WO2004087207A2 - Method for inducing apoptosis and aneuploidy regression in cancer cells - Google Patents

Method for inducing apoptosis and aneuploidy regression in cancer cells Download PDF

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WO2004087207A2
WO2004087207A2 PCT/US2004/009270 US2004009270W WO2004087207A2 WO 2004087207 A2 WO2004087207 A2 WO 2004087207A2 US 2004009270 W US2004009270 W US 2004009270W WO 2004087207 A2 WO2004087207 A2 WO 2004087207A2
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egfr
erbb2
therapeutic agent
inhibits
protein
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WO2004087207A3 (en
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Özge ALPER
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Georgetown University
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • Cancer is the second leading cause of death in the United States. Half of all men and one-third of all women in the US will develop cancer during their lifetimes. Today, millions of people are living with cancer or have had cancer. The sooner a cancer is found and treatment begins, the better are the chances for survival.
  • ovarian cancer is cancer that begins in the cells that constitute the ovaries, including surface epithelial cells, germ cells, and the sex cord-stromal cells. Cancer cells that metastasize from other organ sites to the ovary (most commonly breast or colon cancers) are not then considered ovarian cancer.
  • ovarian cancer accounts for 4 percent of all cancers among women and ranks fifth as a cause of their deaths from cancer.
  • the American Cancer Society statistics for ovarian cancer estimate that there will be 25,400 new cases and 14,300 deaths in 2003. The death rate for this disease has not changed much in the last 50 years.
  • stage III the upper abdomen
  • stage IV the upper abdomen
  • Breast cancer is the most common form of cancer in women in the United States, after skin cancer. Both its cause and its cure remain undiscovered. Based on the current life expectancy for women in the United States, one out of nine women will develop breast cancer in her lifetime - a risk that was one out of 14 in 1960.
  • breast cancer and ovarian cancer account for one-third of all cancers in women and approximately one-quarter of cancer-related deaths in females.
  • Cancer is an abnormal state in which uncontrolled proliferation of one or more cell populations interferes with normal biological functioning.
  • the proliferative changes are usually accompanied by other changes in cellular properties, including reversion to a less differentiated, more developmentally primitive state.
  • the development of cancer consists of multiple, sequential, and interrelated steps that lead to the generation of an autonomous clone with aggressive growth potential. These steps include sustained growth and unlimited self-renewal through a process of growth signal self-sufficiency, decreased sensitivity to growth suppressive signals, and resistance to apoptosis. Genetic or cytogenetic events that initiate aberrant growth sustain cells in a prolonged "ready" state by preventing apoptosis.
  • Another feature of many tumors is the remodeling ofthe surrounding tissues. Local invasive infiltration and growth are key features in many tumors, which are accompanied by remodeling ofthe vasculature and the destruction ofthe surrounding normal tissues.
  • MMPs matrix metalloproteinases
  • zinc dependent proteolytic enzymes that cleave extracellular matrix (collagen, laminin, firbronectin, etc) as well as non-matrix substrates (growth factors, cell surface receptors, etc).
  • MMPs matrix metalloproteinases
  • the deregulation of MMPs is involved in many diseases, such as tumor metastasis, rheumatoid arthritis, and periodontal disease.
  • the present invention relates to methods of inducing apoptosis and aneuploidy regression in cancer cells overexpressing EGFR and/or erbB2 receptor, methods of therapy useful to treat an individual who suffers from cancer or a precancerous condition, pharmaceutical and therapeutic agents and compositions useful in the methods.
  • the invention is a method of inducing apoptosis and aneuploidy regression in aneuploid cancer cells comprising contacting the cells with an agent that blocks signaling through the EGFR and erbB2 pathway(s) in sufficient quantity to block the signaling.
  • the invention is a method of inducing apoptosis and aneuploidy regression in an individual, in aneuploid cells that overexpress EGFR and erbB2, comprising administering to the individual a therapeutically effective amount of an agent that blocks signaling through the EGFR and erbB2 pathways(s).
  • therapeutic agent or anti-tumor therapeutic agent refers to agents (compounds, molecules) that induce apoptosis or aneuploidy regression, or both, in cancer cells.
  • a therapeutically effective amount of an agent is one which induces apoptosis in aneuploid cancer cells and/or aneuploidy regression in such cells, particularly in near-diploid cancer cells and survival of diploid cells.
  • a variety of therapeutic agents can be used in a method ofthe present invention, such an agent may inhibit the interaction of EGFR and/or erbB2 with their respective ligands; inhibit expression of EGFR and/or erbB2; or inhibit EGFR and/or erbB2 activity (e.g., by inhibiting clustering or phosphorylation of EGFR and/or erbB2, or inhibiting a downstream signaling event that occurs upon binding of EGFR/erbB2 to their ligand(s)).
  • an agent may inhibit the interaction of EGFR and/or erbB2 with their respective ligands; inhibit expression of EGFR and/or erbB2; or inhibit EGFR and/or erbB2 activity (e.g., by inhibiting clustering or phosphorylation of EGFR and/or erbB2, or inhibiting a downstream signaling event that occurs upon binding of EGFR/erbB2 to their ligand(s)).
  • Therapeutic agents can be, for example, an antibody (monoclonal or polyclonal) or a fragment thereof, such as an antibody that is specifically immunoreactive with EGFR or erbB2; a protein, such as a mutated form or a mimic of EGFR or erbB2, a fragment of EGFR or erbB2, an extracellular domain of EGFR/erbB2 or a fusion protein; a nucleic acid, such as an antisense nucleic acid of EGFR/erbB2 or an RNAi construct of EGFR/erbB2; or a small organic molecule, such as a peptidomimetic.
  • an antibody monoclonal or polyclonal
  • a fragment thereof such as an antibody that is specifically immunoreactive with EGFR or erbB2
  • a protein such as a mutated form or a mimic of EGFR or erbB2, a fragment of EGFR or erbB2, an extracellular domain of
  • One or more therapeutic agents that induce apoptosis or regression of aneuploidy in cancer cells overexpressing EGFR and erbB2 can be administered, simultaneously or sequentially, in a method ofthe present invention.
  • Pharmaceutical compositions which comprise at least one therapeutic agent that induces apoptosis and/or regression of aneuploidy in cancer cells that overexpress EGFR and erbB2 and a suitable carrier, are also the subject of this invention and are useful in the present methods.
  • compositions comprise at least one therapeutic agent that induces apoptosis in aneuploid cancer cells that overexpress EGFR and erbB2 and at least one therapeutic agent that induces aneuploidy regression in aneuploid cancer cells, particularly near-diploid cancer cells, that overexpress EGFR and erbB2.
  • the method comprises administering at least one additional type of agent, referred to as a chemotherapeutic agent, in combination with a therapeutic agent.
  • a chemotherapeutic agent for example one or more chemotherapeutic agent(s) that affects growth of metastasis tumor cells in an additive or synergistic manner with the therapeutic agent can be administered.
  • the chemotherapeutic agent can be, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin,
  • ionizing radiation can also be administered and/or surgical treatment provided.
  • the method ofthe present invention is useful to treat a variety of types of cancers, such as breast cancer, ovarian cancer, skin cancer (for example, melanoma), cancer ofthe GI mucosa (such as colon cancer); neuro/glioblastoma, leukemia, such as acute promyelocytic leukemia (APL), adenocarcinoma, including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer.
  • APL acute promyelocytic leukemia
  • adenocarcinoma including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer.
  • the present invention also relates to packaged pharmaceutical compositions comprising a pharmaceutical composition comprising at least one therapeutic agent that induces apoptosis and/or regression of aneuploidy in cancer cells that overexpress EGFR and erbB2 and a suitable carrier and instructions and/or a label for administration ofthe pharmaceutical composition for the treatment of patients.
  • a further embodiment ofthe invention is a method of treating a patient suffering from cancer in which the cancerous cells overexpress EGFR and erbB2 by administering to the patient a therapeutically effective amount of a therapeutic agent that induces apoptosis and/or aneuploidy regression in the cancer cells.
  • any ofthe therapeutic agents, or a pharmaceutical composition comprising such a therapeutic agent or agents can be admimstered, alone or in conjunction with a chemotherapeutic agent(s), radiation therapy, and/or surgery.
  • One or more therapeutic agent can be admimstered, simultaneously or sequentially.
  • the present invention also encompasses use of therapeutic agent or agents in the manufacture of medicament for the treatment of cancer in which cancer cells overexpressEGFR and erbB2.
  • the therapeutic agent(s) used in the manufacture of the medicament can be any ofthe therapeutic agents described herein.
  • Figure 1 A shows status of EGFR and c-erbB-2 phosphorylation in selected parental 8A1 cells; control vector-transfected Ev-Cv cells containing vector for EGFR and vector for c-erbB-2; single AS-EGFR-transfected E2 cells; single AS-c- erbB-2-transfected C7 cells; and double AS-EGFR/AS-cerbB-2-transfected E-C32.d and E-C32.2 cells.
