチドを製造することができる。 動物個体を用いて本発明のポリぺプチドを製造する方法としては、 例えば公知 の方法 [American Journal of Clinical Nutrition, fiS, 639S (1996)、 American Journal of Clinical Nutrition, β3, 627S (1996)、 Bio/Technology, S, 830 (1991)] に準じて遺伝子を導入して造成した動物中に本発明のポリべプチドを生産する方 法があげられる。 If the transformant is an individual animal or plant, breed it according to the usual method. Alternatively, the polypeptide can be produced by producing or accumulating the polypeptide, and collecting the polypeptide from the animal or plant individual. As a method for producing the polypeptide of the present invention using an animal individual, for example, known methods [American Journal of Clinical Nutrition, fiS, 639S (1996), American Journal of Clinical Nutrition, β3, 627S (1996), Bio / Technology, S, 830 (1991)] to produce the polypeptide of the present invention in an animal constructed by introducing a gene.
動物個体の場合は、 例えば、 本発明のポリペプチドをコードする DNAを導入 したトランスジエニック非ヒト動物を飼育し、 該ポリべプチドを該動物中に生 成 -蓄積させ、 該動物中より該ポリペプチドを採取することにより、 該ポリぺプ チドを製造することができる。 該動物中の生成 ·蓄積場所としては、 例えば、 該 動物のミルク （特開昭 63-309192)、 卵等をあげることができる。 この際に用いら れるプロモー夕一としては、 動物で発現できるものであればいずれも用いること ができるが、 例えば、 乳腺細胞特異的なプロモー夕一であるひカゼインプロモ一 夕一、 カゼインプロモーター、 ラクトグロプリンプロモー夕一、 ホエー酸性 プロティンプロモー夕一等が好適に用いられる。  In the case of an animal individual, for example, a transgenic non-human animal into which DNA encoding the polypeptide of the present invention has been introduced is bred, and the polypeptide is produced and accumulated in the animal. By collecting the polypeptide, the polypeptide can be produced. Examples of the place of production and accumulation in the animal include milk (eg, JP-A-63-309192) and eggs of the animal. As the promoter used at this time, any promoter that can be expressed in an animal can be used.For example, a promoter specific to mammary gland cells, a casein promoter, a casein promoter, A lactoglobulin promoter and a whey acid protein promoter are preferably used.
植物個体を用いて本発明のポリペプチドを製造する方法としては、 例えば本発 明のポリべプチドをコードする DNAを導入したトランスジヱニヅク植物を公知 の方法 [組織培養， 20 (1994)、 組織培養， 21 (1995)、 Trends in Biotechnology, 15, 45 (1997)] に準じて栽培し、 該ポリペプチドを該植物中に生成 ·蓄積させ、 該植 物中より該ポリぺプチドを採取することにより、 該ポリぺプチドを生産する方法 があげられる。  As a method for producing the polypeptide of the present invention using a plant individual, for example, a transgenic plant into which DNA encoding the polypeptide of the present invention has been introduced can be prepared by a known method [tissue culture, 20 (1994)]. Tissue culture, 21 (1995), Trends in Biotechnology, 15, 45 (1997)], producing and accumulating the polypeptide in the plant, and collecting the polypeptide from the plant. By doing so, a method for producing the polypeptide can be mentioned.
本発明の形質転換体により製造されたポリペプチドを単離精製するためには、 通常の酵素の単離精製法を用いることができる。例えば本発明のポリぺプチドが、 細胞内に溶解状態で発現した場合には、 培養終了後、 細胞を遠心分離により回収 し、 水系緩衝液にけん濁後、 超音波破砕機、 フレンチプレス、 マントンガウリン ホモゲナイザー、 ダイノミル等により細胞を破碎し、 無細胞抽出液を得る。 該無 細胞抽出液を遠心分離することにより得られる上清から、 通常の酵素の単離精製  In order to isolate and purify the polypeptide produced by the transformant of the present invention, a general enzyme isolation and purification method can be used. For example, when the polypeptide of the present invention is expressed in a dissolved state in the cells, the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Menton. The cells are disrupted using a Gaulin homogenizer, Dynomill, etc. to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, normal enzyme isolation and purification
Z7 法、 即ち、 溶媒抽出法、 硫安等による塩析法、 脱塩法、 有機溶媒による沈殿法、 ジェチルァミノェチル （DEAE)-セファロース、 DIAION HPA-75 (三菱化学社製） 等のレジンを用いた陰ィオン交換ク口マトグラフィ一法、 S-Sepharose FF (Pharmacia社製）等のレジンを用いた陽ィォン交換ク口マトグラフィ一法、ブチ ルセファロース、 フェニルセファロ一ス等のレジンを用いた疎水性クロマトグラ フィ一法、 分子篩を用いたゲルろ過法、 ァフィ二ティ一クロマトグラフィー法、 クロマトフォーカシング法、 等電点電気泳動等の電気泳動法等の手法を単独ある いは組み合わせて用い、 精製標品を得ることができる。 Z7 Methods, ie, solvent extraction, salting out with ammonium sulfate, desalting, precipitation with organic solvents, resins such as getylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical Corporation), etc. A method using anion-exchange mouth chromatography using a resin, a method using a resin such as S-Sepharose FF (manufactured by Pharmacia), and a resin using a resin such as butyl sepharose and phenyl sepharose. Hydrophobic chromatography method, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing, etc. are used alone or in combination. A purified sample can be obtained.
また、 該ポリペプチドが細胞内に不溶体を形成して発現した場合は、 同様に細 胞を回収後、 破砕し、 遠心分離を行うことにより、 沈殿画分としてポリペプチド の不溶体を回収する。 回収したポリぺプチドの不溶体を蛋白質変性剤で可溶化す る。 該可溶化液を希釈または透析し、 該可溶化液中の蛋白質変性剤の濃度を下げ ることにより、 該ポリペプチドを正常な立体構造に戻す。 該操作の後、 上記と同 様の単離精製法により該ポリぺプチドの精製標品を得ることができる。  When the polypeptide is expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to collect the insoluble form of the polypeptide as a precipitate fraction. . The insoluble form of the recovered polypeptide is solubilized with a protein denaturant. The polypeptide is returned to a normal tertiary structure by diluting or dialyzing the lysate and reducing the concentration of the protein denaturant in the lysate. After this operation, a purified preparation of the polypeptide can be obtained by the same isolation and purification method as described above.
本発明のポリべプチド、 あるいは該ポリぺプチドに糖鎖の付加されたポリぺプ チド等の誘導体が細胞外に分泌された場合には、 培養上清に該ポリべプチドある いは該ポリペプチドの誘導体を回収することができる。 即ち、 該培養物を上記と 同様の遠心分離等の手法により処理することにより培養上清を取得し、 該培養上 清から、 上記と同様の単離精製法を用いることにより、 精製標品を得ることがで ぎる。  When the polypeptide of the present invention or a derivative such as a polypeptide having a sugar chain added to the polypeptide is secreted extracellularly, the polypeptide or the polypeptide is added to the culture supernatant. Derivatives of the peptide can be recovered. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. You can get it.
このようにして取得されるポリペプチドとして、 例えば、 配列番号 1記載のァ ミノ酸配列の 327番目から 340番目、配列番号 3記載のァミノ酸配列の 335番目 から 348番目、 配列番号 5記載のアミノ酸配列の 229番目から 309番目および 289番目から 309番目、配列番号 7記載のアミノ酸配列の 231番目から 310番目 および 290番目から 310番目、および配列番号 9記載のァミノ酸配列 227番目か ら 306番目および 286番目から 306番目からなるポリぺプチドをあげることがで さる。 2 . 本発明の DNAの調製 Examples of the polypeptide thus obtained include, for example, amino acids 327 to 340 of the amino acid sequence of SEQ ID NO: 1, amino acids 335 to 348 of the amino acid sequence of SEQ ID NO: 3, and amino acids of SEQ ID NO: 5. 229 to 309 and 289 to 309 of the sequence; 231 to 310 and 290 to 310 of the amino acid sequence described in SEQ ID NO: 7, and the amino acid sequence 227 to 306 of SEQ ID NO: 9 It is better to give polypeptides consisting of 286th to 306th. 2. Preparation of DNA of the present invention
本発明の DNAは、 モレキュラー 'クロ一ニング第 2版やカレント ■ プロトコ ールズ 'イン ·モレキュラー ·バイオロジー等に記載された方法等を用い、 例え ば以下の方法により、 ヒトまたは非ヒト動物の組織または細胞から調製すること ができる。  The DNA of the present invention can be prepared by using the method described in Molecular 'Cloning Second Edition or Current Protocols' in Molecular Biology, etc. Alternatively, it can be prepared from cells.
ヒトまたは非ヒト動物の組織または細胞としては、 Aktまたは Hsp90が発現し ている組織または細胞であればいかなるものでもよいが、 例えば、 脳、 精巣、 心 臓、 肺などがあげられる。  The human or non-human animal tissue or cell may be any tissue or cell in which Akt or Hsp90 is expressed, and examples include brain, testis, heart, lung and the like.
ヒトまたは非ヒト動物の組織または細胞から全 RNAまたは mRNAを調製する。 調製した全 RNAまたは mRNAから cDNAラィブラリ一を作製する。  Total RNA or mRNA is prepared from tissues or cells of a human or non-human animal. Prepare a cDNA library from the prepared total RNA or mRNA.
本発明のポリべプチドのアミノ酸配列に基づいて、 デジエネレイティブプライ マ一を作製し、 作製した cDNAライブラリ一を鎵型として PCR法にて本発明の ポリべプチドをコ一ドする遺伝子断片を取得する。  A gene fragment which encodes the polypeptide of the present invention by PCR using the prepared cDNA library as a 鎵 type by preparing a degiene primer based on the amino acid sequence of the polypeptide of the present invention To get.
取得した遺伝子断片をプロ一プとして用い、 cDNAライブラリーをスクリー二 ングし本発明のぺプチドをコ一ドする本発明の DNAを取得することができる。 ヒ トまたは非ヒ ト動物の組織または細胞の mRNA は市販のもの （例えば Clontech社製） を用いてもよいし、 前述のごとくヒトまたは非ヒト動物の組織ま たは細胞から調製してもよい。 ヒトまたは非ヒト動物の組織または細胞から全 RNAを調製する方法としては、チォシアン酸グァニジン一トリフルォロ酢酸セシ ゥム法 [Methods in Enzymology，l ，3 (1987)]、酸性チォシアン酸グァニジン · フエノール · クロ口ホルム （AGPC) 法 [Analytical Biochemistry, 1Q2, 156 (1987)、 実験医学、 S, 1937 (1991)] などがあげられる。  Using the obtained gene fragment as a probe, the cDNA library can be screened to obtain the DNA of the present invention encoding the peptide of the present invention. The mRNA of human or non-human animal tissues or cells may be a commercially available one (for example, manufactured by Clontech), or may be prepared from human or non-human animal tissues or cells as described above. . As methods for preparing total RNA from tissues or cells of human or non-human animals, guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, l, 3 (1987)], guanidine acid thiocyanate, phenol-clo Oral form (AGPC) method [Analytical Biochemistry, 1Q2, 156 (1987), Experimental Medicine, S, 1937 (1991)].
また、 全 ϋΝΑから poly (A)， RNAとして mRNAを調製する方法としては、 オリゴ （dT) 固定化セルロースカラム法 （モレキュラー 'クロ一ニング第 2版）等 があげられる。  As a method for preparing mRNA as poly (A) or RNA from the whole, there is an oligo (dT) -immobilized cellulose column method (Molecular 'Cloning 2nd edition) and the like.
