WO2007114239A1 - AGENT ANTI-CANCÉREUX COMPRENANT UN INHIBITEUR de DGKα - Google Patents

AGENT ANTI-CANCÉREUX COMPRENANT UN INHIBITEUR de DGKα Download PDF

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WO2007114239A1
WO2007114239A1 PCT/JP2007/056841 JP2007056841W WO2007114239A1 WO 2007114239 A1 WO2007114239 A1 WO 2007114239A1 JP 2007056841 W JP2007056841 W JP 2007056841W WO 2007114239 A1 WO2007114239 A1 WO 2007114239A1
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dgka
inhibitor
cells
antibody
dgk
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PCT/JP2007/056841
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English (en)
Japanese (ja)
Inventor
Fumio Sakane
Kenji Yanagisawa
Hideo Kanoh
Kowichi Jimbow
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Sapporo Medical University
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Priority to JP2008508605A priority Critical patent/JP5422204B2/ja
Publication of WO2007114239A1 publication Critical patent/WO2007114239A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to an anticancer agent, particularly a therapeutic agent for melanoma. More specifically, the present invention relates to a method for inducing apoptosis of cancer cells such as melanoma and an apoptosis inducer.
  • melanoma is known to have the highest malignancy and high mortality among all cancers.
  • NF nuclear factor
  • - ⁇ B is always activated along with breast cancer, colon cancer, spleen cancer and ovarian cancer.
  • Ivanov VN Ivanov VN, et al, Oncogene. 2003; 22: 3152-3161: Soengas MS, and Lowe SW. Oncogen e. 2003; 22: 3138-3151.
  • NF- ⁇ B is one of the apoptosis inhibitors, this increase in activity results in reduced effectiveness of chemotherapy. Therefore, there is currently no very effective chemotherapy for melanoma, and in principle the treatment relies on surgical excision.
  • TNF-a Tumor necrosis factor-a
  • its receptor activity are key apoptotic pathways that ultimately activate caspase-8 to cause cell death (Morgan M, et al., J Cell Biol. 2002; 157: 975- 984),-, but after activation of TNF- ⁇ receptor, activation of NF- ⁇ B occurs and is also known to exhibit an anti-apoptotic effect (Chen G, et al, Science. 2002; 296: 1634-1635). Therefore, in the signal transduction system after TNF- ⁇ receptor activation, TNF-a-dependent cell death is controlled by the balance between apoptosis and anti-apoptotic signals!
  • NF- ⁇ B was originally identified as a transcription factor specific for B cells that binds to the DNA sequence GGGG ACTTTCC (SEQ ID NO: 1) present in the ⁇ enhancer of immunoglobulins.
  • SEQ ID NO: 1 DNA sequence GGGG ACTTTCC
  • NF- ⁇ B is a homo- or heterodimer formed by molecular forces such as p65 (RelA) and p50 (NF-K Bl) belonging to the Rel family, and is an inhibitor in unstimulated cells. It is inactivated by forming a complex with the I ⁇ B molecule in the cytoplasm and blocking its nuclear translocation (Baldwin AS. J Clin Invest. 200 1; 107: 241-246).
  • Various stimuli including TNF- ⁇ phosphorylate ⁇ B.
  • I ⁇ B is degraded by the proteasome, and NF- ⁇ is translocated to the nucleus and is thought to regulate the expression of various genes. It has been.
  • NF- ⁇ B has been shown to be associated with cell apoptosis and canceration, and has attracted attention from medical aspects such as immune function, inflammation and viral infection, and its role in onset (Ivanov VN, et al , Oncogene. 2003; 22: 3152-3161; Baeuerle PA, and Henkel TF Annu Rev Immunol. 1994; 12: 141-179; Verma IM, et al., Genes Dev. 1995; 9: 2723-2735).
  • Diacylglycerol kinase is an enzyme that phosphorylates diacylglycerol to produce phosphatidic acid.
  • the substrate diacylglycerol has already been established as an activator of conventional protein kinase C and nobel protein kinase C.
  • protein kinase C chimaerin, Unc-1 3
  • Ras Ras It is known to bind to and activate the CI (zinc finger) domain of guar nucleotide releasing proteins (Hurley JH, et al "Protein Sci. 1997; 6: 477-480; Kazanietz MG. Mol Pharmacol.
  • reaction product phosphatidic acid has also been reported to regulate the activity of various signal transduction enzymes, including phosphatidylinositol 5 kinase N Raf-1 (English D. Cell Signal. 1996; 8: 341-347; Exton JH. Biochim Biophys Acta. 1994; 1212: 26-4 2).
  • DGK is not present in unicellular yeast, and only a few limited isoforms have been found in relatively simple multicellular organisms such as nematodes and fruit flies. . Therefore, many of the DGK isoforms are thought to be involved in the unique functions of multicellular organisms such as cell carcinogenesis, development 'differentiation, immune system, and nervous system construction.
