WO2008063773A9 - Traitement combinatoire avec les molécules inhibitrices du récepteur du facteur de croissance épidermique et le gène-7 associé à la différenciation des mélanomes - Google Patents

Traitement combinatoire avec les molécules inhibitrices du récepteur du facteur de croissance épidermique et le gène-7 associé à la différenciation des mélanomes

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Publication number
WO2008063773A9
WO2008063773A9 PCT/US2007/080962 US2007080962W WO2008063773A9 WO 2008063773 A9 WO2008063773 A9 WO 2008063773A9 US 2007080962 W US2007080962 W US 2007080962W WO 2008063773 A9 WO2008063773 A9 WO 2008063773A9
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nucleic acid
mda
protein
molecule
cancer
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PCT/US2007/080962
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WO2008063773A3 (fr
WO2008063773A2 (fr
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Jeffrey Settleman
Paul B Fisher
Luni Emdad
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Univ Columbia
Jeffrey Settleman
Paul B Fisher
Luni Emdad
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Publication of WO2008063773A2 publication Critical patent/WO2008063773A2/fr
Publication of WO2008063773A9 publication Critical patent/WO2008063773A9/fr
Publication of WO2008063773A3 publication Critical patent/WO2008063773A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the combined use of (i) a molecule which inhibits the Epidermal Growth Factor Receptor and (ii) a Melanoma Differentiation Associated Gene-7 molecule in methods which inhibit the proliferation and/or survival of cancer cells.
  • mda-7 Melanoma differentiation associated gene-7 (mda-7) (Jiang et al., 1995) is a secreted cytokine belonging to the interleukin (IL)-IO family designated as IL-24 (Pestka et al., 2004). Multiple independent studies demonstrate that delivery of mda-7/IL-24 by a replication incompetent adenovirus, Ad.mda-7, or as a GST-MD A- 7 fusion protein selectively kills diverse cancer cells.
  • IL-7 interleukin-IO family designated as IL-24 (Pestka et al., 2004).
  • mda-7/IL-24 does not induce toxicity in normal endothelial and epithelial cells, fibroblasts, melanocytes and astrocytes (Ekmekcioglu et al., 2001; Ellerhorst et al., 2002; Fisher, 2005; Fisher et al., 2003; Gupta et al., 2006; Huang et al., 2001; Jiang et al., 1996; Lebedeva et al., 2005; Lebedeva et al., 2002; Madireddi et al., 2000b; Mhashilkar et al., 2001; Saeki et al., 2000; Saeki et al., 2002; Sarkar et al, 2006; Sarkar et al, 2002a; Sarkar et al, 2002b; Sauane et al, 2003; Su et al, 2001; Su et al., 1998).
  • mda-7/IL-24 possesses potent anti-angiogenic, immunostimulatory and bystander activities (Fisher, 2005; Fisher et al., 2003; Gupta et al., 2006; Lebedeva et al., 2005).
  • the sum of these attributes makes mda-7/IL-24 a significant candidate for cancer gene therapy (Fisher, 2005).
  • Ad.mda-7 has been successfully used for a Phase I clinical trial for advanced carcinomas and melanomas and has shown promising results in tumor growth inhibition and induction of cancer apoptosis (Cunningham et al., 2005; Fisher, 2005; Fisher et al., 2003; Lebedeva et al., 2005; Tong et al., 2005).
  • Ad.mda-7 induces growth suppression and apoptosis in histologically diverse cancer cells containing single or multiple genetic defects, including alterations in p53, pl6/INK4a and/or Rb (Emdad et al., 2006; Huang et al., 2001; Jiang et al., 1996; Lebedeva et al., 2002; Lebedeva, 2006; Mhashilkar et al., 2001; Saeki et al., 2000; Su et al., 2001; Su et al., 1998).
  • Ad.mda-7 is equally effective in inducing apoptosis in breast and lung carcinoma and melanoma cells containing wtp53, mutp53 or which are null for p53 expression (Lebedeva et al., 2002; Madireddi et al., 2000b; Saeki et al., 2000; Saeki et al., 2002; Su et al, 1998).
  • M4 a shorter version of MDA-7, referred to as M4, containing amino acid residues 104-206 of the wild type sequence, displays similar cancer-specific apoptosis inducing properties as the complete molecule (Gupta et al., 2006b; U.S. Patent Application Publication No. 20060292157).
  • EGFR Epidermal Growth Factor Receptor
  • MAbs monoclonal antibodies
  • gefitinib One EGFR inhibitor currently being evaluated in clinical phase II/III trials in cancer patients is gefitinib (having the tradename Iressa®).
  • Gefitinib is an orally active selective reversible inhibitor of the EGFR tyrosine kinase. Growth inhibiting activity of gefitinib has been demonstrated in vitro and in vivo in tumor models that express functional EGFRs, including prostate, breast, ovarian, colon, small cell and non- small cell lung cancers (Ciardiello and Tortora, 2001). However, in contrast to initial expectations, recent data suggests that the EGFR protein expression level does not a priori determine cancer cell sensitivity to EGFR targeted therapies (Sirotnak, 2003).
  • erlotinib Another EGFR inhibitor, erlotinib (Tarceva® OSI-774) has been shown to have potent antitumor effects against NSCLC (Haber and Settleman, 2005; Kwak et al., 2005; Shepherd et al., 2005).
  • Erlotinib is a selective, orally available low molecular weight inhibitor that binds competitively to the ATP -binding site at the kinase domain of EGFR.
  • Erlotinib is approved in the US, Europe and several other countries as a monotherapy for the treatment of patients with locally advanced or metastatic NSCLC after failure of chemotherapy .
  • EGFR mutations may also confer additional resistance to erlotinib and gefitinib in terms of response and thus may account for limited efficacy observed in some clinical trials (Gatzemeier et al., 2004; Haber and Settleman, 2005; Herbst et al., 2004; Kwak et al., 2005).
  • One of the main reasons undermining the success of EGFR inhibitors in the first generation of clinical trials of targeted therapies in lung cancer is the complexity of changes occurring during cancer development and progression (Fisher, 1984.; Leszczyniecka et al., 2001).
  • the present invention relates to methods of treating a cancer associated with aberrant EGFR-activity comprising administering, to a subject in need of such treatment, an effective amount of a combination of an EGFR inhibitor and a MD A-7 molecule.
  • the combined use of these agents allows effective therapy of cancers that may be relatively resistant to either agent alone.
  • the EGFR inhibitor is a EGFR tyrosine kinase inhibitor.
  • the MD A-7 molecule is a nucleic acid, for example, but not limited to, a nucleic acid encoding wild-type MD A-7 protein (SEQ ID NO:2) or a functional equivalent thereof, such as the wild-type protein absent its signal sequence or a truncated molecule, such as M4 (amino acids 104 to 206 of SEQ ID NO:2).
  • the MD A-7 molecule is a protein, for example, but not limited to, a wild-type MD A-7 protein (SEQ ID NO:2) or a functional equivalent thereof, such as the wild-type protein absent its signal sequence or a truncated molecule, such as M4 (amino acids 104 to 206 of SEQ ID NO:2).
  • SEQ ID NO:2 wild-type MD A-7 protein
  • M4 truncated molecule
  • combined therapy using an MD A-7 molecule and an EGFR inhibitor may be used to treat either non-small cell lung cancer (NSCLC) or pancreatic cancer. 4.
  • NSCLC non-small cell lung cancer
  • FIG. IA-B GFP, and MDA-7/IL-24, protein expression following infection of human NSCLC cells with adenoviruses.
  • A Variable infectivity of NSCLC cells by adenovirus as determined by GFP expression. The indicated cells were infected with the indicated pfu/cell of Ad.GFP, and 24 h after infection GFP expression was analyzed by FACS analysis.
  • B Infection of NSCLC cells with Ad.mda-7 results in variable amounts of intracellular MDA-7/IL-24 protein. Cells were infected with the indicated concentrations of Ad.mda-7 virus and total cell lysates were prepared 48 h after infection. The expression of MDA-7/IL-24 and EF l ⁇ proteins were evaluated by Western blot analysis.
  • FIG. 2A-B Effect of infection of NSCLC cells with Ad.mda-7 on cell viability and apoptosis induction.
  • A Infection with Ad.mda-7 results in a decrease in cell viability in NSCLC cells. Cells were infected with the indicated viruses and cell viability was assessed by MTT assay at 5 d post-infection. A significant decrease in cell viability was evident in the H-2030 and H- 1650 cell lines. Results are the average from at least three experiments + S. D.
  • FIG. 3A-B Infection with Ad.mda-7 induces upregulation of PKR and PARP cleavage.
  • A Cells were infected with the indicated viruses, total cellular extracts were prepared at 48 h postinfection and the levels of PKR and EF l ⁇ were analyzed by Western blot analysis.
  • B Cells were infected with the indicated viruses and total cellular extracts were prepared at 48 h postinfection. Western Blot analysis was performed with the antibodies against PARP and EF l ⁇ . Arrowhead indicates the cleaved fragment of PARP.