さらに、 Fast Track mRNA Isolation Kit (Invitrogen社製)、 Quick Prep mRNA Purification Kit (Pharmacia社） などのキヅ トを用いることにより mRNAを調 製することができる。 調製したヒトまたは非ヒト動物の組織または細胞 mRNAから cDNAライプラ リーを作製する。 cDNAライブラリ一作製法としては、 モレキュラー 'クロ一二 ング第 2版、 カレント ·プロトコ一ルズ 'イン 'モレキュラー 'バイオロジー、 A Laboratory Manual, 2nd Ed. (1989)等に記載された方法、 あるいは市販のキ ヅ ト、 例え is: Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies社)、 ZAP-cDNA Synthesis Kit (STRATAGENE社） を 用いる方法などがあげられる。 Furthermore, mRNA can be prepared by using a kit such as the Fast Track mRNA Isolation Kit (Invitrogen) or the Quick Prep mRNA Purification Kit (Pharmacia). A cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA. Methods for preparing a cDNA library include methods described in Molecular 'Clothing Second Edition, Current Protocols' In 'Molecular' Biology, A Laboratory Manual, 2nd Ed. (1989), or commercially available. Examples of such kits include a method using the is: Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) and a ZAP-cDNA Synthesis Kit (STRATAGENE).
cDNAライブラリーを作製するためのクローニングベクタ一としては、 大腸菌 K12株中で自立複製できるものであれば、 ファージベクター、 プラスミ ドベクタ —等いずれでも使用できる。 具体的には、 ZAP Express [STRATAGENE社製、 Strategies, 5, 58 (1992)]、 pBluescript II SK (+) [Nucleic Acids Research, 11, 9494 (1989)]、 Lambda ZAP II (STRATAGENE社)、入 gtl0、 A gtll [DNA cloning, A Practical Approach, 1, 49 (1985)]、 人 riplEx (Clontech 社製）、 Λ ExCell (Pharmacia社製)、 pT7T318U (Pharmacia社製)、 pcD2 [Mol. Cell. Biol., S， 280 (1983)] および pUCl8 [Gene, , 103 (1985)] 等があげられる。  As a cloning vector for preparing a cDNA library, any phage vector, plasmid vector or the like can be used as long as it can replicate autonomously in E. coli K12 strain. Specifically, ZAP Express [Stratagene, Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 11, 9494 (1989)], Lambda ZAP II (Stratagene), gtl0, A gtll [DNA cloning, A Practical Approach, 1, 49 (1985)], human riplEx (Clontech), Λ ExCell (Pharmacia), pT7T318U (Pharmacia), pcD2 (Mol. Cell. Biol) ., S, 280 (1983)] and pUC18 [Gene,, 103 (1985)].
cDNAを組み込んだベクターを導入する大腸菌としては、 大腸菌に属する微生 物であればいずれでも用いることができる。 具体的には、 Escherichia coli XL1- Blue MRF' [STRATAGENE社、 Strategies, 5, 81 (1992)]、 Escherichia coli C600 [Genetics, SS, 440 (1954)]、 Escherichia coli Y1088 [Science, 222, 778 (1983)]、 Escherichia coli Y1090 [Science, 222, 778 (1983)]、 Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)]、 Kschericliia coli K802 [J. Mol. Biol., 16, 118 (1966)] および Escherichia coli JM105 [Gene, , 275 (1985)] 等が用いられる。  As the Escherichia coli to which the vector into which the cDNA is incorporated is introduced, any microorganisms belonging to Escherichia coli can be used. Specifically, Escherichia coli XL1-Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, SS, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 ( 1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Kschericliia coli K802 [J. Mol. Biol., 16, 118 (1966)] and Escherichia coli JM105 [Gene,, 275 (1985)].
この cDNAライブラリ一を、 そのまま以降の解析に用いてもよいが、 不完全長 cDNAの割合を下げ、 なるべく完全長 cDNAを効率よく取得するために、 オリゴ キヤヅプ法 [Gene,道' 171 (1994)、 Gene，纖， 149 (1997)、蛋白質核酸酵素， , 603 (1996)、 実験医学， 11， 2491 (1993)、 cDNAクローニング、 羊土社 （1996)、 遺伝子ライプラリーの作製法、羊土社 （1994)] を用いて調製した cDNAライブラ リーを以下の解析に用いてもよい。 本発明のポリペプチドのァミノ酸配列に基づいて、 該ァミノ酸配列をコードす ることが予測される塩基配列の 5'末端および 3'末端の塩基配列に特異的なデジェ ネレイティブプライマ一を作製し、 作製した cDNA ライブラリ一を铸型として PG 法 [PCR Protocols, Academic Press (1990)] を用いて DNAの増幅を行うこ とにより本発明のポリペプチドをコードする遺伝子断片を取得することができる 取得した遺伝子断片が本発明のポリペプチドをコードする DNAであることは、 通常用いられる塩基配列解析方法、 例えばサンガー （Sanger) らのジデォキシ法 [Pro Natl. Acad. Sci. USA, 24, 5463 (1977)] あるいは ABIPRISM377 DNAシ ークェンサ一 （PE Biosystems社製） 等の塩基配列分析装置を用いて分析するこ とにより、 確認することができる。 This cDNA library may be used as it is for subsequent analyses, but in order to reduce the proportion of incomplete-length cDNA and obtain full-length cDNA as efficiently as possible, the oligocap method [Gene, Michi'171 (1994) , Gene, Fiber, 149 (1997), Protein Nucleic Acid Enzyme, 603 (1996), Experimental Medicine, 11, 2491 (1993), cDNA Cloning, Yodosha (1996), Gene Ripplery, Yodosha ( 1994)] may be used for the following analyses. Based on the amino acid sequence of the polypeptide of the present invention, a degenerative primer specific to the nucleotide sequence at the 5 'end and 3' end of the nucleotide sequence predicted to encode the amino acid sequence is prepared. Then, a gene fragment encoding the polypeptide of the present invention can be obtained by amplifying DNA using the prepared cDNA library type I as a type II and PG method [PCR Protocols, Academic Press (1990)]. That the obtained gene fragment is a DNA encoding the polypeptide of the present invention can be determined by a commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Pro Natl. Acad. Sci. USA, 24, 5463 ( 1977)] or by using a base sequence analyzer such as ABIPRISM377 DNA Sequencer (manufactured by PE Biosystems).
該遺伝子断片を DNAをプローブとして、 ヒトまたは非ヒト動物の組織または 細胞に含まれる mRNAから合成した cDNAあるいは cDNAライプラリーに対し てコロニーハイプリダイゼ一ションゃプラークハイプリダイゼーシヨン （モレキ ユラ一 ·クロ一ニング第 2版） を行うことにより、 本発明の DNAを取得するこ とができる。  Using the gene fragment as a probe for DNA, cDNA or cDNA libraries synthesized from mRNAs contained in tissues or cells of human or non-human animals are subjected to colony hybridization / plaque hybridization (Molecular Cloning). Inning (2nd edition), the DNA of the present invention can be obtained.
また、 本発明をコードする遺伝子断片を取得するために用いたプライマーを用 い、 ヒ 卜または非ヒ ト動物の組織または細胞に含まれる mRNAから合成した cDNAあるいは cDNAライブラリーを錶型として、 PCR法を用いてスクリーニン グを行うことにより、 本発明の DNAを取得することもできる。  Further, using the primers used to obtain the gene fragment encoding the present invention, a cDNA or cDNA library synthesized from mRNA contained in tissues or cells of a human or non-human animal, and The DNA of the present invention can also be obtained by performing screening using the method.
取得した本発明の DNAの塩基配列を末端から、 通常用いられる塩基配列解析 方法、 例えばサンガ一 （Sanger) らのジデォキシ法 [Pro Natl. Acad. Sci. USA, 24, 5463 (1977)] あるいは ABIPRISM377 DNAシークェンサ一 （PE Biosystems 社製） 等の塩基配列分析装置を用いて分析することにより、 該 DNAの塩基配列 を決定する。  The nucleotide sequence of the obtained DNA of the present invention is analyzed from the end by a nucleotide sequence analysis method usually used, for example, the dideoxy method of Sanger et al. [Pro Natl. Acad. Sci. USA, 24, 5463 (1977)] or ABIPRISM377. The base sequence of the DNA is determined by analysis using a base sequence analyzer such as a DNA sequencer (manufactured by PE Biosystems).
決定した cDNAの塩基配列をもとに、 BLAST等の相同性検索プログラムを用 いて、 GenBank、 EMBLおよび DDBJなどの塩基配列データベースを検索する ことにより、 デ一夕ベース中の遺伝子の中で本発明のポリべプチドをコードして いる遺伝子を決定することができる。 決定された DNAの塩基配列に基づいて、 フォスフォアミダイ ト法を利用した パーキン 'エルマ一社の DNA合成機 Model 392等の DNA合成機で化学合成する ことにより、 本発明の DNAを取得することもできる。 By searching the base sequence database such as GenBank, EMBL and DDBJ using a homology search program such as BLAST based on the determined base sequence of the cDNA, the present invention The gene encoding the polypeptide can be determined. Based on the determined DNA base sequence, the DNA of the present invention is obtained by chemically synthesizing with a DNA synthesizer such as Perkin 'Elma's DNA synthesizer Model 392 using the phosphoramidite method. You can also.
3. 本発明のポリペプチドを認識する抗体の調製 3. Preparation of an antibody that recognizes the polypeptide of the present invention
本発明のポリべプチドまたは該ポリべプチドの部分断片ポリべプチドの精製標 品、 あるいは本発明のポリペプチドの一部のアミノ酸配列を有するペプチドを抗 原として用いることにより、 ポリクロ一ナル抗体、 モノクローナル抗体等、 本発 明のポリペプチドを認識する抗体 （以下、 「本発明の抗体」 と略記する） を作製す ることができる。 また、 本発明の抗体は、 それらの抗体断片も包含し、 ヒト化抗 体等の遺伝子組換え抗体およびその抗体断片であってもよい。  A purified antibody of the polypeptide of the present invention or a partial fragment of the polypeptide, or a peptide having a partial amino acid sequence of the polypeptide of the present invention is used as an antigen to obtain a polyclonal antibody, Antibodies that recognize the polypeptide of the present invention, such as monoclonal antibodies (hereinafter, abbreviated as “antibodies of the present invention”) can be produced. The antibody of the present invention also includes those antibody fragments, and may be a recombinant antibody such as a humanized antibody and an antibody fragment thereof.
(1) ポリクローナル抗体の作製 (1) Preparation of polyclonal antibody
本発明のポリぺプチドまたは該ポリべプチドの部分断片ポリぺプチドの精製檩 品、 あるいは本発明のポリべプチドの一部のアミノ酸配列を有するぺプチドを抗 原として用い、 動物に投与することによりポリク口一ナル抗体を作製することが できる。  A purified product of the polypeptide of the present invention or a partial polypeptide of the polypeptide, or a peptide having a partial amino acid sequence of the polypeptide of the present invention is used as an antigen and administered to animals. Thus, a polyclonal antibody can be prepared.
投与する動物として、 ゥサギ、 ャギ、 ラヅト、 マウス、 ハムスター等を用いる ことができる。  Animals to be administered include egrets, goats, rats, mice, hamsters, and the like.
該抗原の投与量は動物 1匹当たり 50〜： 100 gが好ましい。  The dose of the antigen is preferably 50 to 100 g per animal.
ペプチドを用いる場合は、 ペプチ ドをスカシガイへモシァニン （keyhole limpet haemocyanin) ゃ牛チログロブリンなどのキヤリァ蛋白質に共有結合さ せたものを抗原とするのが望ましい。 抗原とするペプチドは、 ペプチド合成機で 合成することができる。  When a peptide is used, it is desirable to use a peptide obtained by covalently binding a peptide to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin. The peptide serving as the antigen can be synthesized using a peptide synthesizer.
該抗原の投与は、 1 回目の投与の後 1〜2週間おきに 3〜： 10回行う。各投与後、 3〜7日目に眼底静脈叢より採血し、該血清が免疫に用いた抗原と反応することを 酵素免疫測定法 [酵素免疫測定法 （ELISA 法） ： 医学書院刊 （1976 年）、 Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] 等で 確認する。 The administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the serum reacts with the antigen used for immunization. Enzyme-linked immunosorbent assay [Enzyme-linked immunosorbent assay (ELISA): published by Medical Shoin (1976 ), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] Confirm.
免疫に用いた抗原に対し、 その血清が充分な抗体価を示した非ヒト哺乳動物よ り血清を取得し、 該血清を分離、 精製することによりポリクローナル抗体を取得 することができる。  A polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum has a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
分離、 精製する方法としては、 遠心分離、 40〜50%飽和硫酸アンモニゥムによ る塩析、 力プリル酸沈殿 [Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory (1988)]、 または DEAE-セファロースカラム、 陰ィオン交換 カラム、 プロティン Aまたは G-カラムあるいはゲル濾過カラム等を用いるクロ マトグラフィ一等を、 単独または組み合わせて処理する方法があげられる。  Methods for separation and purification include centrifugation, salting out with 40-50% saturated ammonium sulfate, force prillic acid precipitation [Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory (1988)], DEAE-Sepharose column, There is a method in which a chromatographic method using an anion exchange column, a protein A or G-column, a gel filtration column, or the like is used alone or in combination.
(2) モノクローナル抗体の作製 (2) Preparation of monoclonal antibody
(a) 抗体産生細胞の調製 (a) Preparation of antibody-producing cells
免疫に用いた本発明のポリべプチドの部分断片ポリべプチドに対し、 その血清 が十分な抗体価を示したラットを抗体産生細胞の供給源として供する。  Rats whose serum shows a sufficient antibody titer against the partial polypeptide of the polypeptide of the present invention used for immunization are used as a source of antibody-producing cells.