  • type I DGK has two EF-node motifs (with Ca 2+ binding ability) in the molecule.
  • Sakane F et al., J Biol Chem. 1991; 266: 7096-7100
  • Sakane F et al., Nature. 1990; 344: 345-348
  • Yamada K et al "Biochem J. 1997; 321: 59 -64
  • type I isozymes research on DGK o; is the most advanced, and several physiological functions have recently been revealed (Kanoh H, et al "J Biochem.
  • this lysozyme suppresses the release of Fas ligand by inhibiting carbachol-induced apoptosis in T lymphocytes expressing muscarinic receptors by experiments using non-specific DGK inhibitors (R59949) and overexpression systems. (Alonso R, et al., J Biol Chem. 2005; 280: 28439-28450).
  • Non-patent literature l Amiri KI, and Richmond A. Cancer Metastasis Rev. 2005; 24:
  • Non-Patent Document 2 Ivanov VN, et al, Oncogene. 2003; 22: 3152-3161
  • Non-Patent Document 3 Soengas MS, and Lowe SW. Oncogene. 2003; 22: 3138-3151
  • Non-Patent Document 4 Morgan M, et al., J Cell Biol. 2002; 157: 975-984
  • Non-Patent Document 5 Chen G, et al., Science. 2002; 296: 1634-1635
  • Non-Patent Document 6 Baeuerle PA, and Henkel T. F. Annu Rev Immunol. 1994; 12
  • Non-Patent Document 7 Verma IM, et al., Genes Dev. 1995; 9: 2723-2735
  • Non-Patent Document 8 Baldwin AS. J Clin Invest. 2001; 107: 241-246
  • Non-Patent Document 9 Hurley JH, et al "Protein Sci. 1997; 6: 477-480
  • Non-Patent Document 10 Kazanietz MG. Mol Pharmacol. 2002; 61: 759-767
  • Non-Patent Document 11 English D. Cell Signal. 1996; 8: 341-347
  • Non-patent document 12 Exton JH. Biochim Biophys Acta. 1994; 1212: 26-42
  • Non-patent document 13 Imai S, et al "J Biol Chem. 2005; 280: 39870-39881
  • Non-patent document 14 Kanoh H, et al., J Biochem. 2002; 131: 629-633
  • Non-Patent Document 15 Sakane F, and Kanoh H. Int J Biochem Cell Biol. 1997; 29:
  • Non-Patent Document 16 Topham MK, and Prescott SM. J Biol Chem. 1999; 274: 11 447-11450
  • Non-Patent Document 17 van Blitterswijk WJ, and Houssa B. Chem Phys Lipids. 1999;
  • Non-patent literature 18 Ito T, et al., J Biol Chem. 2004; 279: 23317-23326
  • Non-patent literature 19 Sakane F, et al "J Biol Chem. 1991; 266: 7096-7100
  • Non-Patent Document 20 Sakane F, et al., Nature. 1990; 344: 345-348
  • Non-Patent Document 21 Yamada K, et al, Biochem J. 1997; 321: 59-64
  • Non-patent literature 22 Alonso R, et al., J Biol Chem. 2005; 280: 28439-28450
  • Non-patent literature 23 Cutrupi S, et al, EMBO J. 2000; 19: 4614-4622
  • Non-Patent Document 24 Outram SV, et al., Immunology. 2002; 105: 391-398 Disclosure of the Invention
  • the present invention provides a therapeutic agent and a therapeutic method for melanoma and other cancers, particularly a cancer cell apoptosis inducer, and a method for screening candidate substances for such therapeutic agents.
  • the purpose is to do.
  • the present inventor found that the ⁇ -isozyme of DGK is expressed in all examined melanoma cells, but not detected in non-cancerous melanocytes. It was also clarified that apoptosis of melanoma cells induced by DGKa force TNF-a is suppressed and that its control is performed via the NF- ⁇ pathway.
  • the present invention provides an anticancer agent containing a DGKa inhibitor as an active ingredient.
  • the present invention provides a cancer cell apoptosis-inducing agent comprising a DGKa inhibitor as an active ingredient.
  • the present invention provides an apoptosis inducer for melanoma cells, which contains a DGKa inhibitor as an active ingredient.
  • the present invention provides an inhibitor of NF- ⁇ B expression comprising a DGKa inhibitor as an active ingredient.