  • FIG. 4A-B Combination of Ad.mda-7 plus gef ⁇ tinib induces enhanced killing and apoptosis in NSCLC cells.
  • A Effect of single and combination treatment with Ad.mda-7 and gef ⁇ tinib on cell viability in human NSCLC cells. The indicated cell type was infected with either Ad.vec, or Ad.mda-7 and either untreated or treated with the indicated concentrations of gefitinib. Cell viability was assessed by MTT assay at 5 d post-infection. Results are the average from at least three experiments + S. D.
  • FIG. 5A-C Combination treatment with Ad.mda-7 and gefitinib increases MDA-7/IL-24 and PKR protein expression, and treatment with siRNA targeting PKR partially protects NSCLC cells from combination treatment of Ad.mda-7 plus gefitinib-mediated apoptosis.
  • NSCLC cells were infected with Ad.vec or Ad.mda-7, followed by treatment with gefitinib at the indicated concentrations. At 48 h after infection, the cells were harvested and immunoblotted for MDA-7/IL-24 protein expression An increase in MDA-7/IL-24 protein expression was observed in cells that were treated with Ad.mda-7 and gefitinib compared with cells that were infected with Ad.mda-7.
  • MDA-7/IL-24 protein expression was not observed in Ad.vec-infected or gefitinib-treated cells. EFlwas used as a protein loading control.
  • B) NSCLC cells were infected with Ad.vec or Ad.mda-7 as described in A, and immunoblotted for PKR. Increased PKR expression was evident, following combination treatment of Ad.mda-7 and gefitinib.
  • FIG. 6 Effect of the combination of Ad.mda-7 and gefitinib on EGFR downstream signaling.
  • NSCLC cells were infected with either Ad.vec or Ad.mda-7 and either untreated or treated with the indicated concentrations of gefitinib.
  • Forty-eight h after infection cells were stimulated with EGF (30 ng/ml) for 15 min and harvested for Western Blot Analysis.
  • Western Blot analysis was performed with antibodies for p-EGFR, p-ERK, p-AKT, total EGFR, total ERK and total AKT.
  • Figure 7 Table 1 : Determination of CAR receptors in human NSCLC cells using monoclonal anti-CAR antibodies and flow cytometry.
  • FIG. 8 Purification of recombinant GST, GST-MD A-7 and GST-M4 proteins. Fusion proteins were expressed after induction with ImM IPTG in E. coli strain BL21. Large scale preparations of bacterial cultures were made and purification of recombinant fusion proteins were performed as described in Materials and Methods. Purified proteins were separated on SDS-PAGE and stained with Coomassie blue dye. GST protein is ⁇ 25-kDa, GST-MD A-7 is ⁇ 45-kDa and GST-M4 is ⁇ 35-kDa. Figure 9A-D. Recombinant GST-MD A-7 and GST-M4 induce apoptotic cell death in various NSCLC cancer cells.
  • (A) H460, (B) H- 1975, (C) H- 1650 and (D) H-446 cells were seeded in 6-well plates at a density of 2 x 10 5 /well and the next day cells were treated with varying concentrations of GST-MD A-7 or GST- M4 ranging from 0.1 ⁇ M to 2 ⁇ M. Similar concentrations of GST were used as a negative control. Twenty h later, cells were trypsinized, washed twice with PBS and stained with allophycocyanin (APC) labeled Annexin-V (BD Biosciences Pharmingen, San Diego, CA) and analyzed by flow cytometry. The amount of apoptotic cells was quantified using the Flow Jo 6.3.1 program.
  • APC allophycocyanin
  • FIG. 1 Figure lOA-C.
  • Panel A EGFR inhibitor erlotinib induces apoptotic cell death in various NSCLC cancer cells.
  • H-1975, H-460, H-1650 and H-446 cells were seeded in 6-well plates at a density of 2 x 105/well and the next day cells were treated with varying concentrations of erlotinib ranging from 0.2 ⁇ M to 40 ⁇ M. Twenty h later, the cells were trypsinized, washed twice with PBS and stained with allophycocyanin (APC) labeled Annexin-V and analyzed by flow cytometry.
  • Panel B and Panel C combinatorial effect of erlotinib and recombinant GST-M4 and GST-
  • MD A-7 proteins were, respectively, on NSCLC cells. 2 x 10 5 /well were plated and next day cells were treated alone or in combination with varying concentrations of Tarceva ranging from 10 ⁇ M to 40 ⁇ M and recombinant GST fusion proteins GST-M4 or GST-MD A-7 at a concentration of 0.1 ⁇ M. The amount of apoptotic cells was quantified using the Flow Jo 6.3.1 program.
  • Figure 1 IA-B Combinatorial action of recombinant GST-MD A-7 or GST-M4 and erlotinib in the colony formation assay and MTT assay.
  • Panel A, H-460, H-1975, H-1650 and H- 446 NSCLC cell lines (1 x 10 3 cells) were treated either with recombinant proteins (0.1 ⁇ M) alone or in combination with EGFR inhibitor erlotinib (10 ⁇ M) and allowed to form colonies for 2 weeks. Media containing protein and erlotinib was replaced every 3-4 days. Colonies > 50 cells were counted and plotted.
  • Panel B Combination of GSTMD A-7 or GST-M4 and erlotinib significantly reduces cell viability of NSCLC cells as quantified by MTT assay.
  • the indicated cell type was seeded (1.5 x 103cells/ well) in 96-well plates and treated with the indicated agents as in colony formation assays. Six days after treatment, cell viability was assessed by MTT assay and plotted.
  • Figure 12A-D A combinatorial effect of recombinant GST-MDA-7 or GST-M4 plus erlotinib on the EGFR signaling pathway in NSCLC cells.
  • the indicated cell types were seeded (2 x 10 6 ) in 60-mm dishes and the next day treated with recombinant proteins alone or in combination with erlotinib for 24 h.
  • Cells were treated with EGF (15 ng/ml) for 15 min and lysed in RIPA buffer and equal amounts of proteins were separated on 8 and 12 % SDS-PAGE and transferred onto nitrocellulose membrane followed by probing with phospho-EGFR and total EGFR in Panel A, phospho-AKT and total AKT in Panel B, and phospho-ERK in Panel C and total ERK in Panel D.
  • FIG 13A-B Treatment with recombinant GST-MDA-7 or GST-M4, but not erlotinib, activated the p38 MAPK signaling pathway.
  • the indicated cell types were seeded (2 xlO 6 ) in 60-mm dishes and next day treated with recombinant proteins alone or in combination with erlotinib for 24 h. Cells were lysed and checked for phospho-p38 (Panel A) and total p38 (Panel B) expression by Western blot analysis.
  • Figure 14 Recombinant GST-MDA-7 or GST-M4 induced apoptotic cell death in pancreatic cancer cells. Cells were seeded in 6-well plates and the next day cells were treated with the indicated concentrations of GST, GST-MD A7 or GST- M4. An Annexin V binding assay was performed 48 hours after the treatments.
  • Figure 15 Dose response of erlotinib in pancreatic cancer cells. Cells were seeded in 96-well plates and the next day the cells were treated with the indicated concentrations of erlotinib. Cell viability was assessed by MTT assay.
  • FIG 16A-B Combinatorial treatment of GST-MDA-7 or GST-M4 and erlotinib significantly increased apoptosis induction and decreased cell viability in pancreatic cancer cells.
  • Cells were treated either with recombinant proteins (100 nM) alone or in combination with erlotinib (5 ⁇ M).
  • Figure 17A-D Combinatorial effect of recombinant GST-MDA-7 or
  • GST-M4 plus erlotinib modulates the EGFR signalling pathway and induced vimentin cleavage in pancreatic cancer cells.
  • the indicated cell types were seeded in 60 mm dishes and the next day treated with recombinant proteins alone (100 nM) or in combination with erlotinib (5 ⁇ M) for 48 hours.
  • Western blot analysis was performed with the indicated antibodies.
  • C. Caspase-3 inhibitors significantly blocked combination treatment mediated apoptosis induction as indicated by annexin V binding assays.
  • FIG. 18A-C Vimentin cleavage induced by combination treatment of recombinant GST-MD A- 7 or GST-M4 plus erlotinib is caspase-3 dependent.
  • AsPC-I cells were seeded in 6 well plates or a 4 well chamber slide and the next day transfected with either control siRNA or caspase 3 siRNA for 48 hours. Then they were treated with recombinant proteins alone or in combination with erlotinib for 24 hours.
  • A. Apoptosis induction was analysed by the annexin V binding assay.
  • B. Western blot analysis was performed with the indicated antibodies.
  • C. Immunofluorescence staining shows vimentin staining (red). Nuclear morphology was shown by DAPI staiing (blue). Arrows indicate apoptotic nuclei.