該抗体価を示したラットに抗原物質を最終投与した後 3〜7 日目に、 脾臓を摘 出する。  The spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
該脾臓を MEM培地 （日水製薬社製） 中で細断し、 ピンセッ トでほぐし、 1,200 rpmで 5分間遠心分離した後、 上清を捨てる。  The spleen is shredded in a MEM medium (manufactured by Nissui Pharmaceutical), loosened with forceps, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
得られた沈殿画分の脾細胞をトリス-塩化アンモニゥム緩衝液 （pH 7.65) で 1 〜2分間処理し赤血球を除去した後、 MEM培地で 3回洗浄し、 得られた脾細胞 を抗体産生細胞として用いる。  The spleen cells in the obtained precipitate fraction are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, washed three times with MEM medium, and the obtained spleen cells are separated from antibody-producing cells. Used as
(b) 骨髄腫細胞の調製 (b) Preparation of myeloma cells
骨髄腫細胞としては、 マウスまたはラッ卜から取得した株化細胞を使用する。 例えば、 8-ァザグァニン耐性マウス （BALB/c由来）骨髄腫細胞株 P3-X63Ag8-Ul (以下、 P3-U1と略す） [Curr. Topics. Microbiol. Immunol., M， 1 (1978)、 Europ. J. Immunol., fi， 511 (1976)]、 SP2/0-Agl4(SP-2) [Nature, 27fi, 269 (1978)]、 P3-X63-Ag8653(653) [J. Immunol., 12Ά, 1548 (1979)]、 P3-X63-Ag8(X63) [Nature, 25fi, 495 (1975)] 等を用いることができる。 これらの細胞株は、 8-ァザ グァニン培地 [RPMI-1640培地にグル夕ミン （1.5 mmol/l)、 2-メルカプトェ夕ノ —ル （5 Χ 10·5 mol/l)、 ジヱン夕マイシン （10 / g/ml) および牛胎児血清 （FCS) (CSL社製、 10%) を加えた培地 （以下、 正常培地という） に、 さらに 8-ァザグァ ニン （15〃g/ml) を加えた培地] で継代するが、 細胞融合の 3〜4日前に正常培地 で培養し、 融合には該細胞を 2 X 107個以上用いる。 As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line P3-X63Ag8-Ul (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol., M, 1 (1978), Europ. J. Immunol., Fi, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 27fi, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunol., 12Ά, 1548 (1979)], P3-X63-Ag8 (X63) [Nature, 25fi, 495 (1975)] and the like can be used. These cell lines include 8 § The Guanin medium [RPMI-1640 medium Guru evening Min (1.5 mmol / l), 2- Merukapute evening Bruno - Le (5 Χ 10 · 5 mol / l), Jiwen evening mycin ( 10 / g / ml) and a medium supplemented with fetal calf serum (FCS) (CSL, 10%) (hereinafter referred to as a normal medium), and further supplemented with 8-azaguanine (15 μg / ml). passaging], but cultured in the normal medium 3 to 4 days before cell fusion, using the cells 2 X 10 7 or more in the fusion.
(c) ハイプリ ドーマの作製 (c) Production of Hypri-Dorma
(a)で取得した抗体産生細胞と (b)で取得した骨髄腫細胞を MEM培地または PBS (リン酸ニナトリウム 1.83 g、 リン酸一力リウム 0.21 g、 食塩 7.65 g 蒸留 水 1リッ トル、 pH 7.2)でよく洗浄し、 細胞数が、 抗体産生細胞：骨髄腫細胞 = 5〜： 10： 1になるよう混合し、 1,200 rpmで 5分間遠心分離した後、 上清を捨てる。 得られた沈殿画分の細胞群をよくほぐし、 該細胞群に、 攪袢しながら、 37°Cで、 108抗体産生細胞あたり、ポリエチレングライコール -1000 (PEG- 1000) 2 g、MEM 2 mlおよびジメチルスルホキシド （DMSO) 0.7 mlを混合した溶液を 0.2〜1 ml 添加し、 さらに 1〜2分間毎に MEM培地 1〜2 mlを数回添加する。 The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) were mixed with MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monolithium phosphate, 7.65 g of sodium chloride, 1 liter of distilled water, pH Wash well in 7.2), mix the cells so that the number of antibody-producing cells: myeloma cells = 5-: 10: 1, centrifuge at 1,200 rpm for 5 minutes, and discard the supernatant. Thoroughly loosened and the resulting precipitate fraction group of cells, in the cell group, while攪袢, at 37 ° C, 10 8 antibody-producing cells per polyethylene of glycol -1000 (PEG- 1000) 2 g, MEM 2 Add 0.2 to 1 ml of a mixed solution of 0.7 ml of dimethylsulfoxide (DMSO) and 0.7 ml of MEM medium several times every 1 to 2 minutes.
添加後、 MEM培地を加えて全量が 50 mlになるように調製する。 該調製液を 900 rpmで 5分間遠心分離後、 上清を捨てる。 得られた沈殿画分の細胞を、 ゆる やかにほぐした後、 メスピぺヅトによる吸込み、 吹出しでゆるやかに HAT培地 [正常培地にヒポキサンチン （10·4 inol/l)、 チミジン （1.5 X 10-5 mol/l) およびアミ ノプテリン （4 X 10·7 mol/1) を加えた培地】 100 ml中に懸濁する。 After the addition, add MEM medium to adjust the total volume to 50 ml. After centrifuging the preparation at 900 rpm for 5 minutes, discard the supernatant. Gently loosen the cells in the obtained precipitate fraction, and slowly inhale and blow out with a medium. HAT medium [Hypoxanthin (10 · 4 inol / l), thymidine (1.5 X 10- 5 mol / l) and amino Noputerin (4 X 10 · 7 medium mol / 1) was added] are suspended in 100 ml.
該懸濁液を 96穴培養用プレートに 100〃1/穴ずつ分注し、 5%C02インキュべ 一夕一中、 37°Cで 7〜： 14日間培養する。 The suspension is dispensed into 96-well culture plates at 100〃 / well, and cultured at 37 ° C. overnight in a 5% CO 2 incubator for 7 to 14 days.
培養後、 培養上清の一部をとりァンチボディィズ [Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Chapter 14 (1988)] 等に述べられてい る酵素免疫測定法により、 本発明のポリベプチドの部分断片ポリぺプチドに特異 的に反応するハイプリ ドーマを選択する。  After culturing, a part of the culture supernatant is taken and subjected to an enzyme immunoassay described in Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Chapter 14 (1988), etc., to obtain a partial fragment of the polypeptide of the present invention. Select a hybridoma that specifically reacts with the peptide.
酵素免疫測定法の具体例として、 以下の方法をあげることができる。 免疫の際、 抗原に用いた本発明のポリべプチドの部分断片ポリべプチドを適当 なプレートにコートし、ハイプリ ドーマ培養上清もしくは後述の (d)で得られる精 製抗体を第一抗体として反応させ、 さらに第二抗体としてピオチン、 酵素、 化学 発光物質あるいは放射線化合物等で標識した抗ラットまたは抗マウスィムノグロ プリン抗体を反応させた後に標識物質に応じた反応を行い、 本発明のポリべプチ ドに特異的に反応するものを本発明のモノクローナル抗体を生産するハイプリ ド —マとして選択する。 The following methods can be given as specific examples of the enzyme immunoassay. At the time of immunization, a partial fragment polypeptide of the polypeptide of the present invention used as the antigen was coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below was used as the primary antibody. And then reacting with an anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radioactive compound, or the like as a second antibody, followed by a reaction according to the labeling substance, and the polypeptide of the present invention. Those that specifically react with are selected as hybridomas producing the monoclonal antibody of the present invention.
該ハイプリ ドーマを用いて、限界希釈法によりクロ一ニングを 2回繰り返し [1 回目は、 HT培地 （HAT培地からアミノプテリンを除いた培地)、 2回目は、 正常 培地を使用する]、安定して強い抗体価の認められたものを本発明のモノクローナ ル抗体を産生するハイプリ ドーマ株として選択する。  Cloning was repeated twice by the limiting dilution method using the hybridoma [the first time using HT medium (medium obtained by removing aminopterin from HAT medium), and the second time using normal medium]. The antibody with a strong antibody titer is selected as a hybridoma strain producing the monoclonal antibody of the present invention.
(d) モノクローナル抗体の調製 (d) Preparation of monoclonal antibody
プリスタン処理 [2,6, 10,14-テトラメチルペン夕デカン （Pristane) 0.5 mlを腹 腔内投与し、 2週間飼育する〗 した 8〜： 10週令のマウスまたはヌードマウスに、 (c)で取得した本発明のポリぺプチドモノクローナル抗体産生ハイプリ ドーマ細 胞 5〜20 X 106細胞/匹を腹腔内に注射する。 10〜21日間でハイプリ ドーマは腹水 癌化する。 Pristane treatment [2,6,10,14-Tetramethylpenedecane (Pristane) 0.5 ml was intraperitoneally administered and bred for 2 weeks. 8 ~: For 10-week old mice or nude mice, (c) 5 to 20 × 10 6 cells / animal of the polypeptide monoclonal antibody-producing polypeptide of the present invention obtained in the above, are injected intraperitoneally. In 10 to 21 days, Hypridoma becomes ascites cancer.
該腹水癌化したマウスから腹水を採取し、 3，000 rpmで 5分間遠心分離して固 形分を除去する。  The ascites is collected from the mouse with ascites tumor and centrifuged at 3,000 rpm for 5 minutes to remove solid components.
得られた上清より、 ポリクローナルで用いた方法と同様の方法でモノクローナ ル抗体を精製、 取得することができる。  From the obtained supernatant, a monoclonal antibody can be purified and obtained in the same manner as in the polyclonal method.
抗体のサブクラスの決定は、 マウスモノクローナル抗体夕ィビングキッ トまた はラヅ トモノクローナル抗体タイピングキットを用いて行う。ポリべプチド量は、 ローリー法あるいは 280 nmでの吸光度より算出する。  The subclass of the antibody is determined using a mouse monoclonal antibody evening kit or a rat monoclonal antibody typing kit. The amount of polypeptide is calculated by the Lowry method or from the absorbance at 280 nm.
4. 本発明のポリペプチドの利用方法 4. Use of the polypeptide of the present invention
( 1 ) 本発明のポリぺプチドを利用した物質のスクリーニング方法 本発明のポリぺプチドを利用して、 該ポリぺプチドの細胞内の Akt活性を制御 する活性を変動させる化合物をスクリーニングすることができる。 具体的には、 本発明のポリペプチドを発現する細胞と被験試料とを接触させ、 Hsp90 と Akt との結合や、 アポトーシスの誘導または抑制を、 定性的または定量的に調べるこ とにより、 細胞内の Akt活性を制御する活性を変動させる化合物を選択すること ができる。 被験物質としては、 本発明のポリペプチドを発現する細胞の培養系に 加えることができるものであれば特に限定されず、 例えば、 低分子化合物、 高分 子化合物、 有機化合物、 無機化合物、 蛋白質、 遺伝子、 ウィルス、 細胞などが挙 げられる。 遺伝子を除く被験物質は、 培養培地中に直接添加すればよい。 (1) A method for screening a substance using the polypeptide of the present invention Using the polypeptide of the present invention, a compound that changes the activity of the polypeptide that regulates intracellular Akt activity can be screened. Specifically, cells expressing the polypeptide of the present invention are brought into contact with a test sample, and qualitatively or quantitatively examined for the binding of Hsp90 to Akt and for the induction or suppression of apoptosis. A compound that changes the activity of controlling the Akt activity can be selected. The test substance is not particularly limited as long as it can be added to the culture system of the cells expressing the polypeptide of the present invention. Examples of the test substance include a low molecular compound, a polymer compound, an organic compound, an inorganic compound, a protein, Genes, viruses, cells, etc. are listed. The test substance excluding the gene may be added directly to the culture medium.
遺伝子を効率的に細胞内に導入する方法としては、 レトロウイルス、 アデノウ ィルス、 アデノ随伴ウィルス、 単純へルぺスウィルス、 レンチウィルス等のウイ ルスべクタ一に乗せて培養系に添加する方法、 またはリボソームなどの人工的な ベジクル構造に封入して培養系に添加する方法などが挙げられる。 その具体的方 法としては、 組換えウィルスベクタ一を用いた遺伝子解析に関する報告 [Pro Natl. Acad. Sci. USA, 22, 6733 (1995); Nucleic Acids Res., 1&, 3587, 1990;Nucleic Acids Res., 2Ά, 3816 (1995)] に記載の方法を挙げることができる。  Examples of a method for efficiently introducing a gene into cells include a method in which the gene is placed on a virus vector such as retrovirus, adenovirus, adeno-associated virus, simple virus, lentivirus, etc., and added to a culture system. Alternatively, a method of encapsulating the vesicles in an artificial vesicle structure such as ribosome and adding to a culture system may be used. As a specific method, a report on gene analysis using a recombinant virus vector [Pro Natl. Acad. Sci. USA, 22, 6733 (1995); Nucleic Acids Res., 1 &, 3587, 1990; Nucleic Acids Res., 2Ά, 3816 (1995)].
Hsp90と Aktとの結合及びアポトーシスの誘導または抑制は、前述した方法に より測定することができる。  The binding between Hsp90 and Akt and the induction or suppression of apoptosis can be measured by the methods described above.
本発明のスクリーニング方法により、 例えば、 本発明のポリペプチドと本発明 のポリぺプチドに結合する Hsp90、 Aktなどの蛋白質との結合を阻害する化合物 を得ることができる。  By the screening method of the present invention, for example, compounds that inhibit the binding of the polypeptide of the present invention to proteins such as Hsp90 and Akt that bind to the polypeptide of the present invention can be obtained.