  • Phosphorylation of EGFR and c-erbB-2 is detected by immunoprecipitation (IP) using the phospho-tyrosine (PTY)-specific 4G10 monoclonal antibody, followed by Western blot (WB) analysis with the EGFR-specific Ab-12 monoclonal antibody, or the c-erbB-2-s ⁇ ecific Ab-18 monoclonal antibody, respectively.
  • IP immunoprecipitation
  • WB Western blot
  • Equal protein-adjusted (normalized) cell lysates were used for all experiments. The data represent replicate analysis.
  • Figure IB (Supplementary CGH Analysis, panels a-c) show chromosomal imbalances detected by CGH in a) 8A1 cells; b) E-C32.d transfectants compared to 8A1 cells that was used as a reference DNA , and c) E-C32.2 transfectants versus 8A1 cells. Losses are shown to the left (red vertical lines), gains are shown to the right (green vertical lines) ofthe chromosome ideograms.
  • Figure 2 shows biological consequences of simultaneous suppression of EGFR and c-erbB-2 in ovarian cancer cells, a, Matrigel outgrowth of parental 8A1 cells, and b, Matrigel outgrowth ofthe E-C32.2 double transfectant simultaneously transfected with AS-EGFR and AS-c-erbB-2.
  • the images are the results of three independent experiments, c, MMP-9 activity in parental 8A1 cells compared to single and double transfectants by zymogram analysis when seeding an equal number of cells. The experiment was repeated three times. Similar result was also obtained with E-C32.d double transfected clone.
  • Figure 3 shows cytogenetic analysis of parental 8A1 cells and double transfectant clones E-C32.d and E-C32.2. Metaphase spreads of a, 8A1 cells (58 chromosomes) after FISH with chromosome 16 (green) and 20 (red) specific painting probes; and b, (61 chromosomes) with chromosome 16q (green) and 20q (red) locus specific probes; c, d, e, multiple figures of apoptosis seen in the clone E- C32.d showing: c, chromosomal pulverization (note the presence of 3 copies of 20q probe).
  • Figure 6 shows apoptosis detected by AnnexinV/PI staining and PARP cleavage, a, AnnexinV/ PI staining was determined in the parental 8A1 cells, the single (E2, C7) and double transfectant (E-C32.d and E-C32.2) clones by FACS analysis.
  • This analysis allowed flow cytometric quantitation of apoptosis occurring in the parental 8A1 cells, and cells transfected individually (E2 (AS- EGFR) and C7 (AS-c-erbB-2) cells) or simultaneously (EC32.d and EC32.2 cells (AS-EGFR/AS-c-erB-2)) with the AS-EGFR and/or the AS-c-erbB-2 constructs.
  • the four proteins are widely expressed in epithelial, mesenchymal and neuronal tissues and play fundamental roles during development (Gassmann et al., 1995, Nature, 378: 390- 394; Lee et al., 1995, Nature, 378: 394-398; Sibilia and Wagner, 1995, J. Biol.
  • a direct consequence of ligand binding to the ECD is the formation of receptor dimers and stimulation ofthe intrinsic kinase activity, which leads to the phosphorylation of tyrosine residues in the intracellular domain ofthe receptors (van der Geer et al, 1994, Annu. Rev. Cell Biol, 10: 251-337). These serve as docking sites for a number of SH2- and PTB-domain containing proteins (Kavanaugh and Williams, 1994, Science, 266: 1862-1865; Cohen, GB.
  • ErbB ligands can be classified into three groups and include: EGF and heparin binding EGF-like growth factor (HB- EGF), which bind ErbB-1 (Savage et al., 1972, J Biol Chem., 241: 7612-7621; Higashiyama et al., 1991, Science, 251: 936-939); betacellulin (BTC), which is a ligand of ErbB-1 and ErbB-4 (Shing et al., 1993, Science, 259: 1604-1607; Beerli and Hynes, 1996, J Biol Chem., 271: 6071-6076; Riese et al.
  • EGF and heparin binding EGF-like growth factor HB- EGF
  • BTC betacellulin
  • ErbB-2 potentiates and prolongs the signal transduction pathways elicited by EGF and ⁇ DF (Beerli et al., 1995, Mol Cell Biol, 15: 6496-6505; Graus-Porta et al; 1995, Mol. Cell. Biol, 15: 1182-1191; Karunagaran et al., 1996, EMBOJ, 15: 254-264).
  • ErbB-2 increases the affinity of both EGF and ⁇ DF for their receptors (Wada et al., 1990, Cell, 61: 1339-1347; Sliwkosky et al, 1994, J. Biol.
  • ErbB-2 is the preferred heterodimerization partner of all ErbB proteins.
  • NDF-activated ErbB-3 or ErbB-4 heterodimerizewith ErbB-1 only when no ErbB-2 is available. If all ErbB receptors are present, NDF receptors preferentially dimerize with ErbB-2.
  • EGF- and BTC-induced activation of ErbB-3 is impaired in the absence of ErbB-2, suggesting that ErbB-2 has a role in the lateral transmission of signals between other ErbB receptors.
  • ErbB-1 activated by all EGF-related peptides couples to SHC, whereas only ErbB-1 activated by its own ligands associates with and phosphorylates Cbl.
  • the current invention is based in part on the discovery that simultaneous targeting of EGFR and erbB2 signaling pathway contributes to increased apoptosis in aneuploid cancer cells and regression of aneuploidy in near-diploid cancer cells, resulting in death of aneuploid cells and rescue of near-diploid cancer cells.
  • apoptosis or "programmed cell death,” refers to the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Apoptosis, is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise ("cellular suicide"). Cells undergoing apoptosis show characteristic morphological and biochemical features.
  • apoptotic bodies membrane bound vesicles
  • apoptotic bodies membrane bound vesicles
  • apoptotic bodies contain ribosomes, morphologically intact mitochondria and nuclear material.
  • these apoptotic bodies are rapidly recognized and phagocytized by either macrophages or adjacent epithelial cells. Due to this efficient mechanism for the removal of apoptotic cells in vivo, no inflammatory response is elicited.
  • the apoptotic bodies as well as the remaining cell fragments ultimately swell and finally lyse. This terminal phase of in vitro cell death has been termed "secondary necrosis.”
  • aneuploid refers to the characteristic of having a chromosome complement that is not an exact multiple ofthe haploid number, with either fewer or more than the normal number of chromosomes in the cell.
  • chromosomes may be present in multiple copies (e.g. Trisomy) or one of a homologous pair may be missing in a diploid cell.
  • aneuploid or all grammatical variations thereof refers to a severe form of chromosomal complement change, such that an aneuploid cell contains on average 5 or more, preferably 8, 10, 20, 40, 50, or even 60 more chromosomes (either in whole or part) than a normal diploid cell.
  • near diploid or all its grammatical variations refers to a less severe form of chromosomal complement change, such that a near diploid cell contains on average no more or no less than 3, preferably 2, or even 1 chromosome(s) (either in whole or part) when compared to a normal diploid cell.
  • c-erb-B2 "erbB2,” or “erbB-2” or “c-erbB-2” or any grammatical variants are used interchangeably.
  • EGFR "erbBl,” “erb Bl,” or any grammatical variants are used interchangeably.
  • Applicant has shown that simultaneous suppression of phosphorylated EGFR and c-erbB2 in human tumor cells that overexpress EGFR and c-erbB2 induces apoptosis of aneuploid cell population and results in survival of near-diploid cells in the population.
  • Applicant has unexpectedly shown genomic rescue of near-diploid cancer.cells and apoptosis of aneuploid cancer cells as a result of simultaneous suppression of phosphorylated EGFR and c-erbB2 in human tumor cells.
  • Simultaneous inhibition of EGFR and erbB2 provides a means of overcoming functional cooperation of these receptors and, surprisingly, inducing apoptosis of aneuploid cells and genomic rescue (regression toward normal/diploid genotype with the result that highly aneuploid cells die and more nearly diploid cells survive.
  • simultaneous targeting of EGFR and erbB2 is a method that results in a trend toward normalization of the karyotype and activation of apoptotic pathways favoring a mechanism involving cell selection.
  • the instant invention provides a method of cancer treatment by simultaneously inhibiting the function of a selected pair of erbB family receptors in cancer cells.
  • the method selectively eliminates highly aneuploid tumor cells by apoptosis (or "programmed cell death"), while rescuing normal or diploid cells and less-severely damaged, near diploid tumor cells, which, as a result, exhibit more benign phenotypes.
  • one aspect ofthe present invention provides a method for treating tumors expressing EGFR and erbB2 receptor, hi one embodiment, the treatment induces apoptosis in aneuploid cells and regression of aneuploidy in near- diploid tumor cells.
  • Such method comprises administering an amount of an EGFR therapeutic agent (at least one agent and an erbB2 therapeutic agent and at least one agent that blocks signaling through the erbB2 pathway) that blocks signaling through the EGFR pathway, sufficient to reduce the aneuploidy and increase apoptosis of cancer cells (e.g., tumor cells).