  • MDA-7 5 1 MDA-7 MOLECULES
  • An MDA-7 molecule is, as defined herein, either a nucleic acid encoding a MDA-7 protein or a MDA-7 protein. 5.1.1 MDA-7 PROTEIN
  • a MDA-7 protein is a protein (which may or may not be glycosylated or otherwise chemically modified) which is structurally and functionally substantially related to wild-type human MDA-7 protein having SEQ ID NO:2, which induces apopotosis of FO-I and MeWo melanoma cells (Sarkar et al., 2002b). M4 has been shown to have apoptosis-promoting activity in DU- 145 prostate cancer cells (Gupta et al., 2006b).
  • One non- limiting example of a MDA-7 protein for use according to the invention is the wild-type human MDA-7 protein having the amino acid sequence set forth as SEQ ID NO:2, Genbank Accession Number U16261.
  • MDA-7 protein for use according to the invention is a protein, the amino acid sequence of which is at least 90 percent or at least 95 percent homologous to the wild type human MDA-7 protein having SEQ ID NO:2, and which exhibits apopotosis-inducing activity against FO-I and/or MeWo and/or DU- 145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • Homology may be determined by standard homology-determining programs, such as BLAST or FASTA.
  • MDA-7 protein for use according to the invention is M4, which has an amino acid sequence consisting essentially of amino acid residues 104 to 206 of SEQ ID NO:2.
  • 104 to 206 means the sequence set forth in SEQ ID NO:2 from amino acid 104 through amino acid 206, inclusive.
  • Another non-limiting example of a MDA-7 protein for use according to the invention is a protein, the amino acid sequence of which is at least 90 percent or at least 95 percent homologous to M4 (the amino acid sequence of which is residues 104 to 206 of SEQ ID NO:2), and which induces apopotosis of FO-I and/or MeWo and/or DU-145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 protein for use according to the invention is a protein comprising amino acids 49 to 206 of SEQ ID NO:2 but lacking, either because they are absent or through substitution, amino acids 1-48 of SEQ ID NO:2, which is the secretory peptide.
  • MDA-7 protein for use according to the invention is a protein, the sequence of which is at least 90 percent or at least 95 percent homologous to amino acids 49 to 206 of SEQ ID NO:2 but lacking, either because they are absent or through substitution, amino acids 1 to 48 of SEQ ID NO:2, which is the secretory peptide, where said protein induces apopotosis of FO-I and/or MeWo and/or DU-145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 proteins which may be used according to the invention are set forth in United States Patent Application Publication No. 20060292157 by Fisher and Gupta, filed December 2, 2005, SN 11/292571.
  • a MDA-7 protein of the invention may comprise or be linked to a molecule that facilitates its biological activity.
  • a molecule may be a secretory signal peptide; where a nucleic acid encoding a MDA-7 protein is introduced into a cell, said secretory peptide would facilitate the secretion of the MDA-7 protein so as to produce a "bystander" effect (Su et al. Proc Natl Acad Sci U S A. 2001; 98:10332-10337).
  • the secretory peptide may be the secretory peptide of wild-type MDA-7 (i.e., residues 1-48), or another naturally occurring or synthetic secretory peptide e.g.
  • the molecule may facilitate cell or tissue compartmentalization; e.g., the molecule may be a KDEL peptide that would favor retention of the variant in the endoplasmic reticulum, or the molecule may facilitate passage across a cell membrane, into the nucleus or through the blood brain barrier.
  • FFAT motif a membrane targeting determinant found in several apparently unrelated lipid binding proteins (Loewen et al., EMBO J. 2003, 22: 2025-2035) may be used to facilitate targeting to the cell membrane.
  • the 15-residue targeting motif of cAMP-dependent protein kinase anchoring protein (d- AKAPI) which targets proteins to either ER or mitochondria depending on interaction with each organelle (Ma and Taylor, J Biol Chem. 2002, 277: 27328-27336) may be used for targeting to both these organelles simultaneously.
  • a MDA-7 protein of the invention may comprise a protein lacking an amino-terminal secretory signal sequence.
  • a MDA-7 protein lacking a secretory peptide may contain an exogenously added N-terminal methionine residue or alternatively start with the glutamine or glycine residues at positions 49 or 50 respectively of the unprocessed native protein (SEQ ID NO:2).
  • the present invention also provides for signal peptide lacking MDA-7 protein containing any additional modification as set forth below, to improve stability or activity.
  • a MDA-7 protein of the invention may comprise elements or be linked to elements that improve its stability or activity. These modifications include but are not limited to N-terminal acetylation or C-terminal amidation, incorporation of D- amino acids or unnatural amino acids including but not limited to ⁇ -alanine, ornithine, hydroxyproline; or substitution at the peptide termini with biotin or long chain alkanes; addition of certain side chain modifications including but not limited to phosphorylation of serine, threonine or tyrosine residues; cyclisation via intramolecular disulphide bond formation; and formation of cyclic amides or radioconjugates.
  • Stabilization of the peptide or protein may be further achieved by, as non- limiting examples, utilization of matrices that enhance delivery, increase stability or achieve controlled release rate such as natural and synthetic biopolymers and cell responsive matrices (Zisch et al., 2003, Cardiovasc Pathol 12: 295-310), or alginate microcapsules (Schneider et al., 2003, J Microencapsul 20:627-636).
  • the MDA-7 protein of the invention may be produced by any method known in the art. Such methods include but are not limited to chemical synthesis and recombinant DNA techniques. With regard to production of MDA-7 variants using recombinant DNA techniques, the present invention provides for nucleic acids encoding said variants. Such nucleic acids may either be nucleic acid fragments of the aforelisted mda-1 nucleic acids encoding the variants, or may be nucleic acids designed, using the genetic code, to encode such variants.
  • an MDA-7 protein of the invention may be comprised in a fusion protein, for example, a GST-fusion, as set forth in the examples sections below.
  • a MDA-7 molecule for use according to the invention may be a nucleic acid encoding a MDA-7 protein, as described above.
  • MDA-7 molecule is a nucleic acid as set forth in SEQ ID NO:1 (GenBank Accession No. U16261; Jiang et al., 1995, Oncogene 11 :2477-2486).
  • MDA-7 molecule is a nucleic acid, the sequence of which is at least 90 percent or at least 95 percent homologous to SEQ ID NO:1 (where homology may be determined using standard programs such as BLAST or FASTA, and see Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), and which encodes a protein that induces apopotosis in FO-I and/or MeWo and/or DU- 145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 Another non-limiting example of such a MDA-7 molecule is a nucleic acid, the entire length of which hybridizes to the entire length of the complement of SEQ ID NO:1 under stringent conditions (as set forth in "Current Protocols in Molecular Biology," Volume 1, Ausubel et al., eds. John Wiley:New York NY pp.
  • a stringent hybridization washing solution may be comprised on 40 mM NaPO4, pH 7.2, 1-2% SDS and 1 mM EDTA), and which encodes a protein that induces apopotosis of FO-I and/or MeWo and/or DU- 145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 molecule is a nucleic acid consisting essentially of that portion of SEQ ID NO:1 from nucleotide 275 to nucleotide 895, which is the coding region.
  • Another non-limiting example of such a MDA-7 molecule is a nucleic acid consisting essentially of a sequence which is at least 90 percent or at least 95 percent homologous to nucleotides 275 to 895 of SEQ ID NO:1 and which encodes a protein that induces apopotosis of FO-I and/or MeWo and/or DU- 145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 molecule is a nucleic acid, the entire length of which hybridizes to the entire length of the complement of nucleotides 275 to 895 of SEQ ID NO:1 under stringent conditions (above) and which encodes a protein that induces apopotosis of FO-I and/or MeWo and/or DU- 145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 molecule is a nucleic acid consisting essentially of that portion of SEQ ID NO:1 from nucleotide 419 to nucleotide 895, which is the portion encoding human wild-type MDA-7 lacking the secretory sequence.
  • a MDA-7 molecule is a nucleic acid which is at least 90 percent or at least 95 percent homologous to nucleotides 419 to 895 of SEQ ID NO:1 and which encodes a protein that induces apopotosis of FO-I and/or MeWo and/or DU-145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 molecule is a nucleic acid, the entire length of which hybridizes to the entire length of the complement of nucleotides 419 to 895 of SEQ ID NO:1, and which encodes a protein that induces apopotosis of FO-I and/or MeWo and/or DU-145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 molecule is a nucleic acid consisting essentially of that portion of SEQ ID NO:1 from nucleotide 584 to nucleotide 895.
  • MDA-7 molecule is a nucleic acid which is at least 90 percent or at least 95 percent homologous to nucleotides 584 to 895 of SEQ ID NO:1, and which encodes a protein that induces apopotosis of FO-I and/or MeWo and/or DU-145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • MDA-7 molecule is a nucleic acid, the entire length of which hybridizes to the entire length of the complement of nucleotides 584 to 895 of SEQ ID NO:1, and which encodes a protein that induces apopotosis of FO-I and/or MeWo and/or DU-145 cancer cells (e.g. in at least 10 percent of cells or in at least 20 percent of cells).