(2) 本発明のポリペプチドの医薬製剤としての利用 (2) Use of the polypeptide of the present invention as a pharmaceutical preparation
本発明のポリぺプチド、 該ポリべプチドと該ポリべプチドに結合する蛋白質と の結合を阻害する化合物、 及び本発明の抗体は、 本発明のポリペプチドに結合す る蛋白質の活性化または活性低下により引き起こされる疾患の治療薬、 予防薬、 または診断薬等の医薬製剤の活性成分 （以下、 「本発明の活性成分」 と略記する） として使用可能である。 ここで、 該ポリペプチドと該ポリペプチドに結合する蛋 白質との結合を阻害する化合物としては、 例えば、 上記スクリーニング方法によ り得られた化合物があげられる。 The polypeptide of the present invention, a compound that inhibits the binding between the polypeptide and a protein that binds to the polypeptide, and the antibody of the present invention can be used to activate or activate a protein that binds to the polypeptide of the present invention. It can be used as an active ingredient (hereinafter abbreviated as “active ingredient of the present invention”) of a pharmaceutical preparation such as a therapeutic drug, a preventive drug, or a diagnostic drug for a disease caused by reduction. Here, the polypeptide and a protein that binds to the polypeptide are Examples of the compound that inhibits the binding to white matter include a compound obtained by the above screening method.
本発明のポリペプチドに結合する蛋白質の活性化または活性低下により引き起 こされる疾病としては、 以下の疾病があげられる。 アポトーシスによる過剰な細 胞死が発症に関わる疾患としては、 例えば劇症肝炎やその他のウィルス疾患、 ァ ルツハイマー病などの神経変性疾患、 放射線障害などがあげられる。 アポト一シ ス抑制が発症に関わる疾患としては、 例えば胃癌、 卵巣癌、 乳癌、 膝癌、 前立腺 癌等の悪性腫瘍や自己免疫疾患などがあげられる。 また、 糖代謝制御シグナルの 異常によって生じる疾患としては、 糖尿病等があげられる。  The diseases caused by the activation or reduction of the activity of the protein binding to the polypeptide of the present invention include the following diseases. Examples of diseases associated with excessive cell death due to apoptosis include fulminant hepatitis and other viral diseases, neurodegenerative diseases such as Alzheimer's disease, and radiation damage. Diseases associated with suppression of apoptosis include, for example, malignant tumors such as stomach cancer, ovarian cancer, breast cancer, knee cancer, and prostate cancer, and autoimmune diseases. In addition, diseases caused by abnormal glucose metabolism control signals include diabetes and the like.
さらに、 本発明の活性成分は、 脳、 心臓などの虚血時における細胞死抑制剤、 抗癌剤や放射線治療時における正常細胞の細胞死からの保護剤、 または抗癌剤等 の既存の治療薬の効果を増強する薬剤として使用される。  Furthermore, the active ingredient of the present invention is effective in inhibiting the effects of existing therapeutic agents such as a cell death inhibitor during ischemia of the brain and heart, an anticancer agent, a protective agent against cell death of normal cells during radiotherapy, and an anticancer agent. Used as a potentiating agent.
抗癌剤としては、 アルキル化剤、 代謝拮抗剤、 抗腫瘍性抗生物質、 植物アル力 ロイ ドがあげられ、 さらに具体的には、 ブスルファン （busulfan)、 シクロフォス ファ ド (cyclophosph amide)、 ィ 、スファ ド 、ifosphamiae メルファフン (melp alan)N 二トロソゥレア (nitrosourea)、 アムサクリン 、amsacrine)ヽ 力ノレ ポプラチン （carboplatin)、 シスプラチン （cisplatin)、 ダカルノ ジン (dacarbazine)N アサンナジン (azacitidine)、 クラドリビン (ciadribine)s シ夕ラ ビン （cytarabine)、 フルダラ ビ ン （fludarabine)、 フノレオロ ウラ シル (fluorouracil)N ヒドロキシゥレア (hydroxyurea) 口づコホリン (leucovorin) メ ト トレキサ一ト （methotrexate^ ァクチノマイシン D (actinomycin D)、 ブレ ォマイシン （bleomycin)、 ダウノルビシン （daunorubicin)、 ドキソルビシン (doxorubicin)、 マイ トマイ シン C (mitomycin C)、 ミ ト キサン ト ロ ン (mitoxantrone)N エト ンド (etoposiae)、 ノ、クリ夕キセソレ (pacritaxel)、 テニ小 シド (teniposide)ヽ ビンプフスナン (vinblastine)、 ビンクリステン (vincristine) ビンデシン （vindesine)、 ゲルダナマイシンおよびラデイシコ一ルがあげられる。 これらの抗癌剤と同時に本発明の活性成分を患者に投与することも可能である し、 別々の容器に封入した組み合わせ （キット） として、 スケジュールを決めて 別々に投与することも可能である。 また、 本発明の活性成分に、 スぺーサーを介 して上記の抗癌剤を結合させて生体へ投与してもよい。 Examples of anticancer drugs include alkylating agents, antimetabolites, antitumor antibiotics, and plant alloids. More specifically, busulfan, cyclophosphamide, cyclophosphamide, and sulfad , Ifosphamiae melphafun (melp alan) N nitrosourea, amsacrine, amsacrine ヽ carplatin, cisplatin, cisplatin, dacarbazine N azacitidine, cladribine Bottles (cytarabine), fludarabine (fludarabine), funooleurouracil (fluorouracil) N -hydroxyperea (hydroxyurea) lipocoholin (leucovorin) methotrexate (methotrexate ^ actinomycin D), bleomycin ), Daunorubicin (danorubicin), doxorubicin (doxorubicin), my toma Shin C (mitomycin C), Mi door hexane collected by filtration down (mitoxantrone) N eth command (etoposiae), Bruno, chestnut evening Kisesore (pacritaxel), tennis small Sid (teniposide)ヽBinpufusunan (vinblastine), Binkurisuten (vincristine) vindesine (Vindesine), geldanamycin and radisicol The active ingredient of the present invention can be administered to a patient simultaneously with these anticancer agents, or a combination (kit) enclosed in separate containers can be used as a schedule. Decide It is also possible to administer separately. Further, the above-described anticancer agent may be bound to the active ingredient of the present invention via a spacer and administered to a living body.
患者への投与方法としては、 活性成分であるポリペプチドをコードする遺伝子 を挿入させた遺伝子治療用に調製した組換えベクターを、 患者から取り出した細 胞に導入させた後、 細胞を生体内に戻す方法、 レトロウイルス、 アデノウイルス、 アデノ随伴ウィルス、 単純へルぺスウィルス、 レンチウィルス、 センダイウィル ス等のウィルスベクターに乗せて生体に投与する方法、 リボソームなどの人工的 なビークル構造に封入して生体に投与する方法、 活性成分であるポリべプチドに 細胞内への導入効率を高めるぺプチド断片、例えば実施例に記載された TATぺプ チド [Science, 2 , 1569-1572 (1999)] などを融合させて生体に投与する方法な どがあげられる。  As a method of administration to a patient, a recombinant vector prepared for gene therapy into which a gene encoding a polypeptide as an active ingredient is inserted is introduced into cells taken out of the patient, and then the cells are introduced into a living body. How to revert, retrovirus, adenovirus, adeno-associated virus, simple virus, lentivirus, Sendai virus, etc., and administer it to a living body, encapsulated in an artificial vehicle structure such as ribosome A peptide fragment that enhances the efficiency of introduction into cells as an active ingredient, such as the TAT peptide described in Examples [Science, 2, 1569-1572 (1999)] Fusion and administration to living organisms.
抗癌剤と本発明の活性成分の投与比、 投与スケジュールは、 患者の年齢、体重、 治療すべき症状の性質もしくは重篤度を考慮して決定される。  The administration ratio and administration schedule of the anticancer agent and the active ingredient of the present invention are determined in consideration of the age and weight of the patient, and the nature or severity of the condition to be treated.
本発明の活性成分は、 医薬製剤として、 単独で、 あるいは任意の他の治療のた めの有効成分との混合物として含有することができる。また、それら医薬製剤は、 活性成分を薬理学的に許容される一種もしくはそれ以上の担体と一緒に混合し、 製剤学の技術分野においてよく知られている任意の方法により製造される。  The active ingredients of the present invention can be contained as pharmaceutical preparations, alone or as a mixture with any other active ingredient for treatment. In addition, these pharmaceutical preparations are prepared by mixing the active ingredient with one or more pharmacologically acceptable carriers and by any method well-known in the technical field of pharmaceuticals.
投与経路は、 治療に際し最も効果的なものを使用するのが望ましく、 経口また は、 例えば静脈内などの非経口をあげることができる。  It is desirable to use the most effective route for treatment, and it can be oral or parenteral, for example, intravenous.
投与形態としては、 錠剤、 散剤、 顆粒剤、 シロップ剤、 注射剤などがある。 経口投与に適当な、 例えばシロップ剤のような液体調製物は、 水、 蔗糖、 ソル ビット、 果糖などの糖類、 ポリエチレングリコ一ル、 プロピレングリコールなど のグリコール類、 ごま油、 オリ一ブ油、 大豆油などの油類、 P-ヒドロキシ安息香 酸エステル類などの防腐剤、 ストロベリーフレーバ一、 ペパーミントなどのフレ 一バー類などを使用して製造できる。 また、 錠剤、 散剤および顆粒剤などは、 乳 糖、 ブドウ糖、 蔗糖、 マンニットなどの賦形剤、 澱粉、 アルギン酸ソーダなどの 崩壊剤、 ステアリン酸マグネシウム、 タルクなどの滑沢剤、 ポリビニールアルコ ール、 ヒドロキシプロピルセルロース、 ゼラチンなどの結合剤、 脂肪酸エステル などの界面活性剤、 グリセリンなどの可塑剤などを用いて製造できる。 Dosage forms include tablets, powders, granules, syrups, and injections. Liquid preparations suitable for oral administration, for example, syrups, include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, sesame oil, olive oil, soybean oil It can be produced by using oils such as, for example, preservatives such as P-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. Tablets, powders, granules, etc. include excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, and polyvinyl alcohol. , Hydroxypropylcellulose, gelatin and other binders, fatty acid esters And a plasticizer such as glycerin.
非経口投与に適当な製剤は、 好ましくは受容者の血液と等張である活性化合物 を含む滅菌水性剤からなる。 例えば、 注射剤の場合は、 塩溶液、 ブドウ糖溶液ま たは塩水とプドゥ糖溶液の混合物からなる担体などを用いて注射用の溶液を調製 する。  Formulations suitable for parenteral administration comprise a sterile aqueous preparation containing the active compound which is preferably isotonic with the blood of the recipient. For example, in the case of an injection, a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of saline and a putose sugar solution.
また、 これら非経口剤においても、 経口剤で例示した希釈剤、 防腐剤、 フレー バー類、 賦形剤、 崩壊剤、 滑沢剤、 結合剤、 界面活性剤、 可塑剤などから選択さ れる 1種もしくはそれ以上の補助成分を添加することもできる。  In addition, these parenteral preparations are also selected from the diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc. exemplified for the oral preparations. One or more auxiliary components can also be added.
本発明の活性成分の投与量および投与回数は、 投与形態、 患者の年齢、 体重、 治療すべき症状の性質もしくは重篤度により異なるが、 通常経口の場合、 成人一 人当り 0.01 mg〜l g、 好ましくは 0.05〜50 mgを一日一回ないし数回投与する。 静脈内投与などの非経口投与の場合、 成人一人当り 0.001〜100 mg、 好ましくは 0.01〜： 10 mgを一日一回ないし数回投与する。 しかしながら、 これら投与量およ び投与回数に関しては、 前述の種々の条件により変動する。  The dose and frequency of administration of the active ingredient of the present invention will vary depending on the mode of administration, the age and weight of the patient, the nature or severity of the condition to be treated, but usually 0.01 mg to lg per adult for oral administration. Preferably, 0.05 to 50 mg is administered once or several times a day. In the case of parenteral administration such as intravenous administration, 0.001 to 100 mg, preferably 0.01 to: 10 mg per adult is administered once or several times a day. However, the dose and the number of administrations vary depending on the various conditions described above.
<図面の簡単な説明 > <Brief description of drawings>
図 1は、 BALB/3T3と 293T細胞内での Hsp90と Aktの結合を、 抗 Akt抗体 による免疫沈降後に抗 Hsp90抗体を用いたウエスタン'ブロッテイングで検出し た実験結果を示す。  FIG. 1 shows the results of an experiment in which binding between Hsp90 and Akt in BALB / 3T3 and 293T cells was detected by Western blotting using an anti-Hsp90 antibody after immunoprecipitation with an anti-Akt antibody.
図 2は、 Aktl欠失変異体を用いて、 Hsp90 ?との結合に必須の領域を解析した 実験結果を示す。  FIG. 2 shows the results of an experiment in which a region essential for binding to Hsp90? Was analyzed using an Aktl deletion mutant.
図 3は、 Hsp90 ?欠失変異体を用いて、 Aktlとの結合に必須の領域を解析した 実験結果を示す。  FIG. 3 shows the results of an experiment in which a region essential for binding to Aktl was analyzed using an Hsp90-deletion mutant.
図 4は、 Aktl欠失変異体遺伝子を細胞に導入したときの、 細胞内での Hsp90 ^と Aktlの結合を阻害ならびに Aktlのリン酸化阻害を解析した実験結果を示す c 図 5は、 Aktl欠失変異体遺伝子を細胞に導入して、細胞内での Hsp90 ?と Aktl との結合を阻害した時の Akt活性の変化を解析した実験結果を示す。 4, when the Aktl deletion mutant gene was introduced into a cell, the c Figure 5 shows the results of an experiment analyzing the inhibition and inhibition of phosphorylation of Aktl the binding of Hsp90 ^ and Aktl in a cell, Aktl missing The results of an experiment analyzing changes in Akt activity when a mutant gene was introduced into cells to inhibit the binding of Hsp90 to Aktl in cells were shown.
