WO2005038052A2 - Methode d'identification de substances mimetiques d'interferons - Google Patents

Methode d'identification de substances mimetiques d'interferons Download PDF

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WO2005038052A2
WO2005038052A2 PCT/EP2004/052550 EP2004052550W WO2005038052A2 WO 2005038052 A2 WO2005038052 A2 WO 2005038052A2 EP 2004052550 W EP2004052550 W EP 2004052550W WO 2005038052 A2 WO2005038052 A2 WO 2005038052A2
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cells
compound
mrna
amount
determined
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PCT/EP2004/052550
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WO2005038052A3 (fr
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Timo Wittenberger
Hans-Peter Hofmann
Volker Gekeler
Cy Aaron Stein
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Altana Pharma Ag
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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Field of Application of the Invention Present invention relates to a method for identifying a compound useful as an interferon mimic.
  • the method of present invention comprises the step of determining the expression rate of certain genes in eukaryotic cells in response to the interaction with a compound.
  • IFN Prior Art Interferons
  • IFN- ⁇ interferon alpha
  • cytokine with pleiotropic cellular functions, including antiviral, antiproliferative, immunomodulatory, and antiangiogenic activities [Gutterman JU. Cytokine therapeutics: lessons from interfero ⁇ -alpha.
  • IFN- ⁇ interferon gamma
  • Interferons and other cytokines are widely used in antiviral or cancer therapy, but suffer from severe side effects if administered systemically. Moreover, synthesis and purification of cytokines applying recombinant DNA and expression vectors are difficult and costly.
  • RNA-DNA heteroduplex nucleic acids So called antisense oligodesoxynucleotides were developed to target RNAs in a sequence specific manner by Watson-Crick base pairing and activating RNaseH finally causing depletion of the gene product of the particular target gene.
  • RNaseH recognizes RNA-DNA heteroduplex nucleic acids.
  • G3139 is a 18mer phosphorothioate oligodesoxyribonucleotide targeted to codons 1-6 of the human Bcl-2 mRNA [Klasa RJ, Gillum AM, Klem RE, Frankel SR. Oblimersen Bcl-2 antisense: fa- ciliating apoptosis in anticancer treatment.
  • Antisense Nucleic Acid Drug Dev 12, 193-213, 2002 Antisense Nucleic Acid Drug Dev 12, 193-213, 2002].
  • This class of oligonucleotides contains a sulfur atom substituted for a non-bridging oxygene atom at each phosphorus atom in the molecule.
  • Phosphorothioate oligonucleotides are relatively nuclease resistant, and have found extensive use in antisense experiments [Chi K, Wallis AE, Lee CH, De Menezes DL, Sartor J, Dragowska WH, Mayer LD. Effects of Bcl-2 modulation with G3139 antisense oligonucleotide on human breast cancer cells are independent of inherent Bcl-2 protein expression.
  • Another antisense oligodesoxynucleotide currently in evaluation for the treatment of cancer is ISIS3521 [Yuen AR, Halsey J, Fisher GA, Holmlund JT, Geary RS, Kwoh TJ, Dorr A and Sikic Bl, Phase I study of an antisense oligonucleotide to protein kinase C- ⁇ (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 5, 3357-3363, 1999].
  • ISIS3531 is designed to specifically bind to protein kinase ⁇ (PKC ⁇ ) messenger RNA by Watson-Crick base pairing thereby inducing RNAseH mediated degradation of the RNA and functionally blockade of the protein kinase.
  • PKC ⁇ is claimed for the use as a medicament for the treatment of malignant cancer such as lung cancer.
  • Phosphorothioate oligonucleotides containing "CpG” subsequence motifs were recently described to strongly activate cells harbouring the toll-like receptor 9 (TLR9) [Krieg A. From bugs to drugs: Therapeutic immu ⁇ omodulation with oligodesoxynucleotides containing CpG sequences from bacterial DNA. Antisense Nucleic Acid Drug Develop 11, 181-188, 2001] which then induces a variety of cytokines including interferons.
  • TLR9 toll-like receptor 9
  • akira S Mammalian toll-like receptors, Curr Opin Immunol 15, 5-11, 2003
  • human plas- macytoide dendritic cells or B-cells such as human plas- macytoide dendritic cells or B-cells [Dalpke A, Zimmermann S, Heeg K. Immunopharmacology of CpG DNA. Biol Chem 383, 1491-1500, 2002] or cell harboring recombinant TLR9.
  • WO 02/22809 discloses a process for high throughput screening of CpG-based immuno-agonist/antagonist, as a screening procedure using cells expressing functional TLR 7, 8 or 9 either from murine or human systems for identification of IS A (immunostimulatory nucleic acids).