  • an MDA-7 molecule which is a nucleic acid may be comprised within a larger molecule.
  • the MDA-7 nucleic acid may be operably linked to a suitable promoter element, such as, but not limited to, the cytomegalovirus immediate early (CMV) promoter, the Rous sarcoma virus (RSV) long terminal repeat promoter, the human elongation factor l ⁇ promoter, the human ubiquitin c promoter, etc. It may be desirable, in certain embodiments of the invention, to use an inducible promoter.
  • CMV cytomegalovirus immediate early
  • RSV Rous sarcoma virus
  • Non- limiting examples of inducible promoters include the murine mammary tumor virus promoter (inducible with dexamethasone), commercially- available tetracycline-responsive or ecdysone -responsive promoters, etc. It may also be desirable to utilize a promoter which is selectively active in the cancer cell to be treated, for example the PEG-3 gene promoter (U.S. No. 6,472,520). Examples of tissue- and cancer cell-specific promoters are well known to those of ordinary skill in the art.
  • RNA molecules comprising a MDA-7-encoding nucleic acid
  • Other elements that may be incorporated into a molecule comprising a MDA-7-encoding nucleic acid include transcription start sites, stop sites, polyadenylation sites, ribosomal binding sites, etc.
  • the MDA-7 molecule which is a nucleic acid, for example together with one or more of the elements listed above, may be comprised in a vector, which may be a virus, a phage, a plasmid, a cosmid, etc.
  • Suitable expression vectors include virus-based vectors and non- virus based DNA or RNA delivery systems.
  • appropriate virus-based vectors include, but are not limited to, those derived from retroviruses, for example Moloney murine leukemia-virus based vectors such as LX, LNSX, LNCX or LXSN (Miller and Rosman, 1989, Biotechniques 7:980-989); lentiviruses, for example human immunodeficiency virus (“HIV”), feline leukemia virus (“FIV”) or equine infectious anemia virus (“EIAV”)-based vectors (Case et al, 1999, Proc. Natl. Acad. Sci. U.S.A.
  • Ad5/CMV-based El- deleted vectors for example Ad5/CMV-based El- deleted vectors (Li et al, 1993, Human Gene Ther. 4:403-409); adeno-associated viruses, for example pSub201 -based AAV2-derived vectors (Walsh et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7257-7261); herpes simplex viruses, for example vectors based on HSV-I (Geller and Freese, 1990, Proc. Natl. Acad. Sci. U.S.A.
  • baculoviruses for example AcMNPV-based vectors (Boyce and Bucher, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2348-2352); SV40, for example SVluc (Strayer and Milano, 1996, Gene Ther. 3:581-587); Epstein-Barr viruses, for example EBV-based replicon vectors (Hambor et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014); alphaviruses, for example Semliki Forest virus- or Sindbis virus-based vectors (Polo et al, 1999, Proc. Natl.
  • vaccinia viruses for example modified vaccinia virus (MVA)-based vectors (Sutter and Moss, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851) or any other class of viruses that can efficiently transduce human tumor cells and that can accommodate the nucleic acid sequences required for therapeutic efficacy.
  • MVA modified vaccinia virus
  • Non- limiting examples of non- virus-based delivery systems which may be used according to the invention include, but are not limited to, so-called naked nucleic acids (Wolff et al., 1990, Science 247:1465-1468), nucleic acids encapsulated in liposomes (Nicolau et al., 1987, Methods in Enzymology 149:157-176), nucleic acid/lipid complexes (Legendre and Szoka, 1992, Pharmaceutical Research 9: 1235- 1242), and nucleic acid/protein complexes (Wu and Wu, 1991, Biother. 3:87-95).
  • naked nucleic acids Wilff et al., 1990, Science 247:1465-1468
  • nucleic acids encapsulated in liposomes Nicolau et al., 1987, Methods in Enzymology 149:157-176
  • nucleic acid/lipid complexes Legendre and Szoka, 1992, Pharmaceutical Research 9: 1235- 12
  • the expression vector is an El -deleted human adenovirus vector of serotype 5, although those of ordinary skill in the art would recognize that many of the different naturally-occurring human Ad serotypes or Ad vectors derived from non-human adenoviruses may substitute for human Ad 5-derived vectors.
  • a recombinant replication-defective Ad.mda-7 virus for use as an mda-1 vector may be created in two steps as described in Su et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:14400-14405.
  • the coding region of the mda-1 gene may be cloned into a modified Ad expression vector p Ad. CMV (Falck-Pedersen et al. , 1994, MoI. Pharmacol. 45:684-689).
  • This vector contains, in order, the first 355 bp from the left end of the Ad genome, the CMV promoter, DNA encoding splice donor and acceptor sites, the coding region of the mda-1 cDNA, DNA encoding a polyA signal sequence from the ⁇ globin gene, and ⁇ 3 kbp of adenovirus sequence extending from within the ElB coding region.
  • the recombinant virus may be created in vitro in 293 cells (Graham et al., 1977, J. Gen. Virol. 36:59-72) by homologous recombination between an m ⁇ i ⁇ -7-containing version of pAd.CMV and plasmid pJM17, which contains the whole of the Ad genome cloned into a modified version of pBR322 (McGrory et al, 1988, Virology 163:614-617).
  • pJM17 gives rise to Ad genomes in vivo, but they are too large to be packaged in mature Ad capsids.
  • This constraint is relieved by recombination with the vector to create a packageable genome (Id.) containing the mda-1 gene.
  • the recombinant virus is replication defective in human cells except 293 cells, which express adenovirus ElA and ElB. Following transfection of the two plasmids, infectious virus may be recovered, and the genomes may be analyzed to confirm the recombinant structure, and then virus may be plaque purified by standard procedures (Volkert and Young, 1983, Virology 125:175-193).
  • the infectivity of an adenovirus vector carrying a MDA-7 molecule may be improved by inserting an Arg-Gly-Asp motif into the fiber know (Ad5-Delta24RGD), as described in Lamfers et al, 2002, Cancer Res. 62:5736-5742.
  • the present invention provides for methods for treatment of cancers associated with aberrant EGFR signaling, for example, but not limited to aberrant EGFR signalling caused by EGFR over expression or an EGFR mutation.
  • EGFR is al70-kd plasma membrane glycoprotein with both extracellular ligand-binding and intracellular protein tyrosine kinase domains and a tyrosine-rich cytoplasmic tail (Cohen et al. 1974, Olayioye et al., 2000).
  • EGFR is a member of a subfamily of 4 closely related cell membrane receptors; EGFR is also referred to as ErbB 1.
  • the present invention encompasses treatment of cancer that targets artificially generated, naturally occurring variants or mutant forms of EGFR.
  • the present invention provides for methods to inhibit any one or more steps involved in EGFR activation.
  • the steps involved in EGFR activation have been well elucidated (Aaronson, 1991).
  • EGFR dimerizes with another available ErbB family monomer, and this dimerization leads to activation of the EGFR tyrosine kinase (TK) domain and subsequent adenosine triphosphate (ATP)-dependent autophosphorylation of adjacent ErbB monomer tyrosine residues (Moyer et al. 1997).
  • TK tyrosine kinase
  • ATP adenosine triphosphate
  • the inhibition or inactivation of EGFR is achieved by specifically inhibiting the TK activity of EGFR.
  • the EGFR signaling pathway contributes to a number of processes important in cancer development and progression such as proliferation, apoptosis, angiogenesis and metastatic tumor spread (Aaronson, 1991).
  • the present invention provides for methods to determine if a cancer possesses aberrant EGFR signaling by measuring the activity of the aforesaid downstream pathways or cancer development processes. By determining the nature of aberrant EGFR signaling as set forth below, the present invention provides for a method to choose the appropriate inhibitory molecule for treatment.
  • the ErbB receptors are expressed in a variety of normal epithelial tissues where they play important roles in cellular proliferation and differentiation (Haley et al., 1987).
  • EGFR overexpression has been noted in breast, non-small cell lung cancer (NSCLC), glioblastomas, head and neck, gastric, genitourinary, and colorectal carcinomas (Woodburn, 1999; Khazaie et al., 1993).
  • Certain EGFR mutations or deletions resulting in constitutive activation of downstream signaling cascades have been found in numerous tumors including glioblastomas (Nishikawa et al., 2004), medulloblastomas (Moscatello et al.
  • EGFRvIII,801EGFR,de2-7EGFR type III EGFR deletion mutant
  • EGFR vIII is exclusively expressed in malignant cells, and evidence suggests that the mutant form may activate signaling pathways different from ligand-stimulated EGFR (Pedersen et al., 2001).
  • Some recurrent tumors have a common secondary mutation in the EGFR kinase domain, T790M, conferring drug resistance.
  • EGFR activation not only promotes cellular proliferation and growth, but also contributes to other processes important for cancer progression, namely angiogenesis and metastases. Both wildtype and mutant EGFR proteins can be preferentially overexpressed in malignant tissues, and likely translates in to cancer progression.