  • a therapeutic agent may exert its inhibitory effect by inhibiting the interaction between EGFR and erbB2 with each other and/or their ligands, inhibiting gene expression of EGFR and erbB2, inhibiting activity of EGFR and erbB2, inhibiting clustering of EGFR and erbB2, inhibiting phosphorylation of EGFR and erbB2, or inhibiting any downstream signaling event upon binding of EGFR and erbB2.
  • the therapeutic agents useful in this method are described in greater detail herein.
  • a therapeutic agent can affect EGFR, erbB2 or both, hi the instances in which a therapeutic agent has both anti- EFGR and anti-erbB2 activity, only a single agent need be administered to induce apoptosis and aneuploid cells and aneuploidy regression in near-diploid cells and, as a result, treat cancer by killing highly aneuploid cells and rescuing non-diploid cells.
  • Two or more therapeutic agents e.g., an anti-EGFR agent and an anti-erbB2 agent; two or more anti-EGFR agents and one anti-erbB2 agent; one anti-erbB2 agent; and two or more of each type of agents can be administered according to the method of present invention.
  • EGFR therapeutic agent or "erbB2 therapeutic agent” is a generic term which include any compound or molecule (agent) which blocks signaling through the EGFR and/or erbB2 pathway.
  • anti- tumor therapeutic agents can inhibit functions (activity or expression) of erbB2 or EGFR, inhibit the interaction of erbB2 or EGFR with each other and/or with their ligands, inhibit the phosphorylation of erbB2 or EGFR, or inhibit any of the downstream signaling events upon binding of ligands for erbB2 or EGFR.
  • substances that act as antagonists of erbB2 or EGFR may be used as anti- tumor therapeutic agents.
  • anti-tumor therapeutic agents include, but are not limited to, a protein, a peptide, a small organic molecule, a peptidomimetic, an antibody, and a nucleic acid.
  • expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
  • expression of a protein coding sequence results from transcription and translation ofthe coding sequence.
  • “Inhibition of gene expression” refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene.
  • Specificity refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination ofthe outward properties ofthe cell or organism (as presented below in the examples) or by biochemical techniques, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
  • biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
  • EGFR and or erbB2 has been implicated in a number of cancers, including: breast cancer (see van de Vijver et al., New Eng. J. Med. 319: 1239-1245, 1988, also see Slamon et al, Science 244: 707-712, 1989; Yu et al., Molec. Cell 2: 581-591, 1998); ovarian cancer (Slamon et al., Science 244: 707-712, 1989); skin cancer (for example, melanoma), see Sibilia et al., Cell 102: 211-220, 2000; cancer ofthe GI mucosa (such as colon cancer, see Pai et al, Nature Med.
  • neuro/glioblastoma Yang-Feng et al., Cytogenet. Cell Genet. 40: 784 only, 1985
  • leukemia such as acute promyelocytic leukemia (APL)
  • APL acute promyelocytic leukemia
  • adenocarcinoma Akiyama et al., Science 232: 1644- 1646, 1986), including adenocarcinoma ofthe salivary gland (Semba et al., Proc. Nat. Acad. Sci. 82: 6497-6501, 1985
  • gastric cancer Flushige et al., Biochem. Biophys. Res.
  • the instant invention can be used to treat a variety of different cancers, especially cancers that express at least one ofthe EGFR or erbB2 receptors as listed above.
  • the therapeutic agent is formulated with a pharmaceutically acceptable carrier.
  • at least one additional chemotherapeutic agent that affects growth ofthe tumor cells in an additive or synergistic manner with a therapeutic agent is administered with the therapeutic agent(s).
  • methods ofthe present invention may further include ionizing radiation or any other cancer therapy (e.g., surgery) known in the art.
  • Another aspect ofthe present invention provides a packaged pharmaceutical.
  • Such packaged pharmaceutical comprises: (i) an EGFR therapeutic agent and an erbB2 therapeutic agent, preferably in therapeutically effective amounts; and (ii) instructions and/or a label for administration ofthe therapeutic agents for the treatment of patients having tumors that express EGFR and erbB2.
  • the term "patient” or “subject” to be treated by the method of the invention can mean either a human or non-human animal.
  • “Animal” refers to mammals, preferably mammals such as humans.
  • the therapeutic agent blocks signaling through the EGFR / erbB2 pathways, for example, by inhibiting the interaction between EGFR and erbB2 with each other and/or their ligands, inhibiting gene expression of EGFR and erbB2, inhibiting activity of EGFR and erbB2, inhibiting clustering of EGFR and erbB2, inhibiting phosphorylation of EGFR and erbB2, or inhibiting any downstream signaling event upon binding of EGFR and erbB2.
  • Another aspect of the present invention provides a method for treating a patient (individual) suffering from a cancer, especially in patient suffering from a cancer in which EGFR and erbB2 are overexpressed.
  • the method comprises treating the patient with therapeutically effective amount of an EGFR therapeutic agent and an erbB2 therapeutic agent.
  • the method may further include assessing the EGFR and erbB2 status of a sample of tumor cells from a patient before said treatment, so that only patients overexpressing both receptors are treated.
  • the EGFR and/or erbB2 status includes, but is not limited to, gene expression of EGFR and/or erbB2, activity of EGFR and/or erbB2 (e.g., protein-protein interaction, clustering, phosphorylation, etc.), and any downstream signaling through the EGFR and/or erbB2 pathway.
  • the present invention also relates to methods of treating an individual (patient) in whom cells overexpress EGFR and erbB2 and, therefore, is in what can be seen to be a precancerous state or condition.
  • a therapeutic agent(s) that inhibits EGFR and a therapeutic agent(s) that inhibits cerbB2 By administering a therapeutic agent(s) that inhibits EGFR and a therapeutic agent(s) that inhibits cerbB2, the progression ofthe precancerous cell to become a cancer cell can be prevented or limited (the progression will occur to a lesser extent that would occur without the treatment).
  • This approach is useful, for example, in individuals at risk for developing a cancer in which EGFR and erbB2 are overexpressed, such as a woman whose family history puts her at risk for developing ovarian or breast cancer.
  • “Overexpression” generally means having a higher expression level than the "normal” level in “normal” cells.
  • “overexpression of EGFR” in a cancer cells means expression of the EGFR protein and/or mRNA is at least 10% more, preferably 20%, 50%, 100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold or more when compared to normal EGFR protein and/or mRNA levels in comparable normal cells.
  • cancer cells are compared to the same types of normal cells.
  • breast carcinoma cancer cells are compared to normal breast epithelial cells, preferably normal breast epithelial cells from the same patient.
  • EGFR and/or erbB2 DNA may be amplified in cancer cells, leading to overexpression of receptor mRNA and/or protein.
  • the quantitation of expression level of mRNA and/or protein can be done using routine methods well-known in the art, such as Northern and Western blots, or commercial kits.
  • Yet another aspect of the present invention provides use of an EGFR therapeutic agent and an erbB2 therapeutic agent in the manufacture of medicament for the treatment of cancer that expresses EGFR and erbB2.
  • the EGFR/erbB2 therapeutic agent(s) block signaling through the EGFR and erbB2 pathway, for example, by inhibiting the interaction between EGFR and or erbB2 with their ligands or with each other, by inhibiting gene expression of EGFR and/or erbB2, by inhibiting activity of EGFR and/or erbB2, by inhibiting clustering of EGFR and/or erbB2, by inhibiting phosphorylation of EGFR and/or erbB2, or by inhibiting any downstream signaling event upon binding of EGFR and/or erbB2 to their ligands.
  • Exemplary cancers include, but are not limited to, breast cancer, ovarian cancer, skin cancer (for example, melanoma), cancer ofthe GI mucosa (such as colon cancer); neuro/glioblastoma, leukemia, such as acute promyelocytic leukemia (APL), adenocarcinoma, including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer.
  • skin cancer for example, melanoma
  • cancer ofthe GI mucosa such as colon cancer
  • neuro/glioblastoma such as leukemia, such as acute promyelocytic leukemia (APL)
  • APL acute promyelocytic leukemia
  • adenocarcinoma including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer.
  • the EGFR/erbB2 therapeutic agents ofthe present invention include any compound, (agent) which blocks the EGFR receptor and erbB2 receptor signaling.
  • therapeutic agents can inhibit functions (activity or expression), interactions between EGFR/erbB2 and their ligands, interactions between the EGFR and the erbB2 receptors, or phosphorylation ofthe EGFR/erbB2 receptors, or inhibit any ofthe downstream signaling events upon binding of EGFR/ erbB2 with their ligands.
  • substances that act as antagonists of EGFR and/or erbB2 may be used as anti-tumor therapeutic agents.
  • Such EGFR/erbB2 therapeutic agents include, but are not limited to, a protein, a peptide, a small organic molecule, a peptidomimetic, an antibody, and a nucleic acid (e.g., antisense, nucleic acid, siRNA or vectors expressing siRNA).
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • the EGFR therapeutic agents and erbB2 therapeutic agents include a polypeptide which is a mutated form, a mimic or a fragment of EGFR and/or erbB2 protein.