  • the object of present invention was to provide a method for identifying compounds having interferon- like activities useful as compounds for treatment of inflammatory disorders and/or cancer.
  • therapeutics for the treatment of inflammatory disorders and/or cancer can be identified by a method which comprises determining the expression rate of certain genes in eukaryotic cells which genes are equal to or resemble those induced by cytokines, such as interferons.
  • Present invention can be used to identify compounds acting as cytokine mimics in tumor cells and also allows the identification of further optimized compounds possessing this quality.
  • the invention relates in a first aspect to a method for identifying an interferon mimic, comprising the steps of: (a) providing a compound, (b) providing eukaryotic cells, (c) incubating the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, MEN1, IFI16, IRF7, SP110, ARHE, MGC14376, DDX21 , PLSCR1, LOC56902, GADD45B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1, PDE4DIP
  • present invention relates to a method for identifying an interferon mimic, comprising the steps of: (a) providing a compound, (b) providing eukaryotic cells, (c) incubating the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, 1SG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, and MEN1 of cells incubated with the compound, and (e) identifying the compound as an interferon mimic.
  • present invention relates to a method for identifying an interferon mimic, comprising the steps of: (a) providing a compound, (b) providing eukaryotic cells, (c) incubating the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1 of cells incubated with the compound, and (e) identifying the compound as an interferon mimic.
  • present invention relates to a method for identifying an interferon mimic, comprising the steps of: (a) providing a compound, (b) providing eukaryotic cells, (c) transfecting the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig5, STAT1, IFI44, OAS1, IFI27, G1P3, 1SG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, MEN1, IFI16, IRF7, SP110, ARHE, MGC14376, DDX21, PLSCR1 , LOC56902, GADD45B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1 , PDE4DIP, KIAA
  • present invention relates to a method for identifying an interferon mimic, comprising the steps of: (a) providing a compound, (b) providing eukaryotic cells, (c) transfecting the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig5, STAT1, IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1 R15A, IFI35, and MEN1 of cells transfected with the compound, and (e) identifying the compound as an interferon mimic.
  • present invention relates to a method for identifying an interferon mimic, comprising the steps of: (a) providing a compound, (b) providing eukaryotic cells, (c) transfecting the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1 of cells transfected with the compound, and (e) identifying the compound as an interferon mimic.
  • the eukaryotic cells used in the methods subject to present invention are tumor cells.
  • the invention relates in a further aspect to a method for identifying an anti-tumor agent, comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) incubating the tumor cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, MEN1 , IFI16, IRF7, SP110, ARHE, MGC14376, DDX21, PLSCR1, LOC56902, GADD45B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1 , PDE4DIP, KI
  • present invention relates to a method for identifying an anti-tumor agent, comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) incubating the tumor cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig5, STAT1 , IFI44, OASI, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, and MEN1 of tumor cells incubated with the compound, and (e) identifying the compound as an anti-tumor agent.
  • present invention relates to a method for identifying an anti-tumor agent, comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) incubating the tumor cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig5, and STAT1 of tumor cells incubated with the compound, and (e) identifying the compound as an anti-tumor agent.
  • present invention relates to a method for identifying an anti-tumor agent, comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) transfecting the tumor cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of of MX1 , IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig5, STAT1 , IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD46A, PPP1 R15A, IFI35, MEN1, IFI16, IRF7, SP110, ARHE, MGC14376, DDX21, PLSCR1, LOC56902, GADD45B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1 , PDE4DIP
  • present invention relates to a method for identifying an anti-tumor agent, comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) transfecting the tumor cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1 R15A, IFI35, and MEN1 of tumor cells transfected with the compound, and (e) identifying the compound as an anti- tumor agent.
  • present invention relates to a method for identifying an anti-tumor agent, comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) transfecting the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1 , IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1 of tumor cells transfected with the compound, and (e) identifying the compound as an anti-tumor agent.