  • agents generally targeting aberrant signaling either due to mutant or wildtype of EGFR, have been developed (section 5.5). The use of broad spectrum anti-EGFR treatment is also provided for herein.
  • the present invention provides for but is not limited to the treatment of cancers involving aberrant EGFR signaling, including but not limited to breast, NSCLC, glioblastomas, head and neck, gastric, pancreatic, genitourinary, prostate and colorectal carcinomas.
  • the present invention encompasses treatment of cancers including those with known mutant EGFR mutations, those that possess structurally wildtype molecules, and those with unidentified alterations that result in aberrant EGFR activity.
  • functional overexpression of EGFR most likely contributes to tumorigenesis and tumor progression through enhanced activation of proliferative and survival pathways as described above.
  • the present invention encompasses treatment of cancers that may involve any mechanism resulting in aberrant EGFR activity, which may directly or indirectly cause oncogenesis.
  • a "cancer associated with aberrant EGFR activity” need not be restricted to the specific cancer in the subject, but rather encompasses the class of cancers associated with aberrant EGFR activity, such as NSCLC, pancreatic, breast, glioblastoma, gastic, genito-urinary, prostate, and colorectal cancers.
  • the present invention provides for a method of treating cancer in a subject using agents targeting the EFGR pathway i.e. EGFR inhibitors.
  • EGFR inhibitors include several classes of molecules such as small molecule inhibitors, antibodies targeting EGFR, including monoclonal antibodies, dual EGFR/VEGFR (Vascular Endothelial Growth Factor Receptor) inhibitors, etc. (Heymach et al, 2006).
  • the present invention provides, in non-limiting embodiments, small molecule selective EGFR tyrosine kinase (TK) inhibitors classified as 4- anilinoquinazo lines (4- AQ; Ciardiello and Tortora, 2001) used in combination with mda-7 nucleic acid or protein.
  • TK small molecule selective EGFR tyrosine kinase
  • 4- anilinoquinazo lines (4- AQ; Ciardiello and Tortora, 2001
  • PD 153035 was the first reported small molecule EGFR receptor inhibitor belonging to the 4-AQ class of TK inhibitors (Fry et al., Science 265:1039-1095, 1994).
  • ErbB-targeted TK inhibitors which may be used according to the invention include Gefitinib (ZD-1839, IressaTM), Erlotinib (OSI-774, Tarceva®), Lapatinib® (GSK572016, GW572016), and Canertinib® (CI- 1033) (Fry, Exp Cell Res 284:131-139, 2003).
  • the present invention provides for the use of these drugs in combination with mda-7 (nucleic acid or protein) for the treatment of susceptible cancers. These compounds share a common 4-AQ core, but they have distinct ErbB inhibition profiles and mechanisms of action.
  • Gefitinib and Erlotinib are potent, selective inhibitors of EGFR (Moyer et al, Cancer Res 57:4838-4848, 1997). These drugs act as competitive inhibitors of ATP binding by the TK (Moyer et al., Cancer Res. 57, 4838-4848, 1997). The effectiveness of these TK inhibitors results both from alterations in the ATP cleft associated with mutations, which lead to enhanced inhibition of the mutant kinase by drugs, and from biological dependence of cancer cells on the increased survival signals transduced by the mutant receptors, a phenomenon described as "oncogene addiction” (Weinstein, I. B. (2002) Science 297, 63-64.).
  • Lapatinib® is a potent inhibitor of both EGFR and ErbB-2 (Intl. Pat. Pub. No. W09935146).
  • Canertinib® was designed to covalently modify an active site cysteine from a template with high initial binding affinity for EGFR.
  • ErbB-2 and ErbB-4 contain the same active site Cys.
  • Canertinib® inhibits all three receptors due to the common target (Smaill et al., J Med Chem 43:1380-1397, 2000).
  • ZD6474 is a small molecule VEGF flk-1/KDR (VEGFR-2) tyrosine kinase inhibitor that also demonstrates inhibitory activity against EGFR TK (Cairdiello et al., Clin Cancer Res. 10(2):784-93, 2004) and may be used in combination with mda-7 according to the invention.
  • VEGFR-2 vascular endothelial growth factor receptor 2
  • Erb receptors or related TKs may be the target of therapy, depending on the inhibitor used in conjunction with mda-7.
  • Combination therapy provided for by the present invention allows for the rational selection of a specific inhibitor depending on the receptor profile of the cancer being treated.
  • Quinazoline derivatives which bear a hetero-arylamino substituent at the 4-position have also been shown to possess receptor tyrosine kinase inhibitory activity (European Patent Application No. 0602851) and may be used according to the invention. Additional aryl- and heteroaryl- compounds inhibit EGF and/or PDGF receptor tyrosine kinase (International Patent Publication No. WO 92/20642). Heteroaryl substitutions at the 6-position of 4-AQ have shown anti-pro liferative activity on cancer cells (U.S. PatentNo. 5,955,464). Derivatives based on 4-AQ chemistry are being continuously developed and tested. The present invention encompasses the use of existing compounds as set forth in the paragraph above, as well as those under development, for therapeutic use in combination with MDA-7 molecules.
  • the present invention encompasses the use of combination cancer therapy with mda-7 and the 4-AQ drugs Gefitinib or Erlotinib.
  • Gef ⁇ tinib an orally active selective reversible inhibitor of the EGFR TK is being used clinically (Heymach et al., 2006). Growth inhibiting activity of Gef ⁇ tinib has been demonstrated in vitro and in vivo in tumor models that express functional EGFRs, including prostate, breast, ovarian, colon, and NSCLC (Ciardiello and Tortora, 2001). Gef ⁇ tinib has been used herein to test efficacy of treatment of wild type and mutant EGFR containing NSCLC lines (section 6.2).
  • the present invention provides for the novel combination of mda-7 with 4- AQ based EGFR TK inhibitors such as Gef ⁇ tinib or Erlotinib as a means to overcome drug resistance and effectively treat NSCLC.
  • Drugs targeting EGFR and based on chemistries distinct from 4-AQ include EKB-569 and HKI-272, based on a 3-quinolinecarbonitrile core (Rnowles et al., J Biol Chem 2006), or AEE788 a broad spectrum TK inhibitor active against VEGFR , ErbB2 and EGFR based on pyrrolo-primidine chemistry (Traxler et al., Cancer Res 64:4931-4941, 2004).
  • the present invention provides for combination therapy using non-4- AQ based TK inhibitors. Clinical trials are being pursued using EGFR TK inhibitors in combination with other biochemical pathway inhibitors such as mTOR, VEGF, Ras/Raf/ERK etc.
  • a combination of Gef ⁇ tinib or Erlotinib with an MDA-7 molecule may also include as a third component, a drug targeting other biochemical pathways such as mTOR, VEGF, Ras/Raf/ERK etc., as set forth above.
  • Monoclonal antibody therapy using fully humanized antibodies directed against EGFR have also entered clinical trials. These include Cetuximab, Panitmumab (ABX-EGF), Matuzumab (EMD72000), Pertuzumab (2C4) and MDX214 (Heymach et al., 2006).
  • the present invention encompasses the use of any of the aforementioned antibodies used in combination with a MDA-7 molecule for treatment of cancer.
  • the present invention provides for methods of treating cancer in a subject in need of such treatment by administration of a therapeutic formulation which combines a MDA-7 molecule with an inhibitor of EGFR.
  • Combination therapy means a therapeutic regimen in which the patient is treated with both agents in the same regimen, but the agents (mda-7 nucleic acid or protein and EGFR inhibitor) may be administered simultaneously or non-simulataneously (for example, the agents may be administered individually, with or without a time interval between administration).
  • Administration may be by any route known in the art, including systemic intravenous or intra-arterial, intra-tumoral (injection into the tumor or a site suspected of containing tumor cells), intra-thecal, pulmonary, intranasal, or by instillation into the site of tumor excision.
  • the therapeutic formulation may combine a MDA-7 molecule with an inhibitor of a pathway that lies downstream of, and is activated by aberrant EGFR signaling.
  • Administration of a MDA-7 molecule encompasses administering a nucleic acid comprising a nucleic acid encoding a MDA-7 protein, in expressible form, or a purified MDA-7 protein in a suitable therapeutic formulation.
  • An EGFR inhibitor molecule may be used according to its standard clinical protocol or may be administered at a dosage optimized for use with combined mda-7 therapy.
  • EGFR inhibitors include but are not limited to small molecule inhibitors such as Gefitinib (ZD-1839, IressaTM), Erlotinib (OSI-774, Tarceva®), Lapatinib® (GSK572016, GW572016), Canertinib® (CI- 1033), EKB-569, HKI-272 and AEE788.
  • the EGFR inhibitor may be an antibody, non-limiting examples of which include Cetuximab, Panitmumab (ABX-EGF), Matuzumab (EMD72000), Pertuzumab (2C4) and MDX214.