  • Such therapeutic agents can bind to natural ligands of EGFR and/or erbB2, and prevent the interaction between EGFR and/or erbB2 with their ligands or with each other.
  • the EGFR therapeutic agents and/or the erbB2 therapeutic agents may include a soluble polypeptide having the amino acid sequence ofthe extracellular domain of EGFR and/or erbB2, or a sufficient portion ofthe extracellular domain, or a polypeptide having an amino acid sequence conferring a similar enough conformation to allow specific binding to ligands of EGFR and/or erbB2. Since the ligand binding region of EGFR and erbB2 are known or can be readily determined using art-recognized techniques (such as in vitro binding assay using fragments ofthe receptors and the natural ligands), such dominant negative mutated form, mimic or fragments can be readily made without undue experimentation.
  • antagonists which comprise the extracellular domain of EGFR and/or erbB2 but do not possess clustering activity can be produced as EGFR and/or erbB2 therapeutic agents.
  • EGFR and/or erbB2 therapeutic agents may be a fusion (hybrid) protein.
  • the extracellular domain of EGFR and/or erbB2 may be fused to the Fc domain of human IgG. Non-clustered forms of these hybrid proteins act as antagonists and thus can be used for anti-tumor purposes.
  • non-clustered forms ofthe hybrid proteins may bind the ligand and/or a wild-type receptor, leading to the formation of a non-functional receptor- ligand complex, thus antagonizing EGFR and/or erbB2 signaling.
  • the extracellular domain that do not possess clustering activity can be identified using a number of routine art-recognized technologies. For example, progressive deletion analysis of smaller and smaller portions ofthe extracellular domain can be used to identify regions ofthe ECD that are required for receptor multimerization. Similarly, Ala scanning mutagenesis may be used to identify point mutations that are detrimental to ligand binding or receptor multimerization (which can be assessed using, for example, yeast two-hybrid assays).
  • the EGFR therapeutic agents and the erbB2 therapeutic agents can be antibodies, such as antibodies that are specifically reactive with EGFR, erbB2, or both.
  • Antibodies may be polyclonal or monoclonal; intact or truncated, e.g., F(ab')2, Fab, Fv; xenogeneic, allogeneic, syngeneic, or modified forms thereof, such as humanized or chimeric antibodies.
  • these antibodies may be encoded by polynucleotides, and expressed upon transfection of such polynucleotides into the target cancer cell.
  • these antibodies or fragments thereof may bind the surface receptors, and antagonize receptor function by receptor intemahzation.
  • immune system ofthe host may recognize and eliminate such Ab- engaged cancer cells, through, for example, natural killer cells (NK cells).
  • NK cells natural killer cells
  • anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form ofthe peptide (e.g., a polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein).
  • an immunogenic form ofthe peptide e.g., a polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein.
  • a full-length or an immunogenic portion of an EGFR polypeptide an erbB2 polypeptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • antibodies ofthe invention are specific for the extracellular portion of the EGFR and/or erbB2 protein, hi another embodiment, antibodies ofthe invention are specific for the intracellular portion or the transmembrane portion ofthe EGFR and/or erbB2 protein.
  • antibody-producing cells can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an EGFR and/or erbB2 polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with an EGFR polypeptide an erbB2 polypeptide or both.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab) 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab) 2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • An antibody ofthe present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an EGFR polypeptide or erbB2 polypeptide conferred by at least one CDR region ofthe antibody. Techniques for the production of single chain antibodies (US Patent No.
  • an antibody ofthe invention is a monoclonal antibody, and in certain embodiments the invention makes available methods for generating novel antibodies.
  • a method for generating a monoclonal antibody that binds specifically to an EGFR polypeptide or erbB2 polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the EGFR polypeptide or erbB2 polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody- producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the EGFR and/or erbB2 polypeptide.
  • antibody-producing cells e.g., cells from the spleen
  • a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the EGFR and/or erbB2 polypeptide.
  • the monoclonal antibody may be purified from the cell culture.
  • an antibody to be used for certain therapeutic purposes will preferably be able to target an antigen on a particular cell type, as opposed to antigen in solution. Accordingly, to obtain antibodies of this type, it maybe desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g., by fluorescence activated cell sorting), or at least confirm that the antibody can bind to EGFR or erbB2 receptor on cell surface.
  • a variety of different techniques are available for testing antibody: antigen interactions to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g.
  • Biacore binding assay Bia-core AB, Uppsala, Sweden
  • sandwich assays e.g. the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland
  • Western blots immunoprecipitation assays and immunohistochemistry.
  • the EGFR therapeutic agents and erbB2 therapeutic agents ofthe present invention comprise a nucleic acid.
  • the invention relates to the use of antisense nucleic acid complementary to polynucleotides encoding EGFR and/or erbB2 polypeptides or variants thereof, to decrease expression of EGFR and/or erbB2.
  • an antisense nucleic acid can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion ofthe cellular mRNA which encodes an EGFR polypeptide or erbB2 polypeptide.
  • the construct is an oligonucleotide (DNA or RNA) which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding an EGFR and/or erbB2 polypeptide.
  • oligonucleotides are optionally modified oligonucleotide which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and is therefore stable in vivo.
  • Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos.
  • RNAi constructs comprise double stranded RNA that can specifically block expression of a target gene.
  • RNAi provides a useful method of inhibiting gene expression in vitro or in vivo.
  • RNAi constructs can comprise either long stretches of dsRNA identical or substantially identical to the target nucleic acid sequence or short stretches of dsRNA identical or substantially identical to only a region of the target nucleic acid sequence.
  • RNAi construct is a generic term including small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form siRNAs.
  • RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts which can produce siRNAs in vivo.
  • the RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions ofthe cell to the nucleotide sequence of at least a portion ofthe mRNA transcript for the gene to be inhibited (i.e., the "target" gene).
  • the double-stranded RNA need only be sufficiently similar to natural RNA that it mediate RNAi under the conditions used (such as physiological condition).
  • the RNAi construct can include sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence.
  • the number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center ofthe siRNA duplex are most critical and may essentially abolish cleavage ofthe target RNA. In contrast, nucleotides at the 3' end ofthe siRNA strand that is complementary to the target RNA do not significantly contribute to specificity ofthe target recognition.
  • Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90%) sequence identity, or even 100% sequence identity, between the inhibitory
  • RNA and the portion ofthe target gene is preferred.
  • the duplex region ofthe RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion ofthe target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C hybridization for 12-16 hours; followed by washing).
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m (melting temperature) of 55°C
  • T m melting temperature
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5 ⁇ or 6 ⁇ SSC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5 ⁇ or 6 ⁇ SSC.
  • SSC is a 0.15M NaCl, 0.015M Na-citrate.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
  • standard hybridization conditions refers to a T m of about 55°C, and utilizes conditions as set forth above.
  • the T m is 60°C; in a more preferred embodiment, the T m is 65°C.
  • “high stringency” refers to hybridization and/or washing conditions at 68°C in 0.2xSSC, at 42°C in 50% formamide, 4 ⁇ SSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
  • Suitable hybridization conditions for oligonucleotides are typically somewhat different than for full- length nucleic acids (e.g., full-length cDNA), because of the oligonucleotides' lower melting temperature. Because the melting temperature of oligonucleotides will depend on the length of the oligonucleotide sequences involved, suitable hybridization temperatures will vary depending upon the oligoncucleotide molecules used.
  • Exemplary temperatures may be 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides) and 60°C (for 23 -base oligonucleotides).
  • Exemplary suitable hybridization conditions for oligonucleotides include washing in 6 ⁇ SSC/0.05% sodium pyrophosphate, or other conditions that afford equivalent levels of hybridization.
  • the double-stranded structure may be formed by a single self- complementary RNA strand or two complementary RNA strands.
  • RNA duplex formation may be initiated either inside or outside the cell.
  • the RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region ofthe RNA are targeted for genetic inhibition.
  • RNAi constructs can be "small interfering RNAs" or "siRNAs.” These nucleic acids are from about 19 to about 30 nucleotides in length, and in specific embodiments 19-23 or 21-23 nucleotides in length.
  • the siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex.
  • the 21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group, hi certain embodiments, the siRNA constructs can be generated by processing of longer double-stranded RNAs, for example, in the presence ofthe enzyme dicer.
  • the Drosophila in vitro system is used, hi this embodiment, dsRNA is combined with a soluble extract derived from Drosophila embryo, thereby producing a combination.
  • the combination is maintained under conditions in which the dsRNA is processed to RNA molecules of from about 19 to about 23 nucleotides or from about 19 to about 23 nucleotides or from about 21 to about 23 nucleotides.
  • the siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs.
  • RNAi constructs can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase ofthe treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro.
  • the RNAi constructs may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties.
  • the phosphodiester linkages of natural RNA may be modified to include at least one of an nitrogen or sulfur heteroatom.
  • RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA.
  • bases may be modified to block the activity of adenosine deaminase.
  • the RNAi construct may be produced enzymatically or by partial total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
  • Methods of chemically modifying RNA molecules can be adapted for modifying RNAi constructs (see, e.g., Heidenreich et al. (1997) Nucleic Acids Res. 25:776- 780; Wilson et al. (1994) J Mol Recog 7:89-98; Chen et al.
  • RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2'-substituted ribonucleosides, a- configuration).
  • At least one strand ofthe siRNA molecules has a 3' overhang of from about 1 to about 6 nucleotides in length, although may be from 2 to 4 nucleotides in length. More preferably, the 3' overhangs are 1-3 nucleotides in length. In certain embodiments, one strand has a 3' overhang and the other strand is blunt-ended or also has an overhang. The length ofthe overhangs may be the same or different for each strand. In order to further enhance the stability ofthe siRNA, the 3' overhangs can be stabilized against degradation, hi one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of uridine nucleotide 3' overhangs by 2 l -deoxythyinidine is tolerated and does not affect the efficiency of RNAi.
  • the absence of a 2' hydroxyl significantly enhances the nuclease resistance ofthe overhang in tissue culture medium and may be beneficial in vivo.
  • the RNAi construct can also be in the form of a long double-stranded RNA.
  • the RNAi construct is at least 25, 50, 100, 200, 300 or 400 bases.
  • the RNAi construct is 400-800 bases in length.
  • the double-stranded RNAs are digested intracellularly, e.g., to produce siRNA sequences in the cell.
  • use of long double-stranded RNAs in vivo is not always practical, presumably because of deleterious effects which may be caused by the sequence-independent dsRNA response.
  • the use of local delivery systems and/or agents which reduce the effects of interferon or PKR are preferred.
  • the RNAi construct is in the form of a hairpin structure (named as hairpin RNA).
  • hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al, Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature. 2002, 418:38-9; McManus et al., RNA. 2002, 8:842-50; Yu et al., Proc Natl Acad Sci U S A, 2002, 99:6047-52).
  • hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.
  • the present invention provides a recombinant vector having the following unique characteristics: it comprises a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene for an RNAi construct of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene f agment in a host cell.
  • RNAi sequences for EGFR and erbB2 are for illustration purposes only. Other similar sequences may be used to achieve similar results. The same approach may be employed to design and produce RNAi sequences for all pairs of target genes ofthe instant invention described above.
  • siRNA sequences for c-erbB-2 (HER2) are for illustration purposes only. Other similar sequences may be used to achieve similar results. The same approach may be employed to design and produce RNAi sequences for all pairs of target genes ofthe instant invention described above.
  • siRNA sequences for c-erbB-2 (HER2) are siRNA sequences for c-erbB-2 (HER2)
  • nts 1840-1860 aactctgagt gcatacagtg c AntiSense 5'-P04 uugagacuca cguaugucac gtt 3' -OH
  • nts 2200-2220 cacatcgttc ggaagcgcac g AntiSense 5'-P04 guguagcaag ccuucgcgug ctt 3' -OH
  • Sense 5' -P04 cacaucguuc ggaagcgcac gtt 3' -OH
  • siRNAs or expression constructs can be constructed by PCR, and the PCR products can then be directly transfected into mammalian cells, resulting in functional expression of siRNAs. This approach should prove useful for identification of other optimal siRNA-target combinations and for multiplexing siRNA expression in mammalian cells.
  • Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • “Host cell” means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell. For example, a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme.
  • Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal).
  • a non-human animal e.g., a transgenic animal or a transiently transfected animal
  • the invention relates to the use of ribozyme molecules designed to catalytically cleave an mRNA transcript to prevent translation of mRNA (see, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225; and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591.
  • the ribozymes ofthe present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and which has been extensively described (see, e.g., Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al, 1986,
  • Cech-type ribozymes such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and which has been extensively described (see, e.g., Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al, 1986,
  • the invention relates to the use of DNA enzymes to inhibit expression of EGFR and/or erbB2 gene.
  • DNA enzymes incorporate some of the mechanistic features of both antisense and ribozyme technologies. DNA enzymes are designed so that they recognize a particular target nucleic acid sequence, much like an antisense oligonucleotide. However much like ribozymes, they are catalytic and specifically cleave the target nucleic acid.
  • the unique or substantially unique target sequence is a G/C rich sequence of approximately 18 to 22 nucleotides. High G/C content helps insure a stronger interaction between the DNA enzyme and the target sequence.
  • the specific antisense recognition sequence that will target the enzyme to the message is divided so that it comprises the two arms ofthe DNA enzyme, and the DNA enzyme loop is placed between the two specific arms.
  • the EGFR and erbB2 therapeutic agents ofthe present invention include a small molecule (e.g., a peptidomimetic).
  • small molecules include, but are not limited to, small peptides or peptide-like molecules (e.g., a peptidomimetic).
  • peptidomimetic includes chemically modified peptides and peptide-like molecules that contain non-naturally occurring amino acids, peptoids, and the like.
  • Peptidomimetics provide various advantages over a peptide, including enhanced stability when administered to a subject. Methods for identifying a peptidomimetic are well known in the art and include the screening of databases that contain libraries of potential peptidomimetics.
  • the Cambridge Structural Database contains a collection of greater than 300,000 compounds that have known crystal structures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)). Where no crystal structure of a target molecule is available, a structure can be generated using, for example, the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)).
  • CONCORD Rule et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)
  • Another database the Available Chemicals Directory (Molecular Design Limited, Informations Systems; San Leandro Calif.), contains about 100,000 compounds that are commercially available and also can be searched to identify potential peptidomimetics of CCL21 or a chemokine receptor.
  • small molecule compounds may encompass numerous chemical classes, although typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, sulfhydryl or carboxyl group.
  • Candidate small molecule compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds can be modified through conventional chemical, physical, and biochemical means.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, and amidification, to produce structural analogs.
  • the present invention also contemplates anti-tumor therapeutic agents obtainable from the screening methods described below.
  • test agents to be assessed for their anti-tumor effects can be any chemical (element, molecule, compound, drug), made synthetically, made by recombinant techniques or isolated from a natural source.
  • test agents can be peptides, polypeptides, peptoids, sugars, hormones, or nucleic acid molecules (such as antisense or RNAi nucleic acid molecules).
  • test agents can be small molecules or molecules of greater complexity made by combinatorial chemistry, for example, and compiled into libraries.
  • Test agents can also be natural or genetically engineered products isolated from lysates or growth media of cells — bacterial, animal or plant ⁇ or can be the cell lysates or growth media themselves. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps.
  • an assay can be carried out to screen for compounds that specifically inhibit binding of EGFR and/or erbB2 to their ligand, or vice- versa, such as by inhibition of binding of labeled ligand- or receptor-Fc fusion proteins to immortalized cells.
  • Compounds identified through this screening can then be tested in animal models of cancer (e.g., tumor xenografts implanted in nude mice) to assess their anti-tumor activity in vivo.
  • samples of cells expressing EGFR and/or erbB2 are contacted with either labeled ligand (e.g., full-length, or a soluble portion thereof, or a fusion protein such as a fusion ofthe extracellular domain and the Fc domain of IgG) or labeled ligand plus a test compound (or group of test compounds).
  • labeled ligand e.g., full-length, or a soluble portion thereof, or a fusion protein such as a fusion ofthe extracellular domain and the Fc domain of IgG
  • test compound or group of test compounds
  • a lesser amount of label (where the label can be, for example, a radioactive isotope, a fluorescent or colormetric label) in the sample contacted with the test compound(s) is an indication that the test compound(s) interferes with binding.
  • the reciprocal assay using cells expressing a ligand e.g., an EGFR and/or erbB2 ligand or a soluble form thereof
  • a ligand e.g., an EGFR and/or erbB2 ligand or a soluble form thereof
  • An assay to identify a substance which interferes with interaction between an EGFR and or erbB2 with their ligand can be performed with the component (e.g., cells, purified protein, including fusion proteins and portions having binding activity) which is not to be in competition with a test compound, linked to a solid support.
  • the solid support can be any suitable solid phase or matrix, such as a bead, the wall of a plate or other suitable surface (e.g., a well of a microtiter plate), column pore glass (CPG) or a pin that can be submerged into a solution, such as in a well.
  • Linkage of cells or purified protein to the solid support can be either direct or through one or more linker molecules.
  • an isolated or purified protein e.g., an EGFR and/or erbB2 receptor
  • a suitable affinity matrix by standard techniques, such as chemical cross-linking, or via an antibody raised against the isolated or purified protein, and bound to a solid support.
  • the matrix can be packed in a column or other suitable container and is contacted with one or more compounds (e.g., a mixture) to be tested under conditions suitable for binding ofthe compound to the protein. For example, a solution containing compounds can be made to flow through the matrix.
  • the matrix can be washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds. Compounds which remain bound can be released by a suitable elution buffer.
  • the elution buffer can comprise a release component or components designed to disrupt binding of compounds (e.g., one or more ligands or receptors, as appropriate, or analogs thereof which can disrupt binding or competitively inhibit binding of test compound to the protein).
  • a release component or components designed to disrupt binding of compounds e.g., one or more ligands or receptors, as appropriate, or analogs thereof which can disrupt binding or competitively inhibit binding of test compound to the protein).