  • a method for identifying an interferon mimic comprising the steps of: (a) providing a compound, (b) providing eukaroytic cells, (c) transfecting the cells of step b) with the compound of step a), (d) growing the cells transfected with the compound and non-transfected control cells, (e) isolating mRNA from transfected cells and non-transfected control cells of step d), (f) labeling isolated mRNA of step e), (g) hybridizing labeled mRNA of step f) onto DNA microarrays, (h) determining the amount of at least 3 mRNA selected from the group consisting of MX1, IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1 , IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, G
  • a method for identifying an interferon mimic comprising the steps of: (a) providing a compound, (b) providing eukaroytic cells, (c) transfecting the cells of step b) with the compound of step a), (d) growing the cells transfected with the compound and non-transfected control cells, (e) isolating mRNA from transfected cells and non-transfected control cells of step d), (f) labeling isolated mRNA of step e), (g) hybridizing labeled mRNA of step f) onto DNA microarrays, (h) determining the amount of at least 3 mRNA selected from the group consisting of MX1, IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG16, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A,
  • a method for identifying an interferon mimic comprising the steps of: (a) providing a compound, (b) providing eukaroytic cells, (c) transfecting the cells of step b) with the compound of step a), (d) growing the cells transfected with the compound and non-transfected control cells, (e) isolating mRNA from transfected cells and non-transfected control cells of step d), (f) labeling isolated mRNA of step e), (g) hybridizing labeled mRNA of step f) onto DNA microarrays, (h) determining the amount of at least 3 mRNA selected from the group consisting of MX1, IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1 bound to DNA microarrays, (i) comparing the amount of label determined in step h) for the cells transfected with the compound with the amount of
  • a method for identifying an anti-tumor agent comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) transfecting the tumor cells of step b) with the compound of step a), (d) growing the tumor cells transfected with the compound and non-transfected control cells, (e) isolating mRNA from transfected tumor cells and non- transfected control cells of step d), (f) labeling isolated mRNA of step e), (g) hybridizing labeled mRNA of step f) onto DNA microarrays, (h) determining the amount of at least 3 mRNA selected from the group consisting of MX1, IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP
  • a method for identifying an anti-tumor agent comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) transfecting the tumor cells of step b) with the compound of step a), (d) growing the tumor cells transfected with the com- pound and non-transfected control cells, (e) isolating mRNA from transfected tumor cells and non- transfected control cells of step d), (f) labeling isolated mRNA of step e), (g) hybridizing labeled mRNA of step f) onto DNA microarrays, (h) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, G
  • a method for identifying an anti-tumor agent comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) transfecting the tumor cells of step b) with the compound of step a), (d) growing the tumor cells transfected with the compound and non-transfected control cells, (e) isolating mRNA from transfected tumor cells and non- transfected control cells of step d), (f) labeling isolated mRNA of step e), (g) hybridizing labeled mRNA of step f) onto DNA microarrays, (h) determining the amount of at least 3 mRNA selected from the group consisting of MX1, IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1 bound to DNA microarrays, (i) comparing the amount of label determined in step h) for the tumor cells transfected with the compound with the amount of label determined for
  • the methods for identifying an interferon mimic or an anti-tumor agent as disclosed herein wherein the compound is identified as an interferon mimic or an anti-tumor agent by comparing the amount of mRNA determined in cells incubated or transfected with a compound with the amount of the same mRNA determined in control cells. Accordingly, this may be particularly done by comparing - on the one hand - the expression rate of certain genes in cells incubated with the compound or cells transfected with the compound with - on the other hand - the expression rate of the same genes of control cells.
  • a method for identifying interferon mimics or antitumor agents as disclosed herein wherein the compound is identified as an interferon mimic or as antitumor agent if (1 ) the amount of least 0 mRNA encoded by the genes selected from the group consisting of MX1 , IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OASI, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R16A, IFI35, MEN1, IFI16, IRF7, SP110, ARHE, MGC14376, DDX21, PLSCR1, LOC66902, GADD46B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1 , PDE4DIP, KIAA1049, PCF11, SNA
  • GADD4 ⁇ A PPP1R15A, IFI36, and MEN1 determined in cells incubated/transfected with the compound exceeds the amount of the mRNA of the same genes determined in control cells by at least 3 times, or (3) the amount of least 3 mRNA encoded by the genes selected from the group consisting of MX1, IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1 determined in cells incubated/transfected with the compound exceeds the amount of the mRNA of the same genes determined in control cells by at least 3 times.
  • a high-throughput screening method for identifying an anti-tumor agent comprising a method of identifying an anti -tumor agent as disclosed herein.
  • a high-throughput screening method for identifying an anti-tumor agent large numbers of compounds are screened in parallel for their capability to differentially influence gene expression of certain genes in a tumor cells compared to the expression of the same genes in control cells.
  • a high-throughput screening method for identifying an anti-tumor agent comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) incubating the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG16, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, MEN1, IFI16, IRF7, SP110, ARHE, MGC14376, DDX21, PLSCR1, LOC56902, GADD4 ⁇ B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1, PDE
  • a high-throughput screening method for identifying an anti-tumor agent comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) incubating the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1, IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig5, STAT1, IFI44, OAS1 , IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, and MEN1 of cells incubated with the compound, and (e) identifying the compound as an anti-tumor agent.