  • typical doses of various EFGR inhibitor therapies are as follows. Gefitinib as a 250 mg tablet may be administered once per day with administration from 14 to 28 days. Erlotinib may be administered in a daily dose between 150 to 300 mg. Lapatinib® may be administered at a daily dose of up to 1,800 mg, Canertinib® may be administered at a daily dose of about 150 mg. EKB-569 may be administered at a daily dose of about 75 mg. HKI-272 may be administered at a daily dose of about 400 mg. Any of the foregoing may also be administered at non-zero lower doses which are effective in combination with an mda-7 nucleic acid or protein.
  • Cetuximab and other anti-EGFR antibodies have been used clinically at 4200-400 mg/m 2 in a single infusion over 60 minutes in weekly doses. These doses may be used in combination therapy according to the invention. In general, a skilled practitioner will have sufficient knowledge and information to adjust a dose appropriate to a subject's tolerance and when used as a combination therapy.
  • the present invention provides for treatment of cancer in a subject in need of such treatment using a combination of each of the above EGFR inhibitor therapies with a suitable dose and formulation of a MDA-7 molecule as set forth in the following paragraphs including but not limited to a naked DNA vector, a viral vector, a liposome formulation, a purified peptide, etc.
  • the nucleic acid encoding a MDA-7 protein may be comprised in a viral vector, operably linked to a promoter element that is inducible or constitutively active in the target cell.
  • the viral vector is a replication-defective adenovirus (as described in section (3) above).
  • a viral vector containing a nucleic acid encoding a MDA-7 protein in expressible form, operably linked to a suitable promoter element may be administered to a population of target cells at a multiplicity of infection (MOI) ranging from 10-100 MOI.
  • MOI multiplicity of infection
  • the amount of a viral vector administered to a subject may be 1 X 10 9 pfu to 1 X 10 12 pfu.
  • a nucleic acid encoding a MDA-7 protein may be introduced into a cell ex vivo and then the cell may be introduced into a subject.
  • a nucleic acid encoding mda-7 may be introduced into a cell of a subject ex vivo and then the cell containing the nucleic acid may be optionally propagated and then (with its progeny) introduced into the subject.
  • a MDA-7 protein may be used in protein/peptide therapy of a subject in need of such treatment.
  • the MDA-7 protein of the invention may be prepared by chemical synthesis or recombinant DNA techniques, purified by methods known in the art, and then administered to a subject in need of such treatment.
  • MDA-7 protein may be comprised, for example, in solution, in suspension, and/or in a carrier particle such as microparticles, liposomes, or other protein- stabilizing formulations known in the art
  • formulations of MDA-7 protein may stabilized by addition of zinc and/or protamine stabilizers as in the case of certain types of insulin formulations.
  • a MDA-7 protein may be linked covalently or non-covalently, to a carrier protein which is preferably non-immunogenic.
  • the MDA-7 protein may be chemically modified; for example, it may be PEGylated.
  • a MDA-7 protein/peptide is administered in an amount which achieves a local concentration in the range of 18 to 50 ng per microliter.
  • a subject may be administered a range of 50-100 mg per kilogram (this may, for example, be used per tumor weight for intra-tumoral injection).
  • the dose range may be between 100 - 2500 mg/treatment or between 1000-2500 mg/day.
  • the concentration achieved in the target tissue/environment of the cells to be treated is between about 10 nM- 500 nM, or between about 5OnM- 200 nM, and preferably about 100 nM).
  • a nucleic acid comprising a nucleic acid encoding a MDA-7 protein combined with a suitable anti-EGFR therapy, as described above, may be introduced into at least one cancer cell of a subject by methods known in the art.
  • a solution comprising an effective amount of the nucleic acid encoding the MDA-7 protein (optionally comprised in a larger nucleic acid) may be introduced (i) into a cavity resulting from the complete or partial surgical excision of a tumor mass, (ii) into a tumor mass by direct intratumoral injection, (iii) into the bloodstream of the subject, or (iv) into the extracellular space, if any, surrounding the tumor.
  • infection of the target cell may be achieved by exposure to approximately 100 plaque-forming units of an adenovirus vector comprising a MDA-7 protein encoding nucleic acid.
  • Clinical protocols as used in the published Phase I studies of mda-7 gene therapy, or derivative protocols, may also be used (Cunningham et al, 2004; Tong et al, 2004).
  • treatment is performed by administering between 2 X 10 10 to 2 X 10 12 Ad.mda-7 viral particles (i.e. 5 X 10 8 to 5 X 10 10 pfu/ml) delivered to the central region of the target tumor.
  • the viral formulation is stored in a saline solution containing 10% glycerol in frozen form below 6O 0 C. Prior to injection the formulation is diluted with 5% glucose to the required viral titre. A marker dye such as Isosulfan blue may be injected with the formulation to precisely localize the injection site. Injections are performed twice weekly for around 3 weeks. Evaluations of treatment are performed by measurement of tumor size, analysis of biopsy tissue for presence of mda-7 gene expression in tumor cells, measurement of apoptosis indices in resected cells, measurement of cytokines, immunological responses etc. (Cunningham et al., 2004; Tong et al., 2004).
  • the anti-EGFR therapies may be delivered to the subject together with or separate from the mda-7 therapy.
  • the route of delivery may be same or a different method may be used depending on suitability to the specific formulation. The choice of route and timing is within the scope of routine practice and knowledge of a person with skill in the related art.
  • the present invention encompasses the use of the aforementioned combinations of a MDA-7 molecule and an EGFR inhibitor for treatment of cancer including but not limited to prostate, breast, ovarian, colon, pancreatic, small cell and NSCL cancer.
  • the present invention encompasses pharmaceutical compositions comprising amounts of a MDA-7 molecule and an EGFR inhibitor which, when used together (simulataneously, concurrently, or sequentially) are effective in treating a cancer, as set forth above.
  • the present invention encompasses therapy kits comprising separate formulations of a MDA-7 molecule and an EGFR molecule in amounts which, when used simulataneously, concurrently, or sequentially, are effective in treating a cancer, as set forth above.
  • NCI-H460 EGFR-WiId type
  • NCI-H1650 in-frame deletion delE746-A750
  • NCI-H1975 L858R + T790M
  • NCI-H2030 EGFR-Wild-type
  • human lung cancer cell lines Kwak et al., 2005; Sordella et al., 2004
  • RPMI- 1640 10% fetal bovine serum, 2mM L-glutamine, 50 U/ml penicillin/streptomycin and ImM sodium pyruvate.
  • control and PKR siRNAs were purchased from Ambion (Austin, TX) and Santa Cruz (Santa Cruz, CA), respectively.
  • CAR receptors were determined as described in Lebedeva et al., 2002. Two methods were used for semi- quantitation of the results. The shift of FLl peak was calculated as difference in peak channels between the controls (cells unstained or stained with non-specific antibody) and the experimental cells stained with anti-CAR antibody relative to the position of the control cell peak channel. The second method used the Kolmogorov-Smirnov (K- S) test for overlaid histograms. The calculation computed the summation of the overlaid curves and determined the greatest difference between the summation curves (K-S statistics). D value indicated the greatest difference between the two curves.
  • K- S Kolmogorov-Smirnov
  • GFP GFP expression by flow cytometry.
  • Cells I x 105 were plated in each well of a 12-well plate. After 24 h the cells were infected with Ad.
  • GFP at a multiplicity of infection (MOI) of 6.25, 12.5, 25, 50, and 100 plaque forming units (pfu)/cell (Emdad et al., 2006).
  • MOI multiplicity of infection
  • the cells were harvested 24 h post-infection by trypsinization, washed with PBS, and resuspended with 400 ⁇ l of PBS. The cells were immediately analyzed for GFP expression by flow cytometry and the percentage of GFP positive cells were determined.
  • Cell viability assay Cells were seeded in 96-well tissue culture plates (5 X 10 3 cells per well) and treated the next day as described in the figure legends. Cell viability was determined by MTT assay as previously described (Sarkar et al., 2002b; Emdad et al., 2006).
  • Annexin V binding assay Cells were trypsinized and washed with complete media and then twice with PBS. The aliquots of the cells (2 x 10 5 ) were resuspended in IX binding buffer (0.5 ml) and stained with APC (allophycocyanin)-labeled Annexin-V (BD Biosciences, Palo Alto, CA) according to the manufacturer's instructions. DAPI was added to the samples after staining with Annexin-V to exclude late apoptotic and necrotic cells. The flow cytometry was performed immediately after staining. Western blot assay. Cell lines were grown on 10-cm plates and protein extracts were prepared with RIPA buffer containing a cocktail of protease inhibitors.
  • a total of 50 ⁇ g of protein was applied to 12% SDS-PAGE and transferred to nitrocellulose/PVDF membranes.
  • the membranes were probed with polyclonal or monoclonal antibodies to mda-7/IL-24, PARP, PKR, phospho-EGFR (Y 1068), phospho-(Ser473) and total Akt, phospho- and total ERKl/2 , and EF l ⁇ .