図 6は、 Aktl欠失変異体遺伝子を細胞に導入して細胞内での Hsp90 ?と Aktl の結合を阻害した時の、 細胞のアポトーシス誘導を DAPIによる核染色で解析し た実験結果を示す。 Figure 6 shows that Aktl deletion mutant gene was introduced into cells and Hsp90? Fig. 4 shows the results of an experiment in which the induction of apoptosis of cells when the binding of A was inhibited was analyzed by nuclear staining with DAPI.
図 Ίは、 Aktl欠失変異体遺伝子を細胞に導入して細胞内での Hsp90 ?と Aktl の結合を阻害した時の、 細胞のアポトーシス誘導をキャスパーゼー 3様活性測定 で解析した実験結果を示す。  Figure 示 す shows the results of an experiment in which the induction of cell apoptosis was analyzed by measuring caspase 3-like activity when the Aktl deletion mutant gene was introduced into cells to inhibit the binding of Hsp90 to Aktl in the cells. .
図 8は、 Aktl欠失変異体遺伝子を細胞に導入して細胞内での Hsp90^と Aktl の結合を阻害した時の、 VP-16よるアポトーシス誘導刺激に対する感受性増強を キャスパーゼの基質である PARPの細胞内切断活性で解析した実験結果を示す。 図 9は、 Hsp90 ?上の Akt結合部位を含んだぺプチドを細胞に導入した際の、 細胞のアポトーシス誘導刺激に対する感受性低下をキャスパーゼの基質である PAKPの細胞内切断活性で解析した実験結果を示す。  Figure 8 shows that when the Aktl deletion mutant gene was introduced into cells to inhibit the binding of Hsp90 ^ to Aktl in the cells, VP-16 increased the sensitivity to apoptosis-inducing stimulus by the caspase substrate PARP. The experimental result analyzed by the intracellular cleavage activity is shown. Fig. 9 shows the results of an analysis of the decrease in sensitivity of cells to apoptosis-inducing stimuli when transfected with a peptide containing an Akt-binding site on Hsp90? Into cells, using the intracellular cleavage activity of PAKP, a caspase substrate. Show.
なお、 図中の符号、 WBはウエスタン 'プロヅティング、 a-Hsp90は抗 Hsp90 抗体、 ひ -Aktは抗 Akt抗体、 Cont. IgGはコント口一ルヒッジ IgG、 IPは免疫沈 降、 pFLAG-akLは FLAG夕グを付けた Aktl全長遺伝子、 pcDNA3.1-Hsp90 ?は V5夕グを付けた Hsp90 βの全長遺伝子、 -FLAGは抗； FLAG tag M2モノクロ —ナル抗体、 ひ -P-Akt (Thr3°8)は抗ホスホ- Akt (Thr308)抗体、 ひ -V5は抗 V5 tag モノクローナル抗体、 ひ- HAは抗 HA tag モノクローナル抗体、 FLAG-Akt は FLAG夕グを付けた Aktl全長蛋白質、 c-Aktは内在性 Aktl全長蛋白質、 1-309 aki は FLAG夕グを付けた Aktl部分配列 （1-309) である。 Symbols in the figure, WB is Western 'printing, a-Hsp90 is an anti-Hsp90 antibody, hi-Akt is an anti-Akt antibody, Cont. IgG is control IgG, IP is immunoprecipitated, and pFLAG-akL is FLAG. Is the full-length Aktl gene with pcg, pcDNA3.1-Hsp90? Is the full-length gene of Hsp90 beta with V5 flag, -FLAG is anti-FLAG tag M2 monoclonal antibody, and -P-Akt (Thr 3 ° 8 ) is an anti-phospho-Akt (Thr 308 ) antibody, HI-V5 is an anti-V5 tag monoclonal antibody, HI-HA is an anti-HA tag monoclonal antibody, FLAG-Akt is a full-length Aktl protein with FLAG ligature, c-Akt Is the endogenous Aktl full-length protein, and 1-309 aki is the Aktl partial sequence (1-309) with FLAG fragment.
<発明を実施するための最良の形態 > <Best mode for carrying out the invention>
以下に、 本発明の実施例を示すが、 これにより本発明の範囲が限定されるもの ではない。 実施例 1  Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited thereto. Example 1
Aktの免疫沈降による細胞内での Hsp90と Aktの結合の確認  Confirmation of binding between Hsp90 and Akt in cells by immunoprecipitation of Akt
10% 牛胎児血清 （FBS) を含む DMEM培地中、 37°C、 5% C02存在下で培養 した、 マウス繊維芽細胞 BALB/3T3 (ATCC CCL-163) ならびにヒト胎児腎細胞 293T細胞 [Pro Natl. Acad. Sci. USA, 00(18), 8392 (1993)] を回収し、 細胞溶 解用緩衝液 [20mM Tris'HCl (pH 7.5)、 0.2% ノニデット （Nonidet) P-40、 10% グリセ口一ル、 1 mM EDTAヽ 1.5 mM MgCl2、 137 mM NaClヽ 50 mM NaF、 1 mM NaV03、 12 mM ? -グリセ口リン酸、 1 mM PMSF、 1 mM ァプロチニン] に懸 濁して細胞溶解液を調製した。 得られた細胞溶解液にコントロールのヒヅジ IgG またはヒヅジ抗 Aktポリクローナル抗体（Upstate Biotechnology社製） を結合 させたプロティン Gァガロースを加え免疫沈降を行なった。免疫沈降物に関して、 抗 Hsp90抗体（StressGen Biotechnologies社製） を用いたウエスタン ·プロッ ティングを行ない Aktと共沈してくる Hsp90の有無を確認した。 結果を図 1に 示す。 BALB/3T3ならび 293Tいずれの細胞においても、細胞内で Aktと Hsp90 が結合していることが確認できた。 実施例 2 In DMEM medium containing 10% fetal bovine serum (FBS), 37 ° C, 5% C0 2 and cultured in the presence, mouse fibroblast BALB / 3T3 (ATCC CCL-163 ) and human embryonic kidney cells 293T cells [Pro Natl. Acad. Sci. USA, 00 (18), 8392 (1993)] were collected and a cell lysis buffer [20 mM Tris'HCl (pH 7.5), 0.2% Nonidet P- suspended in glycerin port phosphate, 1 mM PMSF, 1 mM Apurochinin] - 40, 10% glycerin port Ichiru, 1 mM EDTAヽ1.5 mM MgCl 2, 137 mM NaClヽ50 mM NaF, 1 mM NaV0 3 , 12 mM? The cells were turbid to prepare a cell lysate. To the obtained cell lysate was added protein G agarose conjugated with control IgG or anti-Akt polyclonal antibody (manufactured by Upstate Biotechnology), and immunoprecipitation was performed. The immunoprecipitate was subjected to western blotting using an anti-Hsp90 antibody (manufactured by StressGen Biotechnologies), and the presence or absence of Hsp90 co-precipitated with Akt was confirmed. The results are shown in Figure 1. In both BALB / 3T3 and 293T cells, it was confirmed that Akt and Hsp90 bound in the cells. Example 2
変異型 Aktを用いた Akt上の Hsp90結合領域の解析 Analysis of Hsp90 binding region on Akt using mutant Akt
全長のヒト Aktl遺伝子 （WT) は、 逆転写 PGR (RT-PCR) により、 ヒト骨肉 腫 Saos-2細胞 (ATCC HTB-85) [J. Cell. Physiol.,凰 290 (2000)] より取得し た。 また取得した遺伝子をもとに、 PCRにて、 種々の Aktl遺伝子の断片 （配列 番号 5記載のアミノ酸配列の 1-433 (AC)、 120-480 (ΔΝ)、 120-433 (ANC)、 1-175、 1-228、 1-331、 205-480、 340-480、 1-246、 1-267、 1-288、 1-309番目に相当す るアミノ酸配列を有する欠失変異体をコ一ドする遺伝子断片） を取得した。 上記 の全長 Aktl遺伝子ならびに欠失変異体は、 pFLAG-CMV-2ベクターへサブクロ 一二ングして細胞発現用べクタ一とした。 pcDNA3.1/GS ベクターにサブクロ一 ニングした V5夕グ付き Hsp90 ?と Hsp90 aの全長遺伝子 （Invitrogen社製） と、 上記 Aktl の全長あるいは欠失変異体を含む発現ベクターを Superfect transfection reagent (Qiagen社製） を用いて 293T細胞にトランスフエクトした 後、 実施例 1と同様の方法で細胞溶解液を調製した。 得られた細胞溶解液から抗 FLAG tag M2モノクローナル抗体（Sigma社製） を用いて全長 Aktl蛋白質ある いはその欠失変異体蛋白質を免疫沈降し、免疫沈降物中の Hsp90 ?または Hsp90 ひの有無を、 抗 V5 tagモノクローナル抗体（Invitrogen社製） を用いたウェス夕 ン -プロヅティングにて検出した。結果を図 2に示す。 この結果、 Aktlの Hsp90 結合領域は、 配列番号 5に記載の Aktlのアミノ酸配列の 229~309番目のアミ ノ酸配列を有する領域であり、 その中でも特に 289〜309番目のアミノ酸配列を 有する領域が強い結合に必要であることが明らかとなった。 実施例 3 The full-length human Aktl gene (WT) was obtained from human osteosarcoma Saos-2 cells (ATCC HTB-85) [J. Cell. Physiol., Phoenix 290 (2000)] by reverse transcription PGR (RT-PCR). Was. In addition, based on the obtained genes, various fragments of the Aktl gene (1-433 (AC), 120-480 (ΔΝ), 120-433 (ANC), -175, 1-228, 1-331, 205-480, 340-480, 1-246, 1-267, 1-288, deletion mutants having amino acid sequences corresponding to positions 1-309 Gene fragment). The above full-length Aktl gene and deletion mutant were subcloned into the pFLAG-CMV-2 vector to obtain a vector for cell expression. The full-length Hsp90? and Hsp90a genes (manufactured by Invitrogen) subcloned into the pcDNA3.1 / GS vector and the expression vector containing the full-length or deletion mutant of Aktl described above were combined with Superfect transfection reagent (Qiagen). Was transfected into 293T cells using the method described in Example 1, and a cell lysate was prepared in the same manner as in Example 1. The full-length Aktl protein or its deletion mutant protein was immunoprecipitated from the obtained cell lysate using an anti-FLAG tag M2 monoclonal antibody (manufactured by Sigma), and Hsp90? Or Hsp90 in the immunoprecipitate was used. The presence or absence of the string was detected by Western blotting using an anti-V5 tag monoclonal antibody (manufactured by Invitrogen). The result is shown in figure 2. As a result, the Hsp90-binding region of Aktl is a region having the amino acid sequence at positions 229 to 309 of the amino acid sequence of Aktl described in SEQ ID NO: 5, and among these, the region having the amino acid sequence at positions 289 to 309 is particularly preferable. It proved to be necessary for strong binding. Example 3
変異型 Hsp90を用いた Hsp90上の Akt結合領域の解析 Analysis of Akt binding region on Hsp90 using mutant Hsp90
全長のヒト Hsp90 ?遺伝子 （WT) は Invitrogen社製より入手した。また、; PCR にて種々の Hsp90 ?遺伝子の断片 （配列番号 1記載の Hsp90 ?のアミノ酸配列の 1-301 1-530 (Mid), 1-635、 534-724 (ΔΝ)Ν 534-680、 1-315、 1-326、 1-340、The full-length human Hsp90 gene (WT) was obtained from Invitrogen. Also,;? Various Hsp90 gene fragment by PCR (SEQ ID NO: 1 1-301 of Hsp90 amino acid sequence set forth 1-530 (Mid), 1-635, 534-724 (ΔΝ) Ν 534-680, 1-315, 1-326, 1-340,
1-355、 1-368、 1-456に相当するアミノ酸配列を有する欠失変異体をコードする 遺伝子断片） を取得した。 これら全長 Hsp90ySならびに Hsp90 3欠失変異体遺伝 子は、 pFLAG-CMV-2ベクタ一にサブクロ一ニングした。 pUSEampベクターに 組み込まれた （His)6 tagマウス Aktl遺伝子は Upstate Biotechnology社より購 入した。 該 （His)6 tagマウス Aktl遺伝子と、 FLAG tag Hsp90 ?の全長あるい は欠失変異体を含む発現べク夕一を Superfect transfection reagent (Qiagen ¾ 製） を用いて 293T細胞にトランスフエクトした後、 実施例 1 と同様の方法で細 胞溶解液を調製した。 得られた細胞溶解液から抗（His)6 tag抗体を用いて Aktl を免疫沈降し、 免疫沈降物中の Hsp90 ?あるいはその欠失変異体の有無を、 抗 FLAG tag M2モノクローナル抗体（Sigma社製）を用いたウエスタン ·ブロッテ イングにて検出した。結果を図 3に示す。 この結果、 Hsp90 ?の Akt結合領域は、 配列番号 1に記載の Hsp90 ?のアミノ酸配列の 327~340番目のアミノ酸配列を 有する領域であることが明らかとなった。 実施例 4 Gene fragments encoding deletion mutants having amino acid sequences corresponding to 1-355, 1-368, and 1-456) were obtained. These full-length Hsp90yS and Hsp903 deletion mutant genes were subcloned into the pFLAG-CMV-2 vector. The (His) 6 tag mouse Aktl gene integrated into the pUSEamp vector was purchased from Upstate Biotechnology. The (His) 6 tag mouse Aktl gene and the expression vector containing the full-length or deletion mutant of FLAG tag Hsp90 are transfected into 293T cells using a Superfect transfection reagent (manufactured by Qiagen II). A cell lysate was prepared in the same manner as in Example 1. Aktl was immunoprecipitated from the obtained cell lysate using an anti- (His) 6 tag antibody, and the presence or absence of Hsp90? Or its deletion mutant in the immunoprecipitate was determined by using an anti-FLAG tag M2 monoclonal antibody (Sigma) ) Was detected by Western blotting. The results are shown in Figure 3. As a result, it was revealed that the Akt-binding region of Hsp90? Is a region having the 327th to 340th amino acid sequences of the amino acid sequence of Hsp90? Described in SEQ ID NO: 1. Example 4