  • Fusion proteins comprising all of, or a portion of, a protein (e.g., an EGFR and/or erbB2 receptor) linked to a second moiety not occurring in that protein as found in nature can be prepared for use in another embodiment ofthe method.
  • Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope).
  • the fusion proteins can be produced by inserting the protein (e.g., an EGFR and/or erbB2 receptor) or a portion thereof into a suitable expression vector which encodes an affinity ligand.
  • the expression vector can be introduced into a suitable host cell for expression. Host cells are disrupted and the cell material, containing fusion protein, can be bound to a suitable affinity matrix by contacting the cell material with an affinity matrix under conditions sufficient for binding ofthe affinity ligand portion ofthe fusion protein to the affinity matrix.
  • a fusion protein can be immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion ofthe fusion protein to the matrix, and is contacted with one or more compounds (e.g., a mixture) to be tested, under conditions suitable for binding of compounds to the receptor or ligand protein portion ofthe bound fusion protein.
  • the affinity matrix with bound fusion protein can be washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds without significantly disrupting binding of specifically bound compounds.
  • Compounds which remain bound can be released by contacting the affinity matrix having fusion protein bound thereto with a suitable elution buffer (a compound elution buffer).
  • a suitable elution buffer a compound elution buffer
  • compound elution buffer can be formulated to permit retention ofthe fusion protein by the affinity matrix, but can be formulated to interfere with binding ofthe compound(s) tested to the receptor or ligand protein portion ofthe fusion protein.
  • a change in the ionic strength or pH ofthe elution buffer can lead to release of compounds
  • the elution buffer can comprise a release component or components designed to disrupt binding of compounds to the receptor or ligand protein portion ofthe fusion protein (e.g., one or more ligands or receptors or analogs thereof which can disrupt binding of compounds to the receptor or ligand protein portion ofthe fusion protein).
  • Immobilization can be performed prior to, simultaneous with, or after contacting the fusion protein with compound, as appropriate.
  • Various permutations ofthe method are possible, depending upon factors such as the compounds tested, the affinity matrix selected, and elution buffer formulation.
  • fusion protein with compound bound thereto can be eluted from the affinity matrix with a suitable elution buffer (a matrix elution buffer).
  • a suitable elution buffer a matrix elution buffer
  • the fusion protein comprises a cleavable linker, such as a thrombin cleavage site
  • cleavage from the affinity ligand can release a portion ofthe fusion with compound bound thereto.
  • Bound compound can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction.
  • other assays can be used for screening for compounds that decrease the expression level (protein or nucleic acid) of EGFR and/or erbB2.
  • Methods of detecting and optionally quantitating proteins can be achieved by techniques such as antibody-based detection assays, hi these cases, antibodies may be used in a variety of detection techniques, including enzyme-linked immunosorbent assays (ELISAs), immunoprecipitations, and Western blots.
  • ELISAs enzyme-linked immunosorbent assays
  • methods of detecting and optionally quantitating nucleic acids generally involve preparing purified nucleic acids and subjecting the nucleic acids to a direct detection assay or an amplification process followed by a detection assay. Amplification may be achieved, for example, by polymerase chain reaction (PCR), reverse transcriptase (RT), and coupled RT-PCR.
  • Detection of nucleic acids is generally accomplished by probing the purified nucleic acids with a probe that hybridizes to the nucleic acids of interest, and in many instances, detection involves an amplification as well.
  • Northern blots, dot blots, microarrays, quantitative PCR, and quantitative RT-PCR are all well known methods for detecting nucleic acids.
  • one or more compounds can be tested simultaneously. Where a mixture of compounds is tested, the compounds selected by the foregoing processes can be separated (as appropriate) and identified by suitable methods (e.g., PCR, sequencing, chromatography). Large combinatorial libraries of compounds (e.g., organic compounds, peptides, nucleic acids) produced by combinatorial chemical synthesis or other methods can be tested (see e.g., Ohlmeyer, M.H.J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S.H. et al., Proc. Natl. Acad. Sci.
  • the instant invention also provides a method to identify agents that inhibits both EGFR and erbB2 receptors.
  • a plurality of agents are first identified as EGFR antagonists using any ofthe above described methods. Then these EGFR antagonists may be further tested, using any ofthe above methods, for their ability to inhibit erbB2.
  • the method can be carried out in reverse (first identify erbB2 antagonists, then identify among these antagonists EGFR inhibitors).
  • the present invention provides methods of treating an individual suffering from cancer through administering to the individual a therapeutically effective amount of an EGFR and/or erbB2 therapeutic agent as described above.
  • the invention provides methods of preventing or reducing the onset of cancer in an individual through administering to the individual a therapeutically effective amount of an EGFR therapeutic agent and a therapeutically effective amount of an erbB2 therapeutic agent. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.
  • one or more EGFR and/or erbB2 therapeutic agents can be administered, together (simultaneously) or at different times (sequentially), hi addition, EGFR and/or erbB2 therapeutic agents can be administered with another type of compounds for treating cancer.
  • the two types of compounds may be administered simultaneously or sequentially.
  • gene therapy may be applicable with the use of nucleic acids encoding EGFR and/or erbB2 therapeutic polypeptides (for example, fragments of EGFR and/or erbB2, see below).
  • an antisense nucleic acid or an RNAi construct can be used for reducing or inhibiting expression of EGFR and/or erbB2.
  • gene therapy is specific for tumor tissues.
  • Methods ofthe present invention can be used to treat a variety of cancer types, including but not limited to: breast cancer, ovarian cancer, skin cancer (for example, melanoma), cancer ofthe GI mucosa (such as colon cancer); neuro/glioblastoma, leukemia, such as acute promyelocytic leukemia (APL), adenocarcinoma, including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer.
  • EGFR and or erbB2 therapeutic agents can be also combined with VEGF inhibitors.
  • compositions In certain embodiments, EGFR and/or erbB2 therapeutic agents of the present invention are formulated with a pharmaceutically acceptable carrier or salt.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention, e.g., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • Such therapeutic agents can be administered alone or as a component of a pharmaceutical formulation (composition).
  • composition may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • the compound included in the pharmaceutical preparation may itself be active, or may be a prodrug.
  • prodrug refers to compounds which, under physiological conditions, are converted into therapeutically active agents.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • compositions ofthe EGFR and/or erbB2 therapeutic agents include those suitable for oral/ nasal, topical, parenteral and/or intravaginal administration.
  • the pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount ofthe compound which produces a therapeutic effect.
  • compositions or compositions include combining an anti-tumor therapeutic agent and a carrier and, optionally, one or more accessory ingredients.
  • the pharmaceutical compositions can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an artemisinin-related compound as an active ingredient.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • an artemisinin-related compound may also be administered as a bolus, electuary or paste.
  • one or more EGFR and/or erbB2 therapeutic agents ofthe present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5)
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tefrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • therapeutic agents or pharmaceutical compositions can be topically, either to skin or to mucosal membranes, such as those ofthe cervix and vagina.
  • the topical pharmaceutical compositions may further include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers.
  • Examples of these are 2-pyrrolidone, N-methyl-2- pynolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an artemisinin-related compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an EGFR and/or erbB2 therapeutic agent, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • compositions suitable for parenteral administration may comprise one or more anti-tumor therapeutic agents in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood ofthe intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance ofthe required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • microorganisms Prevention ofthe action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, hi addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • isotonic agents such as sugars, sodium chloride, and the like into the compositions, hi addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
  • Injectable depot forms are made by forming microencapsule matrices of one or more anti-tumor therapeutic agents in biodegradable polymers such as polylactide-polyglycohde. Depending on the ratio of drug to polymer, and the nature ofthe particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable pharmaceutical compositions are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • biodegradable polymers such as polylactide-polyglycohde.
  • Depot injectable pharmaceutical compositions are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • compositions for intravaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds ofthe invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum
  • the subject methods ofthe invention can be used alone.
  • the subject methods may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor).
  • proliferative disorders e.g., tumor
  • such methods can be used in prophylactic cancer prevention, prevention of cancer recunence and metastases after surgery, and as an adjuvant of other conventional cancer therapy.
  • conventional cancer therapies e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery
  • an EGFR / erbB2 therapeutic agent which blocks signaling through the EGFR and/or erbB2 receptor (as described above).
  • a wide variety of conventional compounds have been shown to have anti- neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies.
  • chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each ofthe treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.
  • an EGFR and/or erbB2 therapeutic agent ofthe present invention When an EGFR and/or erbB2 therapeutic agent ofthe present invention is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such therapeutic agent may enhance the therapeutic effect ofthe anti-neoplastic agent or overcome cellular resistance to such anti- neoplastic agent. This may allow decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti- neoplastic agent in resistant cells.
  • Pharmaceutical compounds that may be used for such combination chemotherapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, gose
  • chemotherapeutic compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5 -fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines,
  • EGFR and/or erbB2 therapeutic agents ofthe invention may be continued while the other therapy is being administered and/or thereafter.