  • a high-throughput screening method for identifying an anti-tumor agent comprising the steps of: (a) providing a compound, (b) providing tumor cells, (c) incubating the cells of step (b) with the compound of step (a), (d) determining the amount of at least 3 mRNA selected from the group consisting of MX1 , IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig5, and STAT1 of cells incubated with the compound, and (e) identifying the compound as an anti-tumor agent.
  • a further aspect of present invention relates to a process for optimizing the interferon mimic activity of a compound which process comprises a method for identifying an interferon mimic as disclosed herein.
  • a further aspect of present invention relates to a process for manufacturing a compound which process comprises a method for identifying an interferon mimic or an antitumor agent as disclosed herein.
  • therapeutics for the treatment of inflammatory disorders and/or cancer can be identified by a method comprising the step of determining the expression rate of certain genes in eukaryotic cells.
  • the expression of these genes is equal to or resemble the same expression pattern as known from cells induced by cytokines, such as interferons.
  • present invention can be used to identify compounds acting as cytokine mimics and also allows the identification of further optimized compounds possessing this quality.
  • Present invention is independent of toll-like receptor expression since the carcinoma cells used for the screening do not express TLR7 or TLR9.
  • the present invention is independent of TLR (toll-like receptor) 7, 8 or 9 expression since the carcinoma cells used for the screening do not express TLR7, TLR8, orTLR9.
  • TLR toll-like receptor
  • present invention refers to a method for identifying interferon mimics and/or anti-tumor agents comprising the steps of analyzing gene expression of certain genes and identifying a compound as an interferon mimic and/or as anti-tumor agent as result of the gene expression profiling.
  • interferon mimic refers to a molecule which does not have the same polypeptide structure as an interferon polypeptide but which induces the same or similar cellular responses (induction of interferon-responsive genes, inhibition of cell growth, apoptosis, cell death) in an eukaryotic cell as known for interferons. It is preferred that the interferon mimic may be an oligonucleotide either single stranded or double stranded, or double stranded with hairpin-loop-stem (fold- back, i.e. partially self-complementary) configuration, a polypeptide other than a known interferon or a small chemical molecule.
  • Interferon mimics which may be mentioned as preferred examples are the phosphorothioate oligodeoxynucleotides G3139 (5'-TCTCCCAGCGTGCGCCAT-3'), ISIS3521 (5'- GTTCTCGCTGGTGAGTTTCA-3').
  • the term "compound” refers to a compound as known in art of pharmaceutical business as the active ingredient of a therapeutic agent or medicament.
  • oligonucleotides polypeptides, proteins, or small chemical molecules.
  • anti-tumor agent refers to a "compound” having anti-tumor activity such as interferon- ⁇ , interferon- ⁇ or established cytotoxics such as taxol, etoposide or cisplatin and which may be determined by MTT assay [Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 65, ⁇ -63, 1985].
  • the term "eukaryotic cell” refers to cells of human origin, preferentially to tumor cells of human origin.
  • carcinoma cells originating from tumors of lung, breast, prostate, intestine or bladder may be mentioned as examples.
  • Particular preferred examples are PC3 prostate cancer cells or T24 bladder cancer cells.
  • tumor cell refers to tumor cells of human origin. In particular, it refers to carcinoma cells originating from tumors of lung, breast, prostate, intestine or bladder. Preferred examples are PC3 prostate cancer cells or T24 bladder cancer cells (A549 lung adenocarci- no a cells).
  • control cells is known in the art of cell and molecular biology and refers to the negative control normally used in biological experiments.
  • control cells are eukaryotic cells fulfilling the following conditions: (1 ) the control cells are of the same origin as the cells used for incubation or transfectio ⁇ with the compound, and (2) the control cells are treated under the same conditions as the cells used for incubation or transfection with the compound, and apart from the condition that (3) the control cells are not contacted with the compound.
  • the term "incubation” refers to the situation that eukaryotic cells get together with a compound. In this situation the cells get in a contact with the compound so that the compound either is incorporated into the cells or linked to a cellular receptor of the cells. Such a situation may be possible by transfecting eukaryotic cells with a compound.
  • transfection refers to the uptake of oligonucleotides into eukaryotic cells either facilitated by certain chemical agents or devices or spontaneously (e.g. in certains blood cells as B cells or monocytes).
  • Facilitated uptake includes the use of lipids as carrier molecules, in particular cationic lipids, e.g. Lipofectin, Lipofectamine 2000 and Oligofectamine from Invitrogen (Grand Island, NY) or Argfectin-50 and ⁇ -Argfectin-40 from atugen (Berlin, Germany).
  • Facilitated uptake also includes e.g. mechanical methods (cell scraping), electroporation and so-called nucleofection using the system from A axa (Cologne, Germany).