  • the DGKa inhibitor is an anti-DGKa antibody, an antisense oligonucleotide against the DGKa gene, a ribozyme and siRNA, and 3- [2- [4- [bis- (4-fluorophenol- L) methylene]-1-piberidyl-l] ethyl] -2,3-dihydro-2-thioxo-4 (1H) quinazolinone (R59949) and 6- [2- [4-[(4-fluorophenyl) phenyl] Methylene] -1-pyberidinyl] ethyl] -7-methyl-5H-thiazolo (3,2-a) pyrimidin-5-one (R59022) selected from the group consisting of
  • the present invention provides a candidate substance for an anticancer agent, an apoptosis inducer for cancer cells, an apoptosis inducer for melanoma cells, or an inhibitor of NF- ⁇ B expression.
  • a method comprising: contacting a test substance with a cell expressing DGK a and determining whether the test substance force inhibits the expression and Z or function of SDGK a.
  • the present invention provides a method for treating cancer and a method for inducing apoptosis of cancer cells by inhibiting DGKo ;.
  • the present invention provides a method for inducing apoptosis of melanoma cells by inhibiting DGK.
  • the present invention is, by a child inhibit DGK a, NF-? Provides a method for inhibiting the expression of K B.
  • Fig. 1 shows the expression of DGKO in ⁇ melanoma cells and melanocytes.
  • Figure 2 shows the effect of overexpression of type I DGK on apoptosis.
  • Figure 3 shows the effect of DGKa knockdown on apoptosis.
  • Fig. 4 shows the effect of DGKa overexpression or knockdown on the nuclear localization of NF- ⁇ B.
  • Figure 5 shows the effect of DGKa overexpression or knockdown on NF- ⁇ B activity
  • Figure 6 shows the effect of NF- ⁇ - inhibitor (MG-132) on NF- ⁇ activity and apoptosis.
  • DGKa plays a role of strongly suppressing apoptosis in melanoma cells.
  • DGKo is known to be expressed less in normal cells except for T lymphocytes and oligodendrocytes.
  • DGKo highly expressed in T lymphocytes by experiments using non-specific DGK inhibitors (R59949, inhibits all type I DGK) and overexpression systems; AICD is a phenomenon specific to T lymphocytes (Alonso R, et al, J Biol Chem. 2005; 280: 28439— 28450; Outram SV, et al., Immunology.
  • DGK a There is no report that is involved in the control of apoptosis.
  • the present invention is the first to elucidate the involvement of DGK in apoptosis of cancer cells including melanoma.
  • a specific knockdown (reduction in expression level) of DGKo could be directly shown to be involved in apoptosis.
  • DGKo In T lymphocytes stably expressing muscarinic receptors, it has been suggested that DGKo; inhibits apoptosis induced by carbazole through suppression of Fas ligand release (Alonso R, et al., J Biol Chem. 2005; 280: 28439-28450).
  • Fas ligand release is the main pathway in apoptosis of melanoma cells. It was strongly suggested that it worked.
  • DGK ⁇ the NF- ⁇ pathway is required for the inhibition of apoptosis by DGK ⁇ in melanoma cells. Since there has been no report on the relationship between the inhibition of Fas ligand release and the activation of NF- ⁇ B so far, DGKa regulates apoptosis through different signaling pathways in different cells. It is thought that there is.
  • DGKa As shown in the Examples below, the endogenous DGKa of AKI cells was mainly localized in the nucleus. In addition, DGKa overexpressed in AKI cells was also observed to be markedly localized in the nucleus, but the localization of DGK j8 and DGK y in the nucleus was limited (data not shown). Therefore, it is suggested that localization to DGKa force S nucleus is important in controlling apoptosis. In T lymphocytes, DGK a is present in the cytoplasm during apoptosis induced by carbachol (even when unstimulated) (Alonso R, et al., J Biol Chem. 2005; 280: 28439-2845 0).
  • melanoma cells differ greatly in that DGKa is localized in the nucleus during TNF- a stimulation (even when unstimulated).
  • the difference in localization between T lymphocytes (cytoplasm) and melanoma cells (in the nucleus) may be a major cause of the different signaling pathways that DGKa uses to inhibit apoptosis.
  • the anticancer agent according to the present invention is characterized by containing a DGKa inhibitor as an active ingredient.
  • DGK a inhibitor means a substance that reduces significantly the DGK Hino expression level or enzymatic activity.
  • the DGKa inhibitor may be an inhibitor specific to DGKa or an inhibitor that can also inhibit other DGK isozymes.
  • Inhibitors that can inhibit DGK include, for example, 3- [2- [4- [bis- (4-fluorophenyl) methylene] -1-piveridyl] ethyl] -2,3-dihydro-2- Thioxo-4 (1H) quinazolinone (also referred to as R59949) and 6- [2- [4-[(4-fluorophenyl) phenylmethylene] -1-pyberidinyl] ethyl] -7-methyl-5H-thiazolo ( 3,2-a) pyrimidin-5-one (also referred to as R59022) is known. Any of these can be used in the present invention.