  • Ad a replication incompetent adenovirus
  • Ads employ Coxsackie-adenovirus receptors (CAR) to enter human cells and the efficiency of Ad infection depends upon the CAR expression level in the target cells (Hidaka et al., 1999; Li et al., 1999; Pearson et al., 1999).
  • CAR expression levels were analyzed in NSCLC cells by FACS analysis. The relative CAR expression was found to be: H- 2030 > H1650 > H-460 > H-1975 (Table 1).
  • Ad.GFP Green Fluorescent Protein
  • Ad.GFP a replication incompetent Ad expressing Green Fluorescent Protein (GFP) under the control of the CMV promoter (Ad.GFP) was used (Emdad et al., 2006) and GFP expression was analyzed by FACS analysis at 24 h post-infection after different multiplicities of infection (m.o.i.'s). In H-2030 cells that have the highest CAR level, > 80% of cells were positive for GFP even at an m.o.i. of 25 pfu/cell ( Figure IA). However, in the other three NSCLC cell lines, to achieve GFP expression in -80% cells, 50-100 pfu/cell of Ad.GFP infection was required.
  • NSCLC cells have variable levels of CAR that regulate efficient Ad transduction and subsequent transgene expression.
  • Further studies determined MDA-7/IL-24 protein levels in NSCLC cells following infection with Ad.mda-7 at different m.o.i.'s. As shown in Figure IB, maximum MDA-7/IL-24 protein expression was apparent in Ad.mda-7-infected H-2030 cells when compared with the other three NSCLC cell lines.
  • Ad.mda-7 results in loss of viability by induction of programmed cell death (apoptosis) (Fisher, 2005; Gupta et al, 2006).
  • the effect of Ad.mda-7 on the growth of EGFR wild-type (H-460, and H- 2030) and EGFR mutated (H-1650, and H-1975) NSCLC cells was analyzed 5 days post-infection by MTT assay ( Figure 2A).
  • Different m.o.i.'s of Ad.mda-7 ranging from 25 to 200 pfu/cell, and as a control 100 and 200 pfu/cell of empty Ad (Ad.vec) was employed.
  • Ad.mda-7 induced a significant decrease in cell viability in two of these cell types (H-1650, and H-2030), as compared to Ad.vec treated cells. In these two cell lines, a decrease in cell viability was evident even with 25 pfu/cell of Ad.mda-7 and with increasing concentrations of the Ad the effect became more pronounced. However, infection with Ad.mda-7 in the other two cell types (H-460 and H-1975) did not produce a significant decrease in viability except when administered at a very high m.o.i. (200 pfu/cell).
  • Annexin V staining and FACS analysis performed 48 h after Ad infection demonstrated that in H- 2030 and H-1650 cells infection with Ad.mda-7 markedly increased the percentage of apoptotic (early and late) cells in a dose-dependent manner, while H-460 and H-1975 cells exhibited a low level of apoptosis that corroborated the results obtained using MTT assays. In contrast, no significant change in the percentage of apoptotic cells was evident in NSCLC cells infected with 100 pfu/cell of Ad.vec. Thus, Ad.mda-7 did not demonstrate any EGFR-selective cytotoxicity, as substantial killing was observed in both EGFR wild-type and mutated cells.
  • Ad.mda-7 induces PKR expression and PARP cleavage in NSCLC cells.
  • Previous studies (Pataer et al., 2002) provided evidence linking the tumor suppressor activity of Ad.mda-7 to up-regulation of double-stranded RNA-dependent protein kinase (PKR) in a p53 -independent manner in NSCLC cells.
  • PPKR RNA-dependent protein kinase
  • PKR mediates anti- tumorigenic activity through activation of specific biochemical pathways resulting in growth inhibition and apoptosis (Jagus et al., 1999).
  • Ad.mda-7 and gefitinib Enhanced in vitro growth inhibitory activity of Ad.mda-7 and gefitinib in NSCLC cells.
  • Gefitinib a selective EGFR inhibitor, is clinically used for treatment of NSCLC (Baselga et al, 2002; Fukuoka et al, 2003). Since Ad.mda-7 alone showed limited effect in inhibiting growth of two of the four NSCLC cell lines tested, it was hypothesized that a combinatorial approach of Ad.mda-7 and gefitinib might be useful in augmenting growth suppression and apoptosis.
  • NSCLC cells were infected with either Ad.vec or Ad.mda-7 alone or in combination with various concentrations of gefitinib (0.02, 0.2, and 2 ⁇ M) and cell viability was analyzed by MTT assays (Figure 4A). Together with Ad.vec, gefitinib induced significant growth inhibition only in H- 1650 cells (EGFR mutated) at the clinically achievable concentration (2 ⁇ M). The other three NSCLC cell lines showed relative resistance to gefitinib that was consistent with previous reports (Janmaat et al., 2006; Kwak et al., 2005). In contrast, gefitinib in combination with Ad.mda-7 induced a significant decrease in cell viability in all four NSCLC cells.
  • Ad.mda-7 induced an additive and a synergistic sensitization to gefitinib treatment in H-460 and H- 1975, and H- 1650 and H-2030 cells, respectively.
  • the combination of Ad.mda-7 and gefitinib significantly enhanced the percentage of apoptotic (early and late) cells corresponding with the results observed by MTT assays ( Figure 4B).
  • Gefitinib increases ectopic MDA-7/IL-24 and Ad.mda-7-induced PKR expression. Since gefitinib potentiated Ad.mda-7-induced apoptosis, the effects of gefitinib on MDA-7/IL-24 protein level following Ad.mda-7 infection was examined. In all four Ad.mda-7-infected NSCLC cells, gefitinib significantly increased the level of ectopic MDA-7/IL-24 protein ( Figure 5A), which was most pronounced when cells were infected with 25 pfu/cell of Ad.mda-7, either alone or in combination with gefitinib.
  • gef ⁇ tinib treatment moderately augmented Ad.mda-7-mediated induction in PKR expression, although gefitinib alone had no effect on PKR induction.
  • PKR siRNA significantly inhibited Ad.mda-7-induced apoptosis in H- 1650 cells and to a lesser extent in H- 1975 cells.
  • PKR siRNA could not efficiently block apoptosis induced by the combination treatment suggesting that other or additional signaling pathways might play more significant roles in the context of the combinatorial treatment.
  • gefitinib treatment showed a significant reduction only in the level of p-ERK, but p-EGFR and p-AKT remained unchanged.
  • Treatment with gefitinib significantly decreased p-AKT in H-2030 (gefitinib-resistant) cells, however no substantial changes in p-EGFR and p-ERK was evident.
  • H- 1975 (gef ⁇ tinib-resistant) cells treatment with gefitinib did not result in any reduction of p-EGFR and p-ERK, however a slight reduction in p-AKT level was observed.
  • EGFR is involved in the pathogenesis or progression of many different types of cancer.
  • EGFR overexpression has been observed in 40% to 80% of tumors from NSCLC patients (Van Schaeybroeck et al., 2006). Consequently, EGFR targeting represents a promising approach for lung cancer therapy.
  • Several preclinical studies suggested that EGFR-targeted therapy with monoclonal antibodies or tyrosine -kinase inhibitors was effective toward various types of cancer.
  • contrasting results have been obtained in 2 large phase III clinical trials on NSCLC, in which the addition of gefitinib to conventional chemotherapy did not improve clinical response (Giaccone et al., 2004; Herbst et al., 2004). The reason for this treatment failure remains unresolved and requires further preclinical research.
  • Activating mutations in the ATP binding site of EGFR confer enhanced sensitivity of EGFR to stimulation by ligand binding (Lynch et al., 2004; Settleman, 2004).
  • Tumor cells with EGFR mutations show markedly enhanced sensitivity to gefitinib-induced cytotoxicity and inhibition of EGFR-downstream signaling pathways (Settleman, 2004). This is thought to result from enhanced binding of ATP and gefitinib to the mutated ATP binding domain (Lynch et al., 2004; Paez et al., 2005).
  • a large number of NSCLC patients do not have mutations in EGFR thus mandating a treatment protocol independent of gefitinib.
  • Ad.mda-7 and gefitinib are attractive from a clinical standpoint.
  • Gefitinib was observed to increase the expression of ectopic MDA-7/IL-24 protein in Ad.mda-7-infected cells, suggesting that the enhanced killing by combinatorial treatment was due to increased production of functional MDA-7/IL-24 protein. This increase might be due to stabilization of mda-7/IL-24 mRNA or protein as has been observed during treatment with IFN- ⁇ + mezerein (Madireddi et al., 2000a), as well as with the drug sulindac (Oida et al., 2005). Further studies are required to elucidate the molecular mechanism by which gefitinib augments MDA-7/IL-24 protein level.
  • PKR upregulation plays a role in Ad.mda-7-mediated apoptosis (Pataer et al., 2002), which was also confirmed by studies using PKR siRNA.