  • labeling refers to the modification of a nucleic acid pool, which represents the total expressed mRNA of a certain biological sample, by chemical or enzymatic reaction with a certain chemical group or isotope to allow for subsequent detection of specific nucleic acid sequences after hybridization on DNA chips/arrays.
  • labeling refers to the modification of a nucleic acid pool, which represents the total expressed mRNA of a certain biological sample, by chemical or enzymatic reaction with a certain chemical group or isotope to allow for subsequent detection of specific nucleic acid sequences after hybridization on DNA chips/arrays.
  • radioactive isotopes can be incorporated into nucleic acids or cDNAs can be modified with fluorescence groups (e.g. Cy3, Cy5) during reverse transcription of mRNA.
  • Affymetrix detection protocol uses incorporation of biotin into cRNA and subsequent reaction with a complex of sfreptavidin, which binds to biotin with very high affinity, and the fluorescence group phycoerythrin to enable detection with a laser scanner.
  • the term "determining the amount of mRNA” refers to any method known in the art to detect mRNA. Examples which may be mentioned are Northern Blotting, RNAse protection assay, reverse transcription and subsequent PCR (RT/PCR), real-time PCR, such as TaqMan PCR from Applied Biosystems (Weiterstadt, Germany) or LightCycler PCR from Roche Diagnostics (Mannheim, Germany) or the Scorpions technology as claimed in US patent US 6,326,145. Also mentioned as useful methods to determine the amount of mRNA are all variations of DNA chip/array systems, in particular nylon membrane based arrays (e.g. Atlas arrays from BD Biosciences Clontech, Heidelberg, Germany), PCR products/DNA oligonucleotides spotted on glass slides or GeneChips from Affymetrix (Santa Clara, CA).
  • RT/PCR reverse transcription and subsequent PCR
  • real-time PCR such as TaqMan PCR from Applied Biosystems (Weiterstadt, Germany) or LightCycler
  • identifying the compound as an interferon mimic refers to a process to analyze whether a compound induces the same cellular phenotype in eukaryotic cells incubated with the compound as known for eukaryotic cells incubated with an interferon.
  • the effects of the compound may be analyzed by determining the effect of the compound on the expression rate of a set of genes in eukaryotic cells incubated with the compound. For example, if the expression profiles of certain genes - after cells have been incubated with the compound - are comparable with the expression profiles of the same genes - after cells have been incubated with an interferon, then the compound is identified as an interferon mimic.
  • compounds are regarded to be an interferon mimic and/or an anti-tumor agent (depending on the method performed), if at least one of the following conditions is met: at least 3 genes from group i) are induced 3-fold or higher as compared to controls, or at least ⁇ genes from group ii) are induced 3-fold or higher as compared to controls, or at least 10 genes from group iii) are induced 3-fold or higher as compared to controls.
  • a compound is regarded to be an interferon mimic and/or an anti-tumor agent, if at least one of the following conditions is met: (1) at least 3 genes from group i) are induced ⁇ -fold or higher as compared to controls, or (2) at least 5 genes from group ii) are induced 5-fold or higher as compared to controls, or (3) at least 10 genes from group iii) are induced 5-fold or higher as compared to controls.
  • high-throughput screening method is known in the pharmaceutical business and it herein refers to a screening method wherein the interferon mimic activity of several compounds is analyzed in parallel.
  • oligonucleotides can be synthesized as described by Sanghvi YS [Sanghvi YS, Andrade M, Deshmukh RR, Holmberg L, Scozzari AN, Cole DL. Chemical synthesis and purification of phosphorothioate antisense oligonucleotides. In: Manual of Antisense Methodology, eds G.Hartmann and S.Endres, Perspectives in Antisense Science, Series ed CA.
  • the compound After synthesis of the compound the compound is subjected to the method as described herein. Depending on the results, the compound is identified as an interferon mimics and/or as an anti-tumor agent.
  • the compound may be compounded in suitable formulations as known in the art. As an example, the compound may be brought into association with at least one carrier, which constitutes one or more accessory ingredients/excipients.
  • interferons and other cytokines are widely used in the pharmaceutical business as antiviral or cancer therapeutic agents.
  • interferon alpha is used for the treatment of hepatitis B and C infection, human papillomavirus infection, hairy-cell leukemia, and Kaposi's sarcoma (a cancer associated with AIDS), multiple myeloma, malignant melanoma, whereas interferon beta is used in the treatment of multiple sclerosis.
  • interferons used as a medicament suffer from severe side effects if administered systemically - a medicinal problem, which has not been solved up to now.
  • synthesis and purification of cytokines applying recombinant DNA and expression vectors is difficult and costly.