  • the anticancer agent of the present invention can induce apoptosis of cancer cells in cancers in which DGKa expression is enhanced, and in particular, NF- ⁇ is active in breast cancer, colon cancer, spleen cancer, ovarian cancer, etc. It is thought that it is useful for the treatment of cancer.
  • DGKa inhibitor of the present invention is an anti-DGKa antibody.
  • anti-DGK o; antibody means an antibody that can bind to DGK o; by an antigen-antibody reaction.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • Polyclonal antibodies that bind to DGKo can be used to immunize animals using DGKo; as a sensitizing antigen according to a method well known in the art, and to obtain the serum strength thereof.
  • Monoclonal antibodies that bind to DGK o are immunized with DGK a as a sensitizing antigen according to methods well known in the art, and the resulting immune cells are removed and fused with myeloma cells. It can be obtained by cloning a hyperpridoma that produces lysozyme and culturing this hyperidoma.
  • the monoclonal antibody of the present invention includes a gene recombinant antibody, a chimeric antibody, a CDR graft produced by a transformant transformed with an expression vector containing an antibody gene in addition to an antibody produced by a hyperidoma. Antibodies, and fragments of these antibodies are included.
  • Recombinant antibodies are anti-DGKo; the ability to produce anti-DGKo antibodies, clone the cDNA encoding the antibody, and insert this into an expression vector to transform animal cells, plant cells, etc. It can be produced by culturing this transformant.
  • a chimeric antibody is an antibody composed of a heavy chain variable region and a light chain variable region of an antibody derived from one animal, and a heavy chain constant region and a light chain constant region of an antibody derived from another animal. Examples of antibody fragments that can bind to DGKa include Fab, F (ab ′) 2, Fab ′, scFv, and diabody.
  • DGK o which is an antigen
  • Human DGK o can be produced recombinantly by conventional methods according to the nucleotide sequence of the gene described in GenBank accession number X62535 and the amino acid sequence encoded by it. The nucleotide sequence and amino acid sequence of the human DGKa gene are shown in SEQ ID NOs: 8 and 9, respectively.
  • the thus produced DGK o is administered as an antigen subcutaneously, intravenously or intraperitoneally to the animal.
  • mammals such as mice, rats, hamsters, rabbits and goats can be used.
  • the antigen is administered together with an appropriate adjuvant such as the ability to bind the antigen to the carrier protein and Freund's complete adjuvant.
  • a partial peptide of DGKa may be used as the antigen.
  • the antigen is administered several times every 13 weeks.
  • the antibody titer is monitored by measuring the amount of immunoglobulin in the serum by ELISA. After the antibody titer has risen sufficiently, immune cells are removed from the animal. Spleen cells are preferably used as immune cells.
  • a hybridoma is produced by fusing antibody-producing immune cells with myeloma cells derived from a mammal of the same species. Various myeloma cell lines suitable for producing hybridomas are commercially available. Fusion is performed according to methods well known in the art, eg, in the presence of PEG, and the fused cells are selected in HAT medium.
  • the culture supernatant is assayed by ELISA, etc., and a hyperidoma producing an antibody that binds to the antigen is selected.
  • the hybridoma producing the target antibody can be cloned by limiting dilution.
  • Monoclonal antibodies can be produced from the supernatant of a culture solution obtained by culturing monoclonal antibody-producing cells, or in mice pretreated with 2, 4, 10, 14-tetramethylpentadecane.
  • the cells can be produced by intraperitoneal administration, collecting mouse ascites from day 7 to day 10 after inoculation, centrifuging, and collecting the supernatant.
  • Monoclonal antibodies can be purified using conventional protein purification methods such as salting out, ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography, and HPLC. Preferably, affinity chromatography on a protein A column is performed.
  • the subclass of the purified monoclonal antibody can be determined by typing using a commercially available mouse monoclonal antibody isotyping kit.
  • the nucleotide sequence of the antibody gene encoding the monoclonal antibody of the present invention can be obtained by analyzing the gene of the obtained monoclonal antibody-producing cell. Extract the total RNA from the monoclonal antibody-producing cell strength, and use this as a cage to prepare a cDNA fragment using reverse transcriptase. Next, the V region of the antibody gene is amplified by PCR using appropriately designed primers, and the base sequence of the cDNA in this region is determined.
  • the gene encoding the antibody of the present invention is incorporated into an appropriate expression vector and introduced into a host cell.
  • host cells Escherichia coli, yeast, mammalian cells, insect cells, plant cells, and the like can be used.
  • mammalian cells such as CHO, COS, and BHK are preferably used.
  • Vectors can be introduced into host cells by, for example, the salt calcium method, the calcium phosphate method, the DEAE dextran method, the method using the cationic ribosome DOT AP (Boehringer Mannheim), the electopore method, the ribofusion method, etc. It can be carried out.
  • Recombinant antibodies can be produced by culturing the resulting transformed host cells and expressing the antibodies.