Akt欠失変異体による Hsp90-Akt結合阻害と Akt活性阻害  Inhibition of Hsp90-Akt binding and Akt activity by Akt deletion mutant
HA夕グを付けた Aktl欠失変異体遺伝子 （pHM6ベクタ一を使用。配列番号 5 記載のアミノ酸配列の 1-309、 1-228、 1-288番目に相当するアミノ酸配列をコ一 ドする遺伝子)、 V5タグを付けた Hsp90 ?の全長遺伝子 （pcDNA3.1-Hsp90 ?)、 ならびに FLAGタグを付けた Aktl全長遺伝子 （pFLAG-Aktl) を 293T細胞に導 入した。 その後、 細胞を血清除去条件下で 24時間培養し、 実施例 1の方法で細 胞溶解液を調製した。 得られた細胞溶解液中の Aktlを抗 FLAG tag M2モノク ローナル抗体（Sigma社製）にて免疫沈降した。免疫沈降物中の Aktlのスレオニ ン 308番のリン酸化を抗ホスホ- Akt (Thr3°8)抗体 （Upstate Biotechnology社製） で、 Aktlの発現を抗 Akt抗体（New England Biolabs社製）で、 Hsp90 5の発現 を抗 V5 tagモノクローナル抗体（Invitrogen社製）で、 Akt欠失変異体の発現を 抗 HA tagモノクローナル抗体（Roche Molecular Biochemicals社製） でそれそ れウエスタン ·プロッテイングを行ない確認した。 結果を図 4に示す。 実施例 2 ならびに実施例 3で明らかとなった Hsp90 ?のァミノ酸配列の 327〜340番目の アミノ酸配列を有する領域に結合する Aktl部分配列 （229〜309番目のアミノ酸 配列） を含む Aktl欠失変異体（1〜309番目のアミノ酸配列、および 1〜288番目 のアミノ酸配列）を細胞に導入した場合にのみ、 Aktlと Hsp90 ?の結合ならびに Aktlのスレオニン 308番のリン酸化が阻害された。 Aktl deletion mutant gene with HA fragment (using pHM6 vector. SEQ ID NO: 5 A gene encoding the amino acid sequence corresponding to positions 1-309, 1-228, and 1-288 of the amino acid sequence described), a full-length Hsp90-tagged gene with a V5 tag (pcDNA3.1-Hsp90-), and The FLAG-tagged Aktl full length gene (pFLAG-Aktl) was introduced into 293T cells. Thereafter, the cells were cultured for 24 hours under serum-free conditions, and a cell lysate was prepared by the method of Example 1. Aktl in the obtained cell lysate was immunoprecipitated with an anti-FLAG tag M2 monoclonal antibody (Sigma). Phosphorylation of threonine 308 No. Aktl of immunoprecipitates anti-phospho - in Akt (Thr 3 ° 8) antibody (Upstate Biotechnology, Inc.), with anti-Akt antibody expression Aktl (New England Biolabs, Inc.), The expression of Hsp905 was confirmed by anti-V5 tag monoclonal antibody (Invitrogen) and the expression of the Akt deletion mutant was performed by anti-HA tag monoclonal antibody (Roche Molecular Biochemicals) and subjected to Western blotting. Fig. 4 shows the results. An Aktl deletion mutation containing the Aktl partial sequence (229-309 amino acid sequence) that binds to the region having the 327th to 340th amino acid sequence of the amino acid sequence of Hsp90 as revealed in Examples 2 and 3. Only when the body (amino acids 1 to 309 and amino acids 1 to 288) were introduced into cells, the binding of Aktl to Hsp90? And the phosphorylation of Aktl at threonine 308 were inhibited.
上記の Hsp90 3と Aktlの結合阻害が Akt活性におよぼす影響を調べる目的で、 FLAG夕グを付けた Aktl全長遺伝子 [pFLAG-CMV2 (WT)] またはそのべクタ —コントロール [pFLAG-CMV2 (Mock)] と、 HA夕グを付けた Aktl欠失変異体 (1〜309番目のアミノ酸配列）遺伝子 [pHM6 (1-309)] またはそのベクターコン トロール [pHM6 (Mock)] を 293T細胞に導入し、細胞を血清除去条件下で 24時 間培養した後に実施例 1の方法で細胞溶解液を調製した。 得られた細胞溶解液に 対して抗 Akt抗体 （New England Biolabs社製） と結合させたプロテイン Gァガ ロースを加え免疫沈降を行なった。 免疫沈降物の Akt活性は基質の一つである GSK3のリン酸化部位の部分べプチドを用いた Akt kinase assay kit (Upstate Biotechnology社製） を用いて測定した。結果を図 5に示す。 Aktlの部分配列 （1 〜309番目のアミノ酸配列） によって Aktlと Hsp90 ?の結合を阻害した時に Akt 活性が低下することが明らかとなった。 実施例 5 In order to investigate the effect of the inhibition of Hsp903 binding to Aktl on Akt activity, the Aktl full-length gene [pFLAG-CMV2 (WT)] with FLAG fragment or its vector—control [pFLAG-CMV2 (Mock) And transfecting the Aktl deletion mutant (amino acid sequence at positions 1 to 309) gene [pHM6 (1-309)] or its vector control [pHM6 (Mock)] with HA fragment into 293T cells, After culturing the cells for 24 hours under serum-free conditions, a cell lysate was prepared according to the method of Example 1. To the obtained cell lysate, protein G agarose conjugated with an anti-Akt antibody (manufactured by New England Biolabs) was added and immunoprecipitation was performed. The Akt activity of the immunoprecipitate was measured using an Akt kinase assay kit (manufactured by Upstate Biotechnology) using a partial peptide at the phosphorylation site of GSK3, one of the substrates. Fig. 5 shows the results. The partial sequence of Aktl (amino acid sequence 1 to 309) revealed that Akt activity was reduced when the binding between Aktl and Hsp90 was inhibited. Example 5
Akt欠失変異体によるアポトーシス誘導作用  Apoptosis induction by Akt deletion mutant
実施例 1〜4で示した、 Aktlの部分配列 （1〜309番目のアミノ酸配列） による Aktlと Hsp90 ?の結合阻害とそれに伴う Akt活性の低下が細胞のアポトーシス 誘導に繋がることを証明する目的で、 FLAGタグを付けた Aktl部分配列 ひ〜 309 番目のアミノ酸配列） あるいはそのベクターコントロール （Mock) を 293T細胞 に導入し、 血清除去あるいは 10%牛胎児血清 （FBS) を含む DMEM培地中で 24 時間培養した後に細胞を 4'，6-ジァミノ- 2-フエニルインドール （DAPI) にて染色 した。 また上記細胞内のキャスパ一ゼ -3 様プロテアーゼ （caspase-3 like protease)活性を Oncogene, ， 1295-1304 (1998)記載の方法で測定した。 具体 的には、 回収した細胞を 10 mM HEPES (pH 7.4)、 2 mM EDTA、 0.1% CHAPS を含む溶解液中で溶解し、 得られた細胞溶解液 5 g蛋白質相当と' DEVD-AMC ^acetyl- JJ- asp artyl-L-glutamyl-L-valyl-L-aspart-7-amino-4-methylcoumariii) (ぺプチド研究所社製） を ICE緩衝液 [10 mM HEPES (pH 7.4)/10% グリセロー ル /2 ηιΜ ジチオスレィ トール] 中で 37°C、 1時間反応させた。 反応終了後、 キヤ スパ一ゼ -3 様プロテア一ゼ活性による AMC の遊離を Hitachi fluorescence spectrophotometer (日立製作所社製） を用いて、励起波長 380 nm、測定波長 460 nmにて測定した。 結果を図 6ならびに図 7に示す。 Aktlの部分配列 （1〜309 番目のアミノ酸配列） 導入によって、 10%牛胎児血清存在下でも Mockと比較し て DAPIで染色される核の凝集や断片化が観察され、 血清非存在下ではその傾向 がさらに顕著であつた。 またキヤスパーゼ活性に関しても、 DAPI染色で見られ るアポトーシス細胞像と相関した亢進がみられ、 アポトーシスシグナルが亢進し ていることが確認された。 以上の結果より、 Aktlの部分配列 ひ〜 309番目のァ ミノ酸配列）により、 Aktlと Hsp90 ?の結合を阻害した時に細胞のアポト一シス が誘導されることが明らかになった。 実施例 6  In order to demonstrate that inhibition of the binding of Aktl to Hsp90? By the partial sequence of Aktl (amino acid sequence at positions 1 to 309) and the resulting decrease in Akt activity, as described in Examples 1 to 4, lead to the induction of cell apoptosis. , FLAG-tagged Aktl partial sequence (H-309th amino acid sequence) or its vector control (Mock) was introduced into 293T cells, and serum-free or in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours After culturing, the cells were stained with 4 ', 6-diamino-2-phenylindole (DAPI). In addition, caspase-3 like protease activity in the above cells was measured by the method described in Oncogene,, 1295-1304 (1998). Specifically, the collected cells were lysed in a lysate containing 10 mM HEPES (pH 7.4), 2 mM EDTA, and 0.1% CHAPS, and the resulting cell lysate was equivalent to 5 g protein and DEVD-AMC ^ acetyl -JJ-asp artyl-L-glutamyl-L-valyl-L-aspart-7-amino-4-methylcoumariii) (Peptide Laboratories) in ICE buffer [10 mM HEPES (pH 7.4) / 10% glycerol / 2 ηιΜ dithiothreitol] at 37 ° C for 1 hour. After the completion of the reaction, the release of AMC due to the activity of the capase-3-like protease was measured using a Hitachi fluorescence spectrophotometer (manufactured by Hitachi, Ltd.) at an excitation wavelength of 380 nm and a measurement wavelength of 460 nm. The results are shown in FIGS. With the introduction of the Aktl partial sequence (amino acids 1 to 309), aggregation and fragmentation of nuclei stained with DAPI were observed even in the presence of 10% fetal calf serum compared to Mock, and in the absence of serum. The trend was even more pronounced. In addition, regarding the aspase activity, an increase correlated with the apoptotic cell image observed by DAPI staining was observed, confirming that the apoptotic signal was enhanced. From the above results, it was revealed that the partial sequence of Aktl (amino acid sequence at position 309 to amino acid) induces apoptosis of cells when the binding between Aktl and Hsp90 is inhibited. Example 6
Akt欠失変異体による VP-16によるアポトーシス刺激に対する感受性増強作用 i Akt deletion mutant enhances sensitivity to apoptosis stimulation by VP-16 i
実施例 1〜4で示した、 Aktlの部分配列 ひ〜 309番目のァミノ酸配列） による Aktlと Hsp90 ^の結合阻害とそれに伴う Akt活性の低下が抗癌剤によるアポト —シス刺激に ¾·する癌細胞の感受性を増加させることを VP-16 を用いて検証し た。 具体的には、 pFLAG-CMV-2ベクターへ Aktl欠失変異体遺伝子 （Aktlのァ ミノ酸配列 1〜309 番目のアミノ酸配列をコードする遺伝子に相当） をサブクロ 一二ングしたものあるいはそのベクターコントロール （Mock) を 293T細胞にト ランスフエク トして、 24 時間後に培地を 30〃g/ml VP-16 を含む無血清培地 (VP- 16 十)、 あるいは VP-16を含まない無血清培地 （VP-16 —）へ置換した。 さ らに 24時間培養後、実施例 1に記載の方法で細胞溶解液を調製した。抗ポリ- (ADP リ ボース） ポ リ メ ラーゼ [poly-(ADP ribose)polymerase (PARP)] 抗体 (Promega社製） を用いて、得られた細胞溶解液のウエスタン ·ブロッティングを 行ない、 キャスパ一ゼ -3による切断によって生じる PARPの 85kDaフラグメン トを検出することにより、 細胞内のアポトーシスシグナルの活性化を解析した。 また、 観察される PAKP切断がキャスパーゼによる切断であることを検証する目 的で、 キャスパーゼ阻害剤である Z-Asp-CH2-DCB (フナコシ社製） を 100〃g/ml の濃度で添加したもの （Z-Asp +) に関しても同様のウエスタン 'プロヅティング を行なった。結果を図 8に示す。 Aktlの部分配列 （1-309) を導入して、 Aktl と Hsp90 ? の結合阻害をした時に、 VP-16刺激によって生じる PARPの切断が増 強されており、 VP-16によるアポトーシス刺激に対する感受性が増加しているこ とが確認された。 実施例 7 Inhibition of the binding of Aktl to Hsp90 ^ by the partial sequence of Aktl (amino acid sequence at position 309 to 309) shown in Examples 1 to 4 and the resulting decrease in Akt activity are associated with apoptosis stimulation by anticancer drugs. VP-16 was used to verify that it increased the sensitivity. Specifically, the pFLAG-CMV-2 vector is subcloned with an Aktl deletion mutant gene (corresponding to the gene encoding the amino acid sequence of amino acids 1 to 309 of Aktl) or its vector control. (Mock) was transfected into 293T cells, and after 24 hours, the medium was changed to a serum-free medium containing 30 μg / ml VP-16 (VP-16x) or a serum-free medium without VP-16 (VP-16). -16 —) After further culturing for 24 hours, a cell lysate was prepared by the method described in Example 1. The resulting cell lysate was subjected to Western blotting using an anti-poly (ADP ribose) polymerase [poly- (ADP ribose) polymerase (PARP)] antibody (manufactured by Promega). The activation of apoptotic signals in cells was analyzed by detecting the 85 kDa fragment of PARP generated by cleavage by -3. Further, PAKP cleavage is observed a purpose of verifying that the cleavage by caspases was added Z-Asp-CH 2 -DCB a caspase inhibitor (Funakoshi) at a concentration of 100〃G / ml The same Western 'plotting was performed for the sample (Z-Asp +). Fig. 8 shows the results. When a partial sequence of Aktl (1-309) was introduced to inhibit the binding between Aktl and Hsp90 ?, the cleavage of PARP caused by VP-16 stimulation was enhanced, and the sensitivity of VP-16 to apoptosis stimulation was increased. It was confirmed that it was increasing. Example 7