  • interferon- ⁇ so far has not entered therapeutic applications because of problematic physico-chemical properties rendering it inappropriate for pharmaceutical applications. Therefore, the present invention for the first time might allow to circumvent this problem.
  • Present invention refers to a method to analyze whether a compound induces the same cellular phenotype in cells incubated with this compound as known for cells incubated with an interferon.
  • the effects of the compound may be analyzed by determining the effect of the compound on the expression rate of a set of genes listed herewith.
  • the method of present invention allows the identification of new compounds, herein referred to as "interferon mimics", useful in the prevention and/or treatment of diseases currently treated with interferons, but lacking the severe side-effects known for interferons.
  • RNA profiling technologies such as oligonucleotide microarray or real-time PCR that in tumor cells compounds such as oligonucleotides, and G3139 in particular, induce a variety of genes fully independent from their sequence specific antisense mode of action.
  • an in depth cluster analysis of affected genes reveals distinct clusters of genes comprising chemokines, growth arrest genes or interferon response genes.
  • a very similar set of genes is affected by treating the tumor cells by interferons, together with the same cellular phenotype as observed after treatment of the cells with oligonucleotides i.e. inhibition of cellular growth and induction of apoptosis.
  • Fig. 1 TaqMan PCR analysis of PC3 cells treated with G3139.
  • PC 3 cells were transfected with 400 nM G3139 for 5 hours using the cationic lipid Lipofectin (15 ⁇ g/ml), washed and further incubated for additional 67 hours.
  • relative expression of GADD45A, GADD45B, MX1, ISG15, STAT1, OAS1 and OAS2 was analysed by TaqMan PCR using primer and probe sequences ordered from Applied Biosystems (Assays-on- Demand program). Expression data in the figure are presented as the mean of 3 replicates +/- S.D. and normalized to results of untreated control PC3 samples with expression levels arbitrarily set to 1.
  • Fig. 1 shows the strong induction of a selection of seven genes responsive to the treatment of prostate PC3 carcinoma cells for 72 hours by the compound G3139 compared to untreated PC3 cells.
  • PC3 human prostate carcinoma cells and T24 human bladder carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).
  • PC3 cells were grown in RPMI (Invitrogen, Grand Island, NY) and T24 cells were grown in McCoy's 5A medium (Invitrogen) containing 10% (v/v) heat inactivated (66°C) fetal bovine serum supplemented with 1% non-essential amino acids, 1% py- ruvate, 25 mM HEPES, 100 U/ml penicillin G sodium and 100 ⁇ g/ml streptomycin sulfate. Stock cultures were maintained at 37°C in a humidified 5% C0 2 incubator. PC3 cells were not used after the 10-12 th passage.
  • oligonucleotide transfection using the cationic lipid Lipofectin (Invitrogen) cells were seeded the day before the experiment in 6-well plates at a density of 2.5 x 10 5 cells per well, to be 60-70% confluent on the day of the experiment. All transfections were performed in Opti-MEM medium (Invitrogen) as per the manufacturer's instructions. The appropriate quantities of reagents were diluted in 100 ⁇ l of Opti-MEM medium to give a final concentration of 1 ⁇ ⁇ g/ml Lipofectin and 400 nM oligonucleotide for PC3 cell transfection and 10 ⁇ g/ml Lipofectin and 1000 nM oligonucleotide for T24 cell transfection.
  • Opti-MEM medium Invitrogen
  • PC3 cells were grown in RP I medium and seeded at 2.5 x 10 5 cells per well in 6- well plates. Cells were treated with 250 units/ml of interferon- ⁇ 1 a or 50 units/ml of interferon ⁇ for 24 or 72 hours followed by isolation of total cellular RNA using RNeasy Kit (Qiagen).
  • RNA isolated from treated PC3 or T24 cells was profiled on GeneChips (Affymetrix U95A chip) according to standard protocols provided by Affymetrix (Santa Clara, CA).
  • Double-stranded cDNA was synthesized with an oligo-dT primer containing a T7 promoter using the Superscript Choice Double- Stranded cDNA Synthesis Kit (Invitrogen).
  • Biotin-labeled cRNA was synthesized from the cDNA by employing T7 RNA polymerase using the BioArray High Yield RNA Transcript Labeling Kit (Enzo, Farmingdale, NY).
  • This cRNA was precipitated with ethanol, purified by the RNeasy protocol (Qiagen), and then fragmented at 94°C, pH 8.1, in the presence of Mg 2+ . Completion of fragmentation was defined when the fragments, as determined by gel electrophoresis, were between 35-200mers in length.
  • Hybridization on an Affymetrix U9 ⁇ A GeneChips was performed for 16 hours at 45°C, after which the chips were placed in the Affymetrix GeneChip Fluidics Station 400.