  • a chimeric antibody is a heavy chain variable region and light chain variable region of an antibody derived from a certain animal (eg, mouse), and a heavy chain constant region and light chain constant of an antibody derived from another animal (eg, human).
  • Chimeric antibodies are obtained by cloning cDNA encoding the heavy chain variable region and light chain variable region of an antibody from a hybridoma producing a monoclonal antibody that binds to DGKa, while determining the heavy chain of an antibody derived from another animal.
  • a cDNA encoding the normal region and the light chain constant region can be cloned, inserted into an appropriate expression vector in combination, and expressed in a host cell for recombinant production.
  • a CDR-grafted antibody is an antibody in which the complementarity determining region (CDR) of an antibody of one animal (eg, mouse) is transplanted to the complementarity determining region of an antibody of another animal (eg, human).
  • the gene encoding the CDR-grafted antibody is CDR1, 2 based on the gene sequences of the heavy and light chain variable regions of the hybridoma-cloned antibody that produces monoclonal antibodies that bind to DGKo; , 3 is designed and replaced with the corresponding CDR1, 2 and 3 sequences in the vector containing the genes encoding heavy and light chain variable regions of antibodies from other animals. Can be obtained.
  • the CDR-grafted antibody can be produced recombinantly.
  • Antibody fragments such as Fab, F (ab ') 2, Fab', scFv, and diabody that can bind to DGKa are obtained by using the anti-DGKa monoclonal antibody of the present invention described above with an enzyme such as papain or trypsin. It can be produced by treating or introducing an expression vector incorporating a gene encoding them into a host cell to obtain a transformant.
  • DGKa inhibitors include expression of DGKa genes (transcription and Z, such as antisense oligonucleotides, ribozymes, molecules that cause RNA interference (RNAi) (eg, dsRNA, siRNA, shRNA, miRNA). Or translation) inhibitors.
  • RNAi RNA interference
  • nucleic acids can bind to and inhibit the expression of the DGKa gene or mRNA encoding DGKa.
  • General methods for controlling gene expression using antisense, ribozyme technology and RNAi technology, or gene therapy methods for expressing exogenous genes in this manner are well known in the art.
  • Antisense oligonucleotide refers to a nucleic acid molecule having a sequence complementary to mRNA encoding DGKa or a derivative thereof. Antisense oligonucleotides specifically bind to mRNA and inhibit protein expression by inhibiting transcription and Z or translation. Coupling may be by Watson's Click or Houdsteen-type base pair complementarity, or by triplex formation.
  • a ribozyme refers to the RNA structure of one or more RNAs that have catalytic properties. Ribozymes generally exhibit endonuclease, ligase or polymerase activity. Various secondary structure ribozymes are known, such as hammerhead and hairpin type ribozymes.
  • RNA interference is a method of silencing a target gene using a double-stranded RNA molecule.
  • DGKa inhibitors can be administered as is, but are usually formulated using pharmaceutical carriers. Any carrier commonly used in the pharmaceutical field can be used as the carrier for the formulation, for example, sterile water, physiological saline, excipients, stabilizers, antioxidants, buffers, surface active agents. Agents, binders and the like are preferably used. In addition, DGKa inhibitors can be encapsulated in microcapsules and polymer gels to provide sustained-release preparations. [0039]
  • the administration route of the anticancer agent of the present invention includes oral administration, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, intracavity administration, and the like. Preferably, it may be administered directly or percutaneously to the disease site, or may be directly injected using a catheter or the like. When the anticancer agent of the present invention is used for melanoma treatment, it can be administered directly or by subcutaneous injection.
  • the dose is appropriately selected according to the type of DGKa inhibitor, the route of administration, the degree of disease, etc., but when administered directly to the disease site, it is usually lO / zg lOmg, preferably 500 g ⁇ 500mg.
  • the dose is usually 1 / z g lmg / Kg, preferably 20 ⁇ g to 20 mg / Kg.
  • the present invention screens candidate substances for anticancer agents, cancer cell apoptosis inducers, melanoma cell apoptosis inducers, or inhibitors of NF- ⁇ B expression from various test substances.
  • Screening can be performed by contacting a test substance with DGKa and determining whether the test substance strength 3 ⁇ 4 GKa expression or enzyme activity is inhibited.
  • the ability of a test substance to inhibit DGKa expression can be assessed by measuring the amount of DGKa mRNA or protein by known methods.
  • Test substance ability 3 ⁇ 4 The ability to inhibit the enzyme activity of GKa can be evaluated by measuring diacylglycerol kinase activity by a known method.
  • Substances identified by these assays as inhibiting DGKo expression or enzyme activity are anti-cancer agents, cancer cell apoptosis inducers, melanoma cell apoptosis inducers, or suppression of NF- ⁇ B expression. It is considered to be a candidate substance for the drug.