Hsp90 欠失変異体によるアポトーシス抑制作用  Apoptosis inhibitory effect of Hsp90 deletion mutant
実施例 1〜3で示した、 Hsp90 ?上の Akt結合領域にあたる配列番号 1記載の アミノ酸配列の 327-340番目のアミノ酸配列を有するアミノ酸ぺプチドのアポト 一シス制御作用を調べる目的で、 細胞内導入用ペプチドとして TAT ペプチド [Science, 2 , 1569-1572 (1999)] を Hsp90 ? (327-340番アミノ酸) C末端に結 合した、 HSP327-TATペプチド （LEFRALLFIPRRAP-GYGRKKRRQKRHG) と ネガティブコントロールとして、 Aktとの結合領域ではない配列番号 1記載のァ ミノ酸配列の 287-300番目のアミノ酸配列を有する TAT結合ペプチドである HSP287-TATペプチド （PIWTRNPDDITQEE-GYGRKKRRQRRRG) を細胞に 添加しアポトーシスへの影響を調べた。具体的には、ヒト繊維芽肉腫細胞 HT1080 (ATCC CCL-121) をぺプチド無処理、 0 . 1〃mol/L、 または 1〃mol/L の HSP327-TATまたは HSP287-TATぺプチドを添加して、 血清除去 DMEM培地 中で 24時間培養した。 その後、 細胞を回収し、 実施例 1に記載の方法で細胞溶 解液を調製した。 抗: PARP抗体 （Promega社製）を用いて、 得られた細胞溶解液 のウエスタン ·ブロヅティングを行ない、 キャスパーゼ -3による切断によって生 じる PARPの 85kDaフラグメントを検出することにより、 細胞内のアポト一シ スシグナルの活性化を解析した。 その結果を図 9 に示す。 この結果、 l ^mol/L の HSP327-TATを添加したもののみ、 PARPの 85kDaフラグメントの生成が抑 制されており血清除去によって起こる HT1080細胞のアポト一シスが抑制されて いることを確認した。 本発明を詳細にまた特定の実施態様を参照して説明したが、 本発明の精神と範 囲を逸脱することなく様々な変更や修正を加えることができることは当業者にと つて明らかである。 In order to examine the apoptosis regulating action of the amino acid peptide having the amino acid sequence at positions 327 to 340 of the amino acid sequence described in SEQ ID NO: 1 corresponding to the Akt binding region on Hsp90 shown in Examples 1 to 3, HSP327-TAT peptide (LEFRALLFIPRRAP-GYGRKKRRQKRHG) in which the TAT peptide [Science, 2, 1569-1572 (1999)] was conjugated to the C-terminus of Hsp90? As a negative control, add HSP287-TAT peptide (PIWTRNPDDITQEE-GYGRKKRRQRRRG), which is a TAT-binding peptide having the amino acid sequence at positions 287-300 of the amino acid sequence shown in SEQ ID NO: 1 that is not the Akt-binding region, and apoptosis. The effect was examined. Specifically, human fibroblast sarcoma cells HT1080 (ATCC CCL-121) were not treated with peptide, and 0.1 mol / L or 1 mol / L of HSP327-TAT or HSP287-TAT peptide was added. And cultured in serum-free DMEM medium for 24 hours. Thereafter, the cells were collected, and a cell lysate was prepared by the method described in Example 1. Anti: Western blotting of the obtained cell lysate using PARP antibody (Promega) was performed, and the 85 kDa fragment of PARP generated by cleavage with caspase-3 was detected. The activation of the cis signal was analyzed. Figure 9 shows the results. As a result, it was confirmed that only the addition of l ^ mol / L HSP327-TAT suppressed the production of the 85 kDa fragment of PARP and suppressed the apoptosis of HT1080 cells caused by serum removal. Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.
本出願は、 2000年 8月 22日出願の日本特許出願 No.2000— 251529に基づくも のであり、 その内容はここに参照として取り込まれる。  This application is based on Japanese Patent Application No. 2000-251529, filed on August 22, 2000, the contents of which are incorporated herein by reference.
<産業上の利用可能性 > <Industrial applicability>
本発明により、 アポトーシス抑制や糖代謝に関与する Aktの活性制御に関わる Hsp90上の機能部位および該 Hsp90と結合する Akt上の機能部位を同定された 結果、 アポトーシス制御や糖代謝制御シグナルの異常によって生じる胃癌、 卵巣 癌、 乳癌、 滕癌、 前立腺癌等の悪性腫瘍、 糖尿病等の疾患に対する予防薬および 治療薬、 脳、 心臓などの虚血時における細胞死抑制剤、 抗癌剤や放射線治療時に おける正常細胞の細胞死からの保護剤、 または既存の治療薬の効果を増強する薬 剤等が提供される, According to the present invention, a functional site on Hsp90 involved in apoptosis suppression and regulation of Akt activity involved in glucose metabolism and a functional site on Akt binding to the Hsp90 were identified. Prophylactic and therapeutic drugs against malignant tumors such as gastric cancer, ovarian cancer, breast cancer, breast cancer, prostate cancer, and prostate cancer, and diseases such as diabetes, cell death inhibitors during ischemia in the brain and heart, normal in anticancer drugs and radiation therapy Drugs that protect cells from cell death or enhance the effects of existing therapies Agent is provided,

Claims

請 求 の 範 囲 The scope of the claims
1 . 細胞内の Akt活性を制御する工程を含有する、 アポトーシスの制御方法。 1. A method for controlling apoptosis, comprising the step of controlling Akt activity in a cell.
2 . アポトーシスの制御がアポトーシスの誘導である請求の範囲 1に記載の 方法。 2. The method according to claim 1, wherein the control of apoptosis is induction of apoptosis.
3 . アポト一シスの制御がアポトーシスの抑制である請求の範囲 1に記載の 方法。 3. The method according to claim 1, wherein the control of apoptosis is suppression of apoptosis.
4 . 細胞内の Akt活性の制御が、 Hsp90と Aktとの結合の制御である、 請 求の範囲 1に記載の方法。 4. The method according to claim 1, wherein the control of Akt activity in the cell is a control of binding between Hsp90 and Akt.
5 . 細胞内の Akt活性の制御が、 Hsp90フアミリーに属する少なくとも一つ の蛋白質またはその部分ポリぺプチドにより行われる、 請求の範囲 1〜4のいず れか 1項に記載の方法。 5. The method according to any one of claims 1 to 4, wherein the control of Akt activity in the cell is performed by at least one protein belonging to Hsp90 family or a partial polypeptide thereof.
6 . Hsp90フアミリーに属する蛋白質が、 Hsp90 5または Hsp90ひである、 請求の範囲 5に記載の方法。 6. The method according to claim 5, wherein the protein belonging to Hsp90 family is Hsp90 5 or Hsp90.
7 . 細胞内の Akt活性の制御が、 配列番号 1記載のアミノ酸配列の 3 2 7番 目から 3 4 0番目または配列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8 番目からなるポリぺプチドにより行われる、 請求の範囲 1〜 4のいずれか 1項に 記載の方法。 7. The regulation of intracellular Akt activity is controlled by a polymorphic sequence consisting of the amino acid sequence of SEQ ID NO: 1 from the 327th to the 340th position or the amino acid sequence of the SEQ ID NO: 3 from the 335th to the 348th The method according to any one of claims 1 to 4, which is performed by using a peptide.
8 . 細胞内の Akt活性の制御が、 配列番号 1記載のアミノ酸配列の 3 2 7番 目から 3 4 0番目または配列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8 番目において 1以上のアミノ酸が欠失、 置換、 挿入または付加されたアミノ酸配 列からなり、 かつ細胞内の Akt活性を制御する活性を有するポリぺプチドにより 行われる、 請求の範囲 1〜4のいずれか 1項に記載の方法。 8. The control of Akt activity in the cell is 1 or more at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3. Amino acid deletion, substitution, insertion or addition of amino acids The method according to any one of claims 1 to 4, wherein the method is performed using a polypeptide comprising a sequence and having an activity of controlling Akt activity in a cell.
9 . 細胞内の Akt活性の制御が、 配列番号 1記載のアミノ酸配列の 3 2 7番 目から 3 4 0番目または配列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8 番目のァミノ酸配列と 6 0 %以上の相同性を有するァミノ酸配列からなり、 かつ 細胞内の Akt活性を制御する活性を有するポリペプチドにより行われる、請求の 範囲 1〜4のいずれか 1項に記載の方法。 9. The regulation of Akt activity in the cell is determined by the amino acid sequence of amino acids 3227 to 340 of the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of amino acids 3335 to 348 of the amino acid sequence of SEQ ID NO: 3 The method according to any one of claims 1 to 4, wherein the method is performed using a polypeptide comprising an amino acid sequence having 60% or more homology with the sequence and having an activity of controlling Akt activity in a cell. .
1 0 . 細胞内の Akt活性の制御が、 Attフアミリーに属する少なくとも一つ の蛋白質の部分べプチドにより行われる、 請求の範囲 2または 4記載の方法。 10. The method according to claim 2 or 4, wherein the control of Akt activity in the cell is performed by a partial peptide of at least one protein belonging to Att family.
1 1 . Aktフアミリーに属する蛋白質が、 Aktl、 Akt2または Akt3である、 請求の範囲 1 0に記載の方法。 11. The method according to claim 10, wherein the protein belonging to Akt family is Aktl, Akt2 or Akt3.
1 2 . 細胞内の Akt活性の制御が、 配列番号 5記載のアミノ酸配列の 2 2 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 2 7番目から 3 0 6番目からなる ポリべプチドにより行われる、 請求の範囲 2または 4に記載の方法。 1 2. The regulation of Akt activity in the cell is regulated by the amino acid sequence of SEQ ID NO: 5 from 229 to 309, the amino acid sequence of SEQ ID NO: 7 from 231 to 310, or The method according to claim 2 or 4, wherein the method is carried out using a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 9 from 227th to 306th.
1 3 . 細胞内の Akt活性の制御が、 配列番号 5記載のァミノ酸配列の 2 8 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 9 0番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 8 6番目から 3 0 6番目からなる ポリぺプチドにより行われる、 請求の範囲 2または 4に記載の方法。 1 3. The regulation of Akt activity in the cell is determined by the amino acid sequence of SEQ ID NO: 5 from 289 to 309, the amino acid sequence of SEQ ID NO: 7 from 290 to 310, 5. The method according to claim 2, wherein the method is performed using a polypeptide consisting of positions 286 to 306 of the amino acid sequence of SEQ ID NO: 9.
1 4 . 細胞内の Akt活性の制御が、 配列番号 5記載のアミノ酸配列の 2 2 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 2 7番目から '3 0 6番目において 1以上のアミノ酸が欠失、 置換、 挿入または付加されたアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有するポリぺプチドにより行われる、請 求の範囲 2または 4に記載の方法。 1 4. The regulation of intracellular Akt activity is from the 229th to 309th amino acid sequence of SEQ ID NO: 5, the 23rd to 310th amino acid sequence of SEQ ID NO: 7, or In the amino acid sequence described in SEQ ID NO: 9 from the 227th position to the '306 position The method according to claim 2 or 4, wherein the method is carried out using a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added, and having an activity of controlling intracellular Akt activity. .