  • Administration ofthe EGFR and/or erbB2 therapeutic agents may be made in a single dose, or in multiple doses, hi some instances, administration ofthe EGFR and/or erbB2 therapeutic agents is commenced at least several days prior to the conventional therapy, while in other instances, administration is begun either immediately before or at the time ofthe administration ofthe conventional therapy.
  • the instant invention also provides expression vectors comprising a nucleotide sequence encoding a subject EGFR and/or erbB2 therapeutic agent
  • nucleotide operably linked to at least one regulatory sequence.
  • operably linked when describing the relationship between two DNA regions simply means that they are functionally related to each other.
  • a promoter or other transcriptional regulatory sequence is operably linked to a coding sequence if it controls the transcription ofthe coding sequence.
  • operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the encoded polypeptide.
  • regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in
  • any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the chimeric polypeptides of this invention.
  • Such useful expression control sequences include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAG or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • T7 promoter whose expression is directed by T7 RNA polymerase
  • the major operator and promoter regions of phage lambda the control regions for fd coat protein
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of inhibitors desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • Transformed cells refers to cells that have spontaneously converted to a state of unrestrained growth, e.g., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control.
  • transformed phenotype of malignant mammalian cells and “transformed phenotype” are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals.
  • the subject gene constructs can be used to cause expression ofthe subject therapeutic agents in target cells, e.g., to produce inhibitors that inhibits the function of EGFR and c-erbB2 receptors.
  • the ex vtvo-derived inhibitors are utilized in a manner appropriate for therapy in general.
  • the inhibitors or vectors encoding inhibitors ofthe invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration, hi such embodiments, a polypeptide inhibitor may by combined with a pharmaceutically acceptable excipient, e.g., a non-pyrogenic excipient.
  • a pharmaceutically acceptable excipient e.g., a non-pyrogenic excipient.
  • the inhibitors ofthe invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the inhibitors may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • the peptides are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the oligomers ofthe invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Alternative means of administration of peptides have been developed.
  • Sustained-release pharmaceutical compositions are advantageous, requiring fewer administrations and, often, lower dosages.
  • Techniques for oral delivery of peptides have been reviewed (Fasano, A. Trends in Biotechnology 1998, 16, 152-157), as have several site- specific means of peptide delivery (Pettit, D.K. et al. Trends in Biotechnology 1998, 16, 343-349). Additional techniques for therapeutic administration of peptides are known to those of skill in the art.
  • Genetic material inhibitors ofthe present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces the desired inhibitor (e.g. RNA).
  • the genetic material is provided by use of an "expression" construct, which can be transcribed in a cell to produce the inhibitor.
  • expression constructs may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively transfecting cells either ex vivo or in vivo with genetic material encoding an inhibitor.
  • Approaches may include insertion ofthe antisense nucleic acid in viral vectors including recombinant retro viruses, adenoviruses, adeno-associated viruses, human immunodeficiency viruses, and herpes simplex viruses- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection ofthe gene construct or calcium phosphate precipitation carried out in vivo.
  • lipofectin cationic liposomes
  • derivatized e.g., antibody conjugated
  • polylysine conjugates e.g., gramacidin S
  • artificial viral envelopes or other such intracellular carriers e.g., calcium phosphate precipitation carried out in vivo.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked.
  • a genomic integrated vector or "integrated vector” which can become integrated into the chromosomal DNA of the host cell.
  • an episomal vector e.g., a nucleic acid capable of extra-chromosomal replication.
  • Vectors capable of directing the expression of genes to that they are operatively linked are refened to herein as "expression vectors.”
  • expression vectors In the present specification, "plasmid” and “vector” are used interchangeably unless otherwise clear from the context.
  • regulatory elements controlling transcription can be generally derived from mammalian, microbial, viral or insect genes.
  • the ability to replicate in a host, usually confened by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated.
  • Vectors derived from viruses, such as retroviruses, adenoviruses, and the like, may be employed.
  • One approach to in vivo introduction of genetic material encoding one ofthe subject inhibitors into a cell is by use of a viral vector containing said genetic material.
  • Infection of cells with a viral vector has the advantage that a large proportion ofthe targeted cells can receive the nucleic acid.
  • inhibitors encoded by genetic material in the viral vector e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
  • Such a strategy may be particularly effective when skeletal muscle cells are the targets ofthe vector (Fisher, KJ. et al. Nature Medicine 1997, 3, 306- 312).
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transfened nucleic acids are stably integrated into the chromosomal DNA ofthe host.
  • a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility ofthe spread of wild-type virus in the cell population.
  • the development of specialized cell lines (termed "packaging cells") which produce only replication- defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
  • recombinant retrovirus can be constructed in which part ofthe retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one ofthe antisense E6AP constructs, rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al.
  • retroviruses include pLJ, pZTP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone manow cells, in vitro and/or in vivo (see for example Eglitis, et al.
  • retroviral vectors as a gene delivery system for genetic material encoding the subject chimeric polypeptides, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction ofthe genetic material, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors. In fact, such limitation on infection can be particularly beneficial in the instant circumstances wherein the normal tissue (e.g., nontransformed cells) sunounding the target cancer cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.
  • the normal tissue e.g., nontransformed cells
  • retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Man et al. (1992) J Gen Virol 73:3251-3255; and Goud et al.
  • Coupling can be in the form ofthe chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating chimeric proteins (e.g., single-chain antibody/e «v chimeric proteins).
  • This technique while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.
  • retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences (such as breast or ovary-specific regulatory sequences) which control expression ofthe genetic material of the retroviral vector.
  • tissue- or cell-specific transcriptional regulatory sequences such as breast or ovary-specific regulatory sequences
  • Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors.
  • the genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155).
  • adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting non- dividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al.
  • virus particle is relatively stable and amenable to purification and concentration, and, as above, can be modified so as to affect the spectrum of infectivity.
  • introduced adenoviral DNA and foreign DNA contained therein is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity ofthe adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).
  • Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts ofthe viral El and E3 genes but retain as much as 80% ofthe adenoviral genetic material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, E. J. Munay, Ed.
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • Cell Biol 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol Cell. Biol. 4:2072-2081 ; Wondisford et al. (1988) Mol. Endocrinol 2:32-39; Tratschin et al. (1984) J. Virol 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
  • viral vector systems that may have application in gene therapy have been derived from herpes virus, vaccinia virus, and several RNA viruses.
  • non- viral methods can also be employed to cause expression of genetic material encoding the subject inhibitors in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non- viral gene delivery systems ofthe present invention rely on endocytic pathways for the uptake of genetic material by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, polylysine conjugates, and artificial viral envelopes.
  • genetic material can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and, optionally, which are tagged with antibodies against cell surface antigens ofthe target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
  • lipofection of papilloma-infected cells can be carried out using liposomes tagged with monoclonal antibodies against PV- associated antigen (see Viac et al. (1978) J Invest Dermatol 70:263-266; see also Mizuno et al.
  • the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180).
  • a gene binding agent such as polylysine
  • genetic material encoding the subject chimeric polypeptides can be used to fransfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g., polylysine (see U.S. Patent 5,166,320).
  • a pharmaceutical preparation ofthe gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction ofthe target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression ofthe gene, or a combination thereof.
  • initial delivery ofthe recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3054-3057).
  • the pharmaceutical preparation can consist essentially ofthe gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals, and can be adapted for release of viral particles through the manipulation ofthe polymer composition and form.
  • biocompatible polymers including hydrogels
  • biodegradable and non-degradable polymers can be used to form an implant for the sustained release of an the viral particles by cells implanted at a particular target site.
  • Such embodiments ofthe present invention can be used for the delivery of an exogenously purified virus, which has been incorporated in the polymeric device, or for the delivery of viral particles produced by a cell encapsulated in the polymeric device.
  • the amount of water, porosity and consequent permeability characteristics can be controlled.
  • the selection ofthe shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response.
  • the generation of such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and the Sabel et al. U.S. Patent No. 4,883,666.
  • a source of cells producing a the recombinant virus is encapsulated in implantable hollow fibers.
  • recombinant virus is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
  • Such fibers can be pre-spun and subsequently loaded with the viral source
  • Inhibitors of the present invention can be designed by using molecular modeling.
  • a computer model of EGFR or c-erbB2 receptor may be used to identify any compounds that might bind EGFR or c-erbB2 receptor in the ligand binding sites.
  • antagonistic compounds mimicking the natural ligands of these receptors might be designed in silica.
  • the nature ofthe inhibitory sequence can be determined by calculation, based on knowledge of a receptor or binding pocket. Other calculational strategies will be known to those skilled in the art. Calculations such as these can be useful for directing the synthesis of inhibitors ofthe present invention in a time- and material-efficient manner, before actual synthesis and screening techniques begin. Methods for screening inhibitors ofthe present invention are well known in the art, independent ofthe use of computer modeling. The use of peptide libraries is one way of screening large numbers of polypeptides at once.
  • the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind a target molecule, such as a receptor protein via this gene product is detected in a "panning assay".