  • Test substances can also be obtained from various libraries such as various synthetic or natural compound libraries, combinatorial libraries, oligonucleotide libraries, peptide libraries and the like.
  • extracts from natural products such as bacteria, fungi, algae, plants, animals, and partially purified products may be used as test substances.
  • NHEM human epidermal melanocytes
  • Wild-type (WT) pig DGK a (Sakane F, et al., Nature. 1990; 344: 345-348), Kinase-dead type (KD) -porcine DGK a (G435D) (Yamada K , et al., Bio chem Biophys Res Commun. 2003; 305: 101-107), wild type rat DGK jS (Goto K, and Kondo H. Proc Natl Acad Sci USA. 1993; 90: 7598-7602), and wild Type human DGKY (Kai M, et al, J Biol Chem.
  • GFP sense 5'-ACGGCAUCAAGGUGAACUUCAAGAU-AG-3 '(SEQ ID NO: 2), GFP antisense; 3'-UA-UCCCGUAGUUCCACUUGAAGUUCUA-5' (SEQ ID NO: 3),
  • lysis buffer 150 mM NaCl, 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 1 mM fluorinated methylmethylsulfol, protease inhibitor cocktail (1 tablet / 50 ml) , Roche Diagnostics)
  • lysis buffer 150 mM NaCl, 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 1 mM fluorinated methylmethylsulfol, protease inhibitor cocktail (1 tablet / 50 ml) , Roche Diagnostics
  • sonication 4 ° C
  • centrifuged at 4 ° C for 5 minutes at 3000 rpm centrifuged at 4 ° C for 5 minutes at 3000 rpm, and the supernatant (lysate) was recovered.
  • a portion of this lysate was used and the amount of protein was quantified by BCA protein assay (Pierce Biotechnology, USA).
  • SDS sodium dodecyl sulfate
  • sample buffer 125 mM Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 10% 2-mercaptoethanol, 0.005% bromophenol blue
  • SDS-polyacrylamide gel electrophoresis PAGE. After SDS-PAGE, transfer was performed (400 mA, 1 hour) onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, USA), and the transfer membrane was blocked with Block Ace (Dainippon Pharmaceutical).
  • Anti-DGKa antibody (Kanoh H, et al., J Biol Chem. 1986; 261: 5597-5602), anti-actin antibody (Santa Cruz Biotechnology, USA) or anti-GFP antibody (Sa nta Cruz Biotechnology) was diluted with Block Ace and allowed to react for 1 hour. After washing, each was reacted with a peroxidase-labeled secondary antibody (Jackson Immunoresearch Laboratories, USA) corresponding to each primary antibody. After washing, ECL Western blotting detection cisam (Amersham Biosciences, UK) was used to emit light, and then exposed to Hyperfilm (Amersham Biosciences) to detect bands. Band intensity was quantified using Image J software (National Institute of Health, USA).
  • RT Reverse transcriptase
  • PCR polymerase chain reaction
  • Superscript First—3 ⁇ 4trana synthesis System (Invitrogen, USAj) was used to synthesize cDNA using 5 g total RNA.
  • This first strand cDNA (equivalent to 500 ng total RNA) was added to Ex Taq polymerase (Takara Bio) and human.
  • DGK ⁇ -specific primer (Yamada K, et al., Biochem Biophys Res Commun.
  • TNF-a (Strathmann Biotec AG, Ger many) is apoptotic. Induced. After further incubation for 24 hours, the cells were fixed with 3.7% formaldehyde. After increasing permeability with 0.1% Triton ⁇ -100, In
  • TdT-mediated dUTP nick end label TUNEL
  • the cells were encapsulated using Vectashield (Vector Laboratories, U.S.A.), and more than 1,000 cells were observed with a confocal laser microscope (Zeiss LSM 510), and positive cells were counted.
  • Cells were seeded on a cover glass coated with poly-L-lysine, and various expression vectors or siRNA were introduced. After 24 hours, 50 ng / ml TNF- ⁇ was added to induce apoptosis. Further After 24 hours of incubation, the cells were fixed with 3.7% formaldehyde. Increased permeability with 0.1% Triton X-100, then reacted with anti-DGKa antibody or anti-NF- ⁇ antibody (Santa Cruz Biotechnology) diluted with 2% ushi serum albumin / PBS for 1 hour . After washing, each antibody was reacted with a secondary antibody (Eugene, USA) conjugated with Alexa Fluor 488 or Alexa Fluor 594 corresponding to each primary antibody. The cells were encapsulated using Vectashield, and subcellular localization was observed with a confocal laser microscope (Zeiss LSM 510).