  • PKR siRNA exerted a marginal effect on the additive/synergistic apoptosis induced by Ad.mda-7 + gefitinib.
  • NCI-H460 EGFR-WiId type
  • NCI-H1975 EGFR mutant
  • L858R+T790M NCIH1650
  • NCI-H446 EGFR-WiId type human lung cancer cell lines
  • M4 having amino acid residues 104 to 206 of MDA7-/IL-24, contains essential domains required for cancer-specific apoptotic activity of this molecule (Gupta et al., 2006b)
  • M4 DNA sequence was amplified by PCR and cloned in BamHI and Notl restriction sites of the vector pGEX-4T2 in frame with GST sequence. Positive clones were confirmed by restriction analysis.
  • BL21 competent cells were transformed by GST-M4 construct and expression of recombinant GST-M4 was determined after induction with ImM IPTG.
  • GST-M4 protein was eluted using 25 mM reduced glutathione in tris buffered saline. Eluted GST-M4 protein was first dialyzed against phosphate buffered saline (PBS) followed by DMEM. Protein was passed through 0.2 ⁇ m filter, quantified, aliquoted and stored in ice at 4oC.
  • PBS phosphate buffered saline
  • Recombinant GST-MDA-7 was purified in a similar manner (Sauane et al., 2004a).
  • Annexin-V binding assays Cells were seeded in 6-well plates (2 x 10 5 cells per well) and the next day were treated with recombinant proteins in 2% serum containing medium for 2 h following which medium was replaced with complete growth medium containing different concentrations of Tarceva. After 24 h cells were trypsinized, washed with complete medium and PBS, resuspended in 500 ⁇ l of binding buffer containing 2.5 mM CaC12 and stained with APC-labeled Annexin-V (BD Biosciences, Palo-Alto, CA) and propidium Iodide for 15 min at room temperature. Flow cytometry was performed immediately after staining (Lebedeva et al, 2003b).
  • Cell viability Assays Cells were seeded in 96-well tissue culture plates (1.5 x 10 cells per well) and next day treated as described in the figure legends. After incubation for specific times, medium was removed and 100 ⁇ l of fresh medium containing 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added to each well and incubated for 4 h in a 5% CO2 incubator at 37°C. The precipitate was solubilized in an equal volume of solubilization solution (0.01 N HCl in 10 % SDS).
  • Membranes were incubated overnight at 4°C in Tris buffered saline - tween (TBS-T) containing different dilutions of primary antibodies: anti-MDA-7/IL- 24 (1 :5000 in 5% BSA); anti-phospho p38 (1 :1000 in 5% milk solution), anti-phospho ERK (1 : 1000 in 5% milk solution) and Total ERK (1 : 1000 in 5% milk solution), anti- phospho AKT (1 : 1000 in 5% milk solution) and Total AKT (1 : 1000 in 5% milk solution) and antiphospho EGFR (1 : 1000 in 5% milk solution) and Total EGFR (1 : 1000 in 5% milk solution).
  • TBS-T Tris buffered saline - tween
  • GST-tagged M4 protein (GST-M4) was expressed and purified from E. coli as described in Materials and Methods.
  • the recombinant protein was soluble having an advantage over insoluble proteins as it could be purified under native conditions while insoluble proteins needed to be purified under denaturing conditions followed by renaturation.
  • recombinant GST-M4 and GST-MD A-7 were purified as ⁇ 35-kDa and ⁇ 47-kDa proteins, respectively ( Figure 8).
  • Some free GST protein of ⁇ 25-kDa was co-purified with soluble GST-MD A-7 and GST-M4.
  • Functional activity of purified recombinant GST-MD A-7 and GST-M4 was determined on various NSCLC cell lines, H460, H-1975, H-446 and H-1650, using Annexin-V binding assays that monitor apoptosis.
  • Cells were treated with different concentrations of the recombinant proteins ranging from 0.1 ⁇ M to 2 ⁇ M for 20 h.
  • Both GST-MD A- 7 and GST-M4 were functional at a concentration of 0.2 ⁇ M in the different cells and GST-M4 was more active than GST-MD A-7 at lower concentrations (Figure 9 A-D). However, at a higher concentration (> 0.8 ⁇ M) both proteins demonstrated similar levels of cytotoxic effects with > 80% apoptotic cells.
  • H-446 and H- 1650 were more sensitive to the recombinant proteins at a lower concentration ( Figure 9D and 9C, respectively) and no apoptotic activity was observed when cells were treated with 2 ⁇ M or less of GST protein alone confirming the apoptosis-specific properties of the MDA-7/IL-24 and M4 fusion proteins.
  • EGFR status of the cells did not affect their sensitivity to GST-MD A-7 and GST-M4, as substantial killing was observed in both EGFR wild type (H460 and H446) and mutated cells (H1650 and H1975).
  • EGFR tyrosine kinase inhibitors such as erlotinib and Gef ⁇ tinib
  • erlotinib and Gef ⁇ tinib are active drugs in advanced NSCLC and erlotinib has demonstrated a survival benefit in these patients.
  • This study evaluated the effects of another EGFR inhibitor, erlotinib, in combination with either GST-M4 or GST-MD A-7 in NSCLC cells.
  • apoptosis induction by erlotinib was assessed alone on the four NSCLC cell lines (H- 1975, H-460, H-446 and H- 1650). Cells were treated with varying doses of erlotinib, ranging from 0.2 ⁇ M to 40 ⁇ M, for 24 hours and the next day apoptosis was analyzed by Annexin-V binding assay. It was observed that erlotinib at a concentration of 10 ⁇ M induced -20% apoptosis in all four NSCLC cell lines and with a higher concentration of erlotinib (40 ⁇ M) -30-35% cells became apoptotic (Figure 10A).
  • GST-M4 and GST-MD A-7 activates the p38 MAPK pathway.
  • Activation of p38 MAPK followed by induction of GADD family genes play a critical role in Ad.mda-7-induced apoptosis in multiple cell types (Sarkar et al., 2002).
  • immunoblot analysis revealed that treatment with GST-MD A-7 and GST-M4 resulted in phosphorylation of p38 MAPK while the total p38 MAPK level remained unchanged.
  • GIn Ala GIn Asp Asn lie Thr Ser Ala Arg Leu Leu GIn GIn GIu VaI 85 90 95
  • VaI Leu lie VaI Ser GIn Leu GIn Pro Ser GIn GIu Asn GIu Met Phe 145 150 155 160
  • Ciardiello F Caputo R, Bianco R, Damiano V, Pomatico G, De Placido S, Bianco AR, Tortora G. 2000. Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD- 1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor. Clin Cancer Res 6(5):2053- 2063.
  • Ciardiello F Tortora G. 2001. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 7(10):2958- 2970.
  • Emdad L Sarkar D, Lebedeva IV, Su ZZ, Gupta P, Mahasreshti PJ, Dent P, Curiel DT, Fisher PB. 2006. Ionizing radiation enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis in human ovarian cancer. J Cell Physiol 208(2):298-306.
  • mda-7/IL-24 Multifunctional cancer-specific apoptosis-inducing cytokine. Pharmacol Ther 111 : 596-628.
  • BiP/GRP78 is an intracellular target for mda- 7/IL-24 induction of cancer-specific apoptosis. Cancer Res. 66(16) : 8182-8191. Haley et al., The human EGF receptor gene: structure of the 110 Kb locus and identification of sequences regulating its transcription. Oncogene Res. 1987, 1 :375- 396.
  • Madireddi MT Dent P, Fisher PB. 2000a. Regulation of mda-7 gene expression during human melanoma differentiation. Oncogene 19(10): 1362-1368. Madireddi MT, Su ZZ, Young CS, Goldstein NI, Fisher PB. 2000b. Mda-7, a novel melanoma differentiation associated gene with promise for cancer gene therapy. Adv Exp Med Biol 465:239-261.
  • mda-7/IL-24 Melanoma Differentiation Associated Gene-7 (mda-7)/ Interleukin-24 (IL-24), mda-7/IL-24: Current perspectives on a unique member of the IL-IO family of cytokines. Anti-Inflammatory & Anti- Allergy Agents in Medicinal Chemistry 5: 259-274.
  • the cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice. Proc Natl Acad Sci U S A 95(24): 14400-14405.

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Abstract

La présente invention concerne des procédés de traitement d'un cancer associé à une activité aberrante de l'EGFR comprenant l'administration, à un sujet qui a besoin d'un tel traitement, d'une quantité efficace d'une association d'un inhibiteur de l'EGFR et d'une molécule de MD A-7. L'utilisation combinée de ces agents permet une thérapie efficace des cancers qui peuvent être relativement résistants à l'un ou l'autre des agents seul.
PCT/US2007/080962 2006-10-10 2007-10-10 Traitement combinatoire avec les molécules inhibitrices du récepteur du facteur de croissance épidermique et le gène-7 associé à la différenciation des mélanomes WO2008063773A2 (fr)

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