1 5 . 細胞内の Akt活性の制御が、 配列番号 5記載のアミノ酸配列の 2 2 9 番目から 3 0 9番目、 配列番号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番 目、 または配列番号 9記載のアミノ酸配列の 2 2 7番目から 3 0 6番目のァミノ 酸配列と 6 0 %以上の相同性を有するァミノ酸配列からなり、 かつ細胞内の Akt 活性を制御する活性を有するポリべプチドにより行われる、 請求の範囲 2または 4に記載の方法。 1 5. The regulation of Akt activity in the cell is regulated by the amino acid sequence of SEQ ID NO: 5 from 229 to 309, the amino acid sequence of SEQ ID NO: 7 from 231 to 310, or A polyamino acid sequence comprising an amino acid sequence having at least 60% homology with the amino acid sequence at positions 227 to 306 of the amino acid sequence set forth in SEQ ID NO: 9, and having an activity of controlling intracellular Akt activity. The method according to claim 2 or 4, wherein the method is performed with a peptide.
1 6 . 細胞内の Akt活性の制御が、 請求の範囲 5から 1 5のいずれか 1項に 記載のポリべプチドの細胞内の Akt活性を制御する活性を変動させる化合物によ り行われる、 請求の範囲 1または 2に記載の方法。 16.The control of intracellular Akt activity is carried out by a compound that varies the activity of the polypeptide according to any one of claims 5 to 15 that regulates intracellular Akt activity. The method according to claim 1 or 2.
1 7 . 該化合物が、 該ポリペプチドと該ポリペプチドに結合する蛋白質との 結合を阻害するポリペプチドである、 請求の範囲 1 6に記載の方法。 17. The method according to claim 16, wherein said compound is a polypeptide that inhibits binding between said polypeptide and a protein that binds to said polypeptide.
1 8 . 細胞内の Akt活性の制御が、 請求の範囲 5から 1 5のいずれか 1項に 記載のポリべプチドを認識する抗体またはその抗体断片により行われる、 請求の 範囲 2または 4に記載の方法。 18. The method according to claim 2 or 4, wherein the control of Akt activity in the cell is performed by an antibody recognizing the polypeptide according to any one of claims 5 to 15 or an antibody fragment thereof. the method of.
1 9 . 配列番号 1記載のアミノ酸配列の 3 2 7番目から 3 4 0番目または配 列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8番目からなるポリべプチド< 1 9. Polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 from 327 to 340 or the amino acid sequence of SEQ ID NO: 3 from 335 to 348 <
2 0 . 配列番号 1記載のアミノ酸配列の 3 2 7番目から 3 4 0番目または配 列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8番目において 1以上のアミ ノ酸が欠失、 置換、 挿入または付加されたアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有するポリペプチド。 20. one or more amino acids are deleted at positions 327 to 340 of the amino acid sequence of SEQ ID NO: 1 or at positions 335 to 348 of the amino acid sequence of SEQ ID NO: 3, Consisting of substituted, inserted or added amino acid sequences, and A polypeptide having an activity of controlling Akt activity.
2 1 . 配列番号 1記載のアミノ酸配列の 3 2 7番目から 3 4 0番目または配 列番号 3記載のアミノ酸配列の 3 3 5番目から 3 4 8番目と 6 0 %以上の相同性 を有するアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有する ポリペプチド。 21. An amino acid having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 from 327 to 340th or the amino acid sequence of SEQ ID NO: 3 with 335 to 348 A polypeptide comprising a sequence and having an activity of controlling Akt activity in a cell.
2 2 . 配列番号 5記載のアミノ酸配列の 2 2 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列 2 2 7番目から 3 0 6番目からなるポリべプチド。 22. Amino acid sequence described in SEQ ID NO: 5 from 229 to 309, SEQ ID NO: 7 from amino acid sequence 23 1 to 310, or amino acid sequence described in SEQ ID NO: 9 2 2 Polypeptides consisting of 7th to 30th.
2 3 . 配列番号 5記載のァミノ酸配列の 2 8 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 9 0番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列 2 8 6番目から 3 0 6番目からなるポリぺプチド。 23. The amino acid sequence described in SEQ ID NO: 5 from amino acids 289 to 309, the amino acid sequence described in SEQ ID NO: 7 from amino acids 309 to 310, or the amino acid sequence described in SEQ ID NO: 9 8 6th to 30th polypeptides.
2 4 . 配列番号 5記載のアミノ酸配列の 2 2 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列 2 2 7番目から 3 0 6番目において 1以上のアミノ酸が欠失、置換、 挿入または付加されたァミノ酸配列からなり、 かつ細胞内の Akt活性を制御する 活性を有するポリべプチド。 24. Amino acid sequence described in SEQ ID NO: 5 from 229 to 309, SEQ ID NO: 7 from amino acid sequence 23 1 to 310, or amino acid sequence described in SEQ ID NO: 9 2 2 A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added at the 7th to 30th positions, and which has an activity of controlling Akt activity in a cell.
2 5 . 配列番号 5記載のアミノ酸配列の 2 2 9番目から 3 0 9番目、 配列番 号 7記載のアミノ酸配列の 2 3 1番目から 3 1 0番目、 または配列番号 9記載の アミノ酸配列の 2 2 7番目から 3 0 6番目のアミノ酸配列と 6 0 %以上の相同性 を有するアミノ酸配列からなり、 かつ細胞内の Akt活性を制御する活性を有する ポリべプチド。 25. The amino acid sequence of SEQ ID NO: 5 from 229 to 309, the amino acid sequence of SEQ ID NO: 7 from 231 to 310, or the amino acid sequence of SEQ ID NO: 9 27 A polypeptide consisting of an amino acid sequence having 60% or more homology with the amino acid sequence from the 7th to the 36th amino acid and having an activity to control intracellular Akt activity.
2 6 . 請求の範囲 1 9 ~ 2 5のいずれか 1項に記載のポリべプチドをコ一ド する DNA。 26. Code the polypeptide according to any one of claims 19 to 25 DNA.
2 7 . 請求の範囲 2 6に記載の DNAとストリンジェントな条件下でハイプ リダイズし、 かつ細胞内の Akt活性を制御する活性を有するポリぺプチドをコ一 ドする DNA。 27. A DNA that hybridizes with the DNA of claim 26 under stringent conditions and encodes a polypeptide having an activity of controlling intracellular Akt activity.
2 8 . 請求の範囲 2 6または 2 7記載の DNAをベクターに組み込んで得ら れる組換え体ベクター。 28. A recombinant vector obtained by incorporating the DNA according to claim 26 or 27 into a vector.
2 9 . 請求の範囲 2 8に記載の組換えベクターを宿主細胞に導入して得られ る形質転換体。 29. A transformant obtained by introducing the recombinant vector according to claim 28 into a host cell.
3 0 . 請求の範囲 2 9に記載の形質転換体を培地に培養し、 培養物中に細胞 内の Akt活性を制御可能なポリぺプチドを生成蓄積させ、該培養物から該ポリべ プチドを採取することを特徴とする細胞内の Akt活性を制御する活性を有するポ リベプチドの製造方法。 30. The transformant according to claim 29 is cultured in a medium, a polypeptide capable of controlling Akt activity in cells is produced and accumulated in the culture, and the polypeptide is removed from the culture. A method for producing a polybeptide having an activity of controlling intracellular Akt activity, which is characterized by being collected.
3 1 . 請求の範囲 1 9〜2 5のいずれか 1項に記載のポリべプチドを発現す る細胞と被験試料とを接触させる工程を含有する、 該ポリべプチドの細胞内の Akt活性を制御する活性を変動させる化合物のスクリーニング方法。 31. The method according to any one of claims 19 to 25, wherein the step of contacting the polypeptide expressing the polypeptide according to any one of claims 19 to 25 with a test sample comprises the step of measuring the intracellular Akt activity of the polypeptide. A method for screening a compound that changes the activity to be controlled.
3 2 . 該化合物が、 該ポリペプチドと該ポリペプチドに結合する蛋白質との 結合を阻害する化合物である、 請求の範囲 3 1に記載の方法。 32. The method according to claim 31, wherein said compound is a compound that inhibits binding between said polypeptide and a protein that binds to said polypeptide.
3 3 . 請求の範囲 1 9〜2 5のいずれか 1項に記載のポリぺプチドを認識す る抗体またはその抗体断片。 33. An antibody or an antibody fragment thereof that recognizes the polypeptide according to any one of claims 19 to 25.
3 4 . 請求の範囲 1 9〜2 5のいずれか 1項に記載のポリペプチド、 該ポリ ぺプチドと該ポリぺプチドに結合する蛋白質との結合を阻害するポリべプチド、 または請求の範囲 3 3に記載の抗体または抗体断片を有効成分として含有する医 薬。 ' 34. The polypeptide according to any one of claims 19 to 25, A medicament comprising, as an active ingredient, a polypeptide that inhibits binding between a peptide and a protein that binds to the polypeptide, or the antibody or antibody fragment according to claim 33. '
3 5 . 請求の範囲 1 9 ~ 2 5のいずれか 1項に記載のポリペプチドに結合す る蛋白質の活性化または活性低下により引き起こされる疾患の治療薬または予防 薬である、 請求の範囲 3 4記載の医薬。 35. The method according to claim 34, which is a therapeutic or prophylactic agent for a disease caused by activation or reduction of the activity of a protein binding to the polypeptide according to any one of claims 19 to 25. The medicament according to claim.
3 6 . 請^の範囲請求項 1 9〜2 5のいずれか 1項に記載のポリペプチドに 結合する蛋白質の活性化または活性低下により引き起こされる疾患の診断薬であ る、 請求の範囲 3 4記載の医薬。 36. A diagnostic agent for a disease caused by activation or decreased activity of a protein binding to the polypeptide according to any one of claims 19 to 25. The medicament according to claim.
3 7 . 請求の範囲 1 9〜2 5のいずれか 1項に記載のポリペプチド、 該ポリ ぺプチドと該ポリぺプチドに結合する蛋白質との結合を阻害するポリべプチド、 または請求の範囲 3 3に記載の抗体あるいは抗体断片、 および抗癌剤を組み合わ せてなる医薬組成物。 37. The polypeptide according to any one of claims 19 to 25, a polypeptide that inhibits binding between the polypeptide and a protein that binds to the polypeptide, or claim 3. A pharmaceutical composition comprising a combination of the antibody or antibody fragment according to 3, and an anticancer agent.
3 8 . 請求の範囲 1 9〜 2 5のいずれか 1項に記載のポリぺプチド、 該ポリ ぺプチドと該ポリベプチドに結合する蛋白質との結合を阻害するポリぺプチド、 または請求の範囲 3 3に記載の抗体あるいは抗体断片、 および抗癌剤を有効成分 として含有する医薬組成物。 38. The polypeptide according to any one of claims 19 to 25, a polypeptide that inhibits binding between the polypeptide and a protein that binds to the polypeptide, or claim 33. A pharmaceutical composition comprising, as an active ingredient, the antibody or antibody fragment described in 1 above and an anticancer agent.
3 9 . 抗癌剤が、 アルキル化剤、 代謝拮抗剤、 抗腫瘍性抗生物質、 植物アル カノィ ドから選ばれる少なくとも一つの抗癌剤である請求の範囲 3 7または 3 8 に記載の医薬組成物。 39. The pharmaceutical composition according to claim 37 or 38, wherein the anticancer agent is at least one anticancer agent selected from an alkylating agent, an antimetabolite, an antitumor antibiotic, and a plant alkanoid.
4 0 . 抗癌剤が、 ブスルファン、 シクロフォスフアミ ド、 ィホスフアミ ド、 メルファラン、 ニトロソゥレア、 アムサクリン、 カルボプラチン、 シスプラチン、 ダカルバジン、 ァザシチジン、 クラドリビン、 シ夕ラビン、 フルダラビン、 フル ォロウラシル、 ヒドロキシゥレア、 ロイコボリン、 メ トトレキサート、 ァクチノ マイシン D、 ブレオマイシン、 ダウノルビシン、 ドキソルビシン、 マイ トマイシ ン C、 ミ トキサントロン、 エトポシド、 パクリ夕キセル、 テニポシド、 ビンプラ スチン、 ビンクリスチン、 ビンデシン、 ゲルダナマイシンおよびラデイシコール から選ばれる少なくとも一つである請求の範囲 3 7〜3 9のいずれか 1項に記載 の医薬組成物。 40. Anticancer drugs include busulfan, cyclophosphamide, diphosphamide, melphalan, nitrosoperrea, amsacrine, carboplatin, cisplatin, Dacarbazine, azacytidine, cladribine, cilavine, fludarabine, fluorouracil, hydroxyperrea, leucovorin, methotrexate, actinomycin D, bleomycin, daunorubicin, doxorubicin, mitomycin C, mitoxantrone, etoposide, paclixelide The pharmaceutical composition according to any one of claims 37 to 39, which is at least one selected from vinplastin, vincristine, vindesine, geldanamycin, and radicicol.
PCT/JP2001/007179 2000-08-22 2001-08-22 Methof of regulating apoptosis and apoptosis-regulatory polypeptide WO2002015925A1 (en)

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