  • PNF- ⁇ B-Luc Vector (Takara Bio-Clontech), which incorporates the NF- ⁇ B promoter sequence and luciferase cDNA downstream as a reporter, is introduced into AKI melanoma cells along with various expression vectors or siRNA. 24 hours after introduction, 50 ng / ml TNF- ⁇ was induced to induce apoptosis. After an additional 12 hours, the cells were thawed with Glo Lysis Buffer (Promega, U.S.A.), and the luciferase substrate (Steady-Glo, Promega) was added and reacted.
  • Glo Lysis Buffer Promega, U.S.A.
  • Luminescence by luciferase was measured using a Wallac 1420 ARVOsx multilabel plate reader (PerkinElmer, U.S.A.). The luciferase activity was corrected by coexpression of pSV-j8-galactosidase control vector (Promega), measuring ⁇ -galatatosidase activity, and determining the expression efficiency.
  • DGK a protein was detected in all melanoma cells examined. However, the other type I isozymes DGK ⁇ and DGK ⁇ mRNA and protein were undetectable under the same conditions as those detected for DGK a. Therefore, it was revealed that only ⁇ -isozyme was expressed in melanoma cells as far as examined.
  • DGKa-expressing cells the results of Western blot and RT-PCR obtained using AKI cells are shown in Figures 1A and 1B, respectively. In Western blotting, a band is also observed at a molecular weight of 60K in addition to 80K, which matches the calculated molecular weight of the cloned DGK a cDNA (Fig. 1A). It is considered to be an alternative splice product of
  • DGKa has a function specific to melanoma cells, so we tried to clarify its function.
  • Melanoma cells are known to be highly resistant to apoptosis induced by various stimuli and drugs. Therefore, we next examined the role of this isozyme in apoptosis of melanoma cells by overexpressing DGKa using AKI cells, or conversely reducing the expression level of endogenous DGKa.
  • FIG. 1 AKI melanoma cells were transfected with a control vector (pEGFP alone), pEGFP-DGK ⁇ -WT, pEGFP-DGKa-KD, pEGFP-DGK j8-WT or pEGFP-DGKy-WT, and TNF- 24 hours after introduction.
  • ⁇ 50 ng / ml was added and incubated for another 24 hours.
  • cell lysates (10 g) were separated by SDS-PAGE and Western blotting was performed using anti-GFP antibodies to confirm the expression of each protein (Fig.
  • FIG. 2A As a result, it was found that overexpression of DGK a -WT (FIG. 2A) markedly inhibits apoptosis induced by TNF-a in AKI cells (FIG. 2B).
  • FIG. 2B In the case of DGK a-KD, which is inactive to the kinase, even though almost the same level of expression was observed (Fig. 2A), this effect was not observed (Fig. 2B). This result indicates that the catalytic activity of DGKa is essential for the inhibition of apoptosis by DGKa.
  • DGKa protein both 80K and 60K bands
  • apoptosis induced by TNF- ⁇ was significantly enhanced by DGKa knockdown (Fig. 3B).
  • DGKy siRNA did not have this effect. This result further supports the specific involvement of a-isozymes in inhibiting apoptosis.
  • control vector pEGFP alone
  • pEGFP-DGK a -WT pEGFP-DGK a -KD
  • TNF-a 50 ng / ml
  • anti-NF- ⁇ B p65
  • the cells were stained (red), and the intracellular localization of NF- ⁇ B (p65) in cells expressing the GFP fusion protein was observed with a confocal laser microscope (Fig. 4A). Bar: 10 ⁇ m.
  • control GFP
  • DGK a DGK y siRNA
  • TNF-a 50 ng / ml
  • Fig. 4C confocal laser microscope
  • Fig. 4D Image J software was used to analyze the fluorescence intensity ratio of NF- ⁇ B (p65) present in the nuclear Z cytoplasm under each condition. The results are shown as the average standard deviation obtained in three independent experiments (each analyzing 20 or more cells).
  • NF- ⁇ B transcriptional activity of NF- ⁇ B actually increased was examined by one luciferase reporter.
  • AKI melanoma cells were transfected with a control vector (pEGFP alone), pEGFP-DGK a-WT, or pEGFP-DGK ⁇ -KD, and TNF- ⁇ (50 ng / ml) was obtained 24 hours after the introduction. After further incubation for 12 hours, cells were collected and luciferase activity was measured (Fig. 5A). The activity was expressed as a relative value with 100% of control cells. The results are expressed as the mean average standard deviation obtained by three independent experiments.
  • GFP control
  • DGKa siRNA was introduced into AKI melanoma cells, and TNF- ⁇ (50 ng / ml) was collected 24 hours after introduction. After further incubation for 12 hours, cells were collected and luciferase activity was measured (Fig. 5B). The activity was expressed as a relative value with control cells taken as 100%. The results are shown as the average standard deviation obtained from three independent experiments. In the DGKa siRNA knockdown, conversely, when combined with the results above the decrease in NF- ⁇ B activity, DGKa positively regulates the transcriptional activity of NF- ⁇ through its catalytic activity. It became clear. It also suggests that there is a close relationship between the suppression of apoptosis by DGKa and the increased activity of NF- ⁇ .