  • SAPE streptavidin-phycoerythrin
  • Real-time TaqMan PCR amplification of cDNA was performed on an ABI Prism 7900HT sequence detection system (Applied Biosystems, Rothstadt, Germany) using AmpliTaq Gold DNA polymerase.
  • TaqMan assays containing forward primer, reverse primer and minor groove binder (MGB)-labeled probes for GADD46A, GADD46B, MX1, ISG1 ⁇ , STAT1, OAS1 and OAS2 were ordered from Applied Biosystems (Assays-on-Demand program). For normalization of RNA concentrations, TaqMan PCR analysis of the 18S ribosomal RNA was employed.
  • PC3 cells were transfected with the oligonucleotides G3139 and ISIS3521. Non-transfected PC3 cells were grown in parallel as controls. All experiments were done in triplicates. mRNA was isolated 72h after transfection, labeled, and hybridzed onto Affymetrix human DNA M icroarrays. After hybridization, arrays were scanned using Affymetrix, and DAT files were generated by Affymetrix MAS 5.0 software. Gene expression levels were calculated from DAT files using GeneData Refiner 1.0 software. The mean expression level of triplicates was calculated. Genes were selected that show consistently increased expression after transfection with oligonucleotides as compared to controls.
  • T24 cells were transfected with ISIS3521. Non-transfected control cells were grown in parallel. Trans- fections and controls were done in triplicates. RNA was isolated 24h after transfection. Expression of marker genes was determined by Affymetrix DNA chip technology (U95A). Expression levels of triplicates were averaged. For each gene, the mean expression in transfected cells was divided by the mean control level to obtain oligonucleotide vs. control ratios. The results are shown in Tables 4-6.
  • the mRNA levels in T24 cells 24 hours after transfection with ISIS3521 were determined applying Affymetrix Genechip technology for the genes MX1 , IFIT1 , W28948 (ESTs), IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1, and compared with the mRNA levels of the same genes in T24 cells grown in parallel without transfection.
  • the ratios of mRNA levels in ISIS3521 transfected cells versus untreated controls were determined for each gene.
  • ISIS3521 fulfills our criteria for being an interferon mimic, which is to induce three or more genes out of the 10 genes in group i) at least five fold. This alone is sufficient to regard ISIS3521 an interferon mimic.
  • Table 4 Gene expression results of group i) genes analysed by Affymetrix GeneChips using RNA isolated from T24 cells after transfection with ISIS3521.
  • the mRNA levels in T24 cells 24 hours after transfection with ISIS3521 were determined applying Affymetrix Genechip technology for the genes MX1, IFIT1, IFITM1, OAS2, TNFAIP3, C1orf29, OASL, cig5, STAT1, IFI44, OAS1, IFI27, G1P3, ISG1 ⁇ , SFRS6, 1FIT4, MX2, ID2, BST2, GADD46A, PPP1 R15A, IFI35, and MEN1 and compared with the mRNA levels of the same genes in T24 cells grown in parallel without transfection. The ratios of mRNA levels in ISIS3521 transfected cells versus untreated controls were determined for each gene.
  • ISIS3521 fulfills our criteria for being an interferon mimic, which is to induce five or more genes out of the 2 ⁇ genes in group ii) at least five fold. This alone is sufficient to regard ISIS3 ⁇ 21 an
  • Table 5 Gene expression results of group ii) genes analysed by Affymetrix GeneChips using RNA isolated from T24 cells after transfection with ISIS3521.
  • the mRNA levels in T24 cells 24 hours after transfection with ISIS3521 were determined applying Affymetrix Genechip technology for the genes MX1 , IFIT1 , IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1 , IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD4 ⁇ A, PPP1R15A, IFI36, MEN1, IFI16, IRF7, SP110, ARHE, MGC14376, DDX21, PLSCR1, LOC ⁇ 6902, GADD45B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1, PDE4DIP, KIAA1049, PCF11, SNAPC1, CDC2L2, UBE2L6, ASNS, and HLA-C and compared with the mRNA levels of the same genes in T24 cells grown in
  • ISIS3521 fulfills our criteria for being an interferon mimic, which is to induce ten or more genes out of the 50 genes in group iii
  • Table 6 Gene expression results of group iii) genes analysed by Affymetrix GeneChips using RNA isolated from T24 cells after transfection with ISIS3521.
  • PC3 cells were transfected with G3139. Non-transfected control cells were grown in parallel. Transfec- tions and controls were done in triplicates. RNA was isolated 72h after transfection. Expression of marker genes was determined by Affymetrix DNA chip technology (U9 ⁇ A).