  • the activity was expressed as a relative value with 100% of control cells.
  • the results are expressed as the average standard deviation obtained by three independent experiments.
  • MG-132 an NF- ⁇ B inhibitor
  • TUNEL staining was performed after 24 hours of incubation. Using a confocal laser microscope, cells expressing GFP fusion protein and positive for TUNEL staining were counted, and the percentage (%) of the total number of GFP positive cells was determined (Fig. 6B). The degree of apoptosis induced was expressed as a relative value with control cells taken as 100%. In experiments that were repeated twice independently, almost the same results were obtained. As a result, it is recognized in FIG.
  • the effect of the DGKa inhibitor of the present invention to suppress cancer is known in the literature (for example, Y. Takei, et al, Cancer Res., 64, 3365 (2004); Y. Minakuchi, et al, Nucleic Acids Res. , 32, el09 (2004); F. Takeshita, et al., Proc. Nat. Acad. Sci. USA., 102, 1 2177 (2005)).
  • Mouse B16F1 melanoma cells are used as skin cancer cells.
  • female C5 7BL / 6J mice (4 weeks old, 10 gm) are used.
  • To form a cancer subcutaneous injection of 0.1 ml phosphate-buffered I ⁇ saline about LxlO 5 amino B16F1 melanoma cells suspended in the left flank of C57BL / 6J mice. The volume of the cancer that forms after 7-10 days is measured.
  • DGKa-specific siRNA The effect of DGKa-specific siRNA is measured as follows. 48 hours after subcutaneous injection of B16F1 melanoma cells, DGKa specific siRNA mixed with atelocollagen (AteloGene TM; Koken) is administered locally (subcutaneously) or systemically (intravenously). Various conditions can be set for the mixing ratio and concentration of DGKa-specific siRNA and AteloGene, and the administration period of DGKa-specific siRNA. Measure the volume of cancer that formed after 5 to 8 days and test the effect of DGK o; specific siRNA.
  • the anticancer agent of the present invention is useful for the treatment of cancer, particularly melanoma.
  • the present invention is also useful for screening candidate substances for anticancer agents.

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Abstract

L'invention concerne un nouvel agent anti-cancéreux. Elle concerne un agent anti-cancéreux, un inducteur d'apoptose dans une cellule cancéreuse, un inducteur d'apoptose dans une cellule de mélanome et un suppresseur de l'expression de NF-ϰB, chacun de ceux-ci comprenant un inhibiteur de DGKα comme principe actif. Des exemples d'inhibiteur de DGKα comprennent un anticorps anti-DGKα et un oligonucléotide, ribozyme ou ARNsi anti-sens pour un gène DGKα. L'invention concerne égalment une méthode pour identifier une substance candidate pour un agent anti-cancéreux, un inducteur d'apoptose dans une cellule cancéreuse, un inducteur d'apoptose dans une cellule de mélanome ou un suppresseur de l'expression de NF-ϰB. La méthode consiste à mettre en contact une substance à examiner avec une cellule capable d'exprimer DGKα et à déterminer si la substance peut inhiber ou pas l'expression et/ou la fonction de DGKa.
PCT/JP2007/056841 2006-03-30 2007-03-29 AGENT ANTI-CANCÉREUX COMPRENANT UN INHIBITEUR de DGKα WO2007114239A1 (fr)

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WO2021130638A1 (fr) 2019-12-24 2021-07-01 Carna Biosciences, Inc. Composés modulant la diacylglycérol kinase
WO2022271659A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases
WO2022271650A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2022271677A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2022271684A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021130638A1 (fr) 2019-12-24 2021-07-01 Carna Biosciences, Inc. Composés modulant la diacylglycérol kinase
US11845723B2 (en) 2019-12-24 2023-12-19 Gilead Sciences, Inc. Diacylglycerol kinase modulating compounds
WO2022271659A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases
WO2022271650A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2022271677A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés de modulation de la diacylglycérol kinase
WO2022271684A1 (fr) 2021-06-23 2022-12-29 Gilead Sciences, Inc. Composés modulant les diacylglycérol kinases
US11926628B2 (en) 2021-06-23 2024-03-12 Gilead Sciences, Inc. Diacylglyercol kinase modulating compounds
US11932634B2 (en) 2021-06-23 2024-03-19 Gilead Sciences, Inc. Diacylglycerol kinase modulating compounds
US11976072B2 (en) 2021-06-23 2024-05-07 Gilead Sciences, Inc. Diacylglycerol kinase modulating compounds
US11999733B2 (en) 2021-06-23 2024-06-04 Gilead Sciences, Inc. Diacylglycerol kinase modulating compounds

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