  • the mRNA levels in PC3 cells 72 hours after transfection with G3139 were determined applying Affymetrix Genechip technology for the genes MX1 , IFIT1 , W28948 (ESTs), IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , and STAT1 , and compared with the mRNA levels of the same genes in PC3 cells grown in parallel without transfection.
  • the ratios of mRNA levels in G3139 transfected cells versus untreated controls were determined for each gene.
  • the mRNA levels in PC3 cells 72 hours after transfection with G3139 were determined applying Affymetrix Genechip technology for the genes MX1 , IFIT1, IFITM1 , OAS2, TNFAIP3, C1 ⁇ rf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG1 ⁇ , SFRS6, IFIT4, MX2, ID2, BST2, GADD45A, PPP1R15A, IFI35, and MEN1 and compared with the mRNA levels of the same genes in PC3 cells grown in parallel without transfection. The ratios of mRNA levels in G3 39 transfected cells versus untreated controls were determined for each gene.
  • G3139 fulfills our criteria for being an interferon mimic, which is to induce five or more genes out of the 2 ⁇ genes in group ii) at least five fold. This alone is sufficient to regard G3139 an interferon mimic.
  • Table 8 Gene expression results of group ii) genes analysed by Affymetrix GeneChips using RNA isolated from T24 cells after transfection with G3139. Wilcoxon signed rank test G3139 vs. C20 Affymetrix: p ⁇ 0.0001
  • the mRNA levels in PC3 cells 72 hours after transfection with G3139 were determined applying Affymetrix Genechip technology for the genes MX1, IFIT1, IFITM1 , OAS2, TNFAIP3, C1orf29, OASL, cig ⁇ , STAT1, IFI44, OAS1, IFI27, G1P3, ISG15, SFRS6, IFIT4, MX2, ID2, BST2, GADD46A, PPP1R15A, IFI35, MEN1, IFI16, IRF7, SP110, ARHE, MGC14376, DDX21, PLSCR1, LOC ⁇ 6902, GADD46B, STC2, ATF3, KIAA0170, Tls/Chop, HNRPA1, PDE4DIP, KIAA1049, PCF11, SNAPC1, CDC2L2, UBE2L6, ASNS, and HLA-C and compared with the mRNA levels of the same genes in PC3 cells grown in parallel without transfection.
  • G3139 fulfills our criteria for being an interferon mimic, which is to induce ten or more genes out of the 60 genes in group iii) at least five fold. This alone is sufficient to
  • Table 9 Gene expression results of group iii) genes analysed by Affymetrix GeneChips using RNA isolated from T24 cells after transfection with G3139.

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Abstract

L'invention concerne une méthode d'identification de substances mimétiques d'interférons.
PCT/EP2004/052550 2003-10-15 2004-10-14 Methode d'identification de substances mimetiques d'interferons WO2005038052A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007032743A2 (fr) * 2005-09-16 2007-03-22 Biovator Technologies Ab Analyse in vitro pour identifier des proteines allergenes
WO2012076846A1 (fr) * 2010-12-10 2012-06-14 The University Of Nottingham Polymorphisme
CN104975011A (zh) * 2015-06-30 2015-10-14 山东省农业科学院畜牧兽医研究所 一种检测鸡体抗病毒蛋白的试剂盒及应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US6331396B1 (en) * 1998-09-23 2001-12-18 The Cleveland Clinic Foundation Arrays for identifying agents which mimic or inhibit the activity of interferons
WO2003006627A2 (fr) * 2001-07-13 2003-01-23 Whitehead Institute For Biomedical Research Facteurs de transcription leucemigenes
WO2004069860A2 (fr) * 2003-02-03 2004-08-19 The Scripps Research Institute Proteines conjuguees a isg15

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US6331396B1 (en) * 1998-09-23 2001-12-18 The Cleveland Clinic Foundation Arrays for identifying agents which mimic or inhibit the activity of interferons
WO2003006627A2 (fr) * 2001-07-13 2003-01-23 Whitehead Institute For Biomedical Research Facteurs de transcription leucemigenes
WO2004069860A2 (fr) * 2003-02-03 2004-08-19 The Scripps Research Institute Proteines conjuguees a isg15

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007032743A2 (fr) * 2005-09-16 2007-03-22 Biovator Technologies Ab Analyse in vitro pour identifier des proteines allergenes
WO2007032743A3 (fr) * 2005-09-16 2007-06-14 Biovator Technologies Ab Analyse in vitro pour identifier des proteines allergenes
WO2012076846A1 (fr) * 2010-12-10 2012-06-14 The University Of Nottingham Polymorphisme
CN104975011A (zh) * 2015-06-30 2015-10-14 山东省农业科学院畜牧兽医研究所 一种检测鸡体抗病毒蛋白的试剂盒及应用

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