  • the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting chimeric polypeptide detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).
  • the peptide library is expressed as chimeric polypeptides on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • coli filamentous phages M13, fd, and fl are most often used in phage display libraries, as either ofthe phage gill or gVIII coat proteins can be used to generate chimeric polypeptides without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02809; Ganard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBOJ 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
  • U.S. Pat. No. 6,420,110 discloses a method for isolating biologically active peptides.
  • a polypeptide inhibitor ofthe present invention may be developed which interacts with a chosen receptor, and inhibits the signaling of said receptor.
  • the inhibition can be readily tested in many suitable in vitro or in vivo models, using such art-recognized techniques as reporter genes and/or selectable markers under the control of promoters responsive to ⁇ GFR and/or c-erbB2 receptors.
  • this method is utilized to identify polypeptide inhibitors which have antagonistic activity with respect to one or more types of cells expressing at least one ofthe EGFR or erbB2 receptors.
  • the chimeric polypeptide library in the display mode, can be panned with the target cells or immobilized EGFR or erbB2 receptor in order to enrich for polypeptides which bind to that cell or receptor.
  • the polypeptide library can also be panned against one or more control cell lines (that does not express any ofthe two EGFR / erbB2 receptors) in order to remove polypeptides which bind the control cells.
  • the polypeptide library which is then tested in the secretion mode can be enriched for polypeptides which selectively bind target cells (relative to the control cells).
  • the display mode can produce a polypeptide library enriched for polypeptides which preferentially bind EGFR + and or erbB2 + tumor cells relative to normal cells, which preferentially bind p53- cells relative to p53+ cells, which preferentially bind hair follicle cells relative to other epithelial cells, or any other differential binding characteristic.
  • the polypeptides are tested for antiproliferative activity against the target cell using any of a number of techniques known in the art. For instance, BrdU or other nucleotide uptake can be measured as an indicator of proliferation.
  • the secretion mode can include negative controls in order to select for polypeptides with specific antiproliferative activity.
  • polypeptides can be isolated from the library based on their ability to induce apoptosis or cell lysis, for example, in a cell-selective manner. Also, this method can be used to identify polypeptides with angiogenic or antiangiogenic activity. For instance, the polypeptide library can be enriched for polypeptides that bind to endothelial cells but which do not bind to fibroblasts. The resulting sub-library can be screened for polypeptides which inhibit capillary endothelial cell proliferation and/or endothelial cell migration.
  • Polypeptides scoring positive for one or both of these activities can also be tested for activity against other cell types, such as smooth muscle cells or fibroblasts, in order to select polypeptides active only against endothelial cells. Furthermore, this method can be used to identify anti-infective polypeptides, for example, which are active as anti-fungal or antibacterial agents.
  • this assay can be used for identifying effectors of a receptor protein or complex thereof.
  • the assay is characterized by the use of a test cell which includes a target receptor or ion channel protein whose signal transduction activity can be modulated by interaction with an extracellular signal, the transduction activity being able to generate a detectable signal.
  • such assays are characterized by the use of a mixture of cells expressing a target receptor protein or ion channel capable of transducing a detectable signal in the reagent cell.
  • the receptor/channel protein can be either endogenous or heterologous.
  • a culture ofthe instant reagent cells will provide means for detecting agonists or antagonists of receptor function.
  • the ability of particular polypeptides to modulate a signal transduction activity of the target receptor or channel can be scored for by detecting up or down- regulation ofthe detection signal.
  • second messenger generation e.g., GTPase activity, phospholipid hydrolysis, or protein phosphorylation patterns as examples
  • an indicator gene can provide a convenient readout.
  • a detection means consists of an indicator gene.
  • polypeptides which induce a signal pathway from a particular receptor or channel can be identified. If a test polypeptide does not appear to induce the activity ofthe receptor/channel protein, the assay may be repeated as described above, and modified by the introduction of a step in which the reagent cell is first contacted with a known activator ofthe target receptor/channel to induce signal transduction, and the test peptide can be assayed for its ability to inhibit the activated receptor/channel, for example, to identify antagonists. In yet other embodiments, peptides can be screened for those which potentiate the response to a known activator ofthe receptor. EXAMPLES
  • Example 1 Assessment of the effect of inhibiting EGFR and erbB2 in cancer cells
  • AS-EGFR cells have reduced ⁇ 6-integrin subunit expression (a well characterized laminin- 1 receptor) and MMP-9 activity and decreased adhesion to laminin-1.
  • transfection of NIH:OVCAR-8 cells with an inducible c-erbB-2-antisense expression vector (AS-c-erbB-2) resulted in inhibition of cell growth and colony formation in soft agar 4 .
  • Applicant describes here use of antisense-EGFR and antisense-c-erbB-2 to study the combined effect of inhibiting both receptors in NIH:OVCAR-8 cells, paying particular attention to 1) the potential loss of invasive phenotype due to decreased EGFR expression, and 2) the potential inhibition of transforming ability due to decreased c-erbB-2 expression.
  • Applicants report that cells with both EGFR and erbB2 inhibition exhibit reduced outgrowth in Matrigel, reduced MMP-9 expression, and a decreased ability to form colonies in soft agar.
  • Applicants cloned NIH:OVCAR-8 cells and analyzed the subtle changes in the clones by CGH analysis (data not shown).
  • the C7 cells expressed decreased level of phosphorylated c-erbB-2 (Fig.l).
  • the parental 8A1 cells and the Ev-Cv control transfectants were polygonal in shape with interconnected cell clusters (Fig. 2a).
  • the E-C32.d and the E-C32.2 double transfectants Applicants observed viable as isolated, spherical, single cells (Fig. 2b).
  • Zymography analysis showed a more than 3 fold decrease in secreted MMP-9 compared to the parental 8A1 cells (30% in E-C32.2 cells versus 100% in 8A1 cells (Fig. 2c).
  • Genomic instability is thought to be a driving force of carcinogenesis, producing many genetic changes that confer growth advantage, invasive and metastatic ability to tumor cells 7 . Reduced malignancy, therefore, may be accompanied by a more stable genetic phenotype.
  • Applicants have compared the chromosomal profiles ofthe single and double antisense-transfected clones to the parental clone by molecular cytogenetic techniques, including fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH).
  • FISH fluorescence in situ hybridization
  • CGH comparative genomic hybridization
  • the parental 8A1 cells had a ploidy range from 42-113, with the major clone having 58 chromosomes indicative of significant chromosomal instability (Fig. 3a and b).
  • Fig. 3a and b the major clone having 58 chromosomes indicative of significant chromosomal instability
  • Applicants observed a reduction in the chromosomal number, with clonal ploidy of 46-52 and 46-50, respectively (Fig. 3f) (Table 1).
  • Consistent with the decrease in chromosome copy number Applicants observed a decrease in ploidy of E-C32.d and E-C32.2 double transfectants by fluorescence activated cell sorting (FACS) analysis (Table 1).
  • FACS fluorescence activated cell sorting
  • Chromosomal analysis revealed that a high percentage of cells (40%) were undergoing apoptosis in the E-C32.d clone, as evidenced by chromosomal pulverization (Fig. 3c) and nuclear fragmentation (Fig. 3d). Chromosomal endoreduplication and non-disjunction (Fig. 3e) were indicated by giant metaphase plates that were either blocked in mitosis or at the point of mitotic exit. In contrast, only 5 % ofthe parental 8 Al cells were apoptotic.
  • Chromosome number from metaphase spreads and DNA index by FACS analysis for each clone is compared with gains and losses from CGH analysis. Each result represents a duplicate experiment.
  • DNA from the parental 8A1 cells was used as a reference for comparison with the E-C32.d and the E-C32.2 double transfectants 8 .
  • CGH analysis of chromosomal imbalances in the parental 8A1 cells revealed a high level of genomic imbalances (Fig. 3g and Fig. IB - Supplementa CGH analysis) as summarized in Table 1.
  • Gain of chromosome 5 is one ofthe earliest genetic events for different human ovarian surface epithelial cells immortalized by HPV16-E6E7 viral oncogenes 9 .
  • Applicants also observed loss of chromosome 8q22-qter in E-C32.2 double transfected cells, while the same region was a gain in the parental 8A1 cells (Table 1).
  • the amplification of c-myc on chromosomal region 8q24 ( ⁇ 30 %) is a common finding in ovarian and other cancers 10 ' 11 .
  • CGH data analysis showed a loss of chromosome 8q22-qter in the E-C32.2 double transfectant (Table 1).
  • a loss of chromosome 20q was also observed (Table 1).
  • ovarian primary carcinomas also have a gain in copy number of chromosome region 20q 12 .
  • This region includes MMP-9 (20ql2.2-13.1), a putative oncogene, EEF1A2 (20ql3), and a recently identified testis-cancer gene BORIS (Brother ofthe Regulator of
  • BORIS is the first gene from the testis-cancer family that has oncogenic properties and maps to 20ql3.2, a hot spot of amplification in many cancers including ovarian cancer 1 .
  • Caspase-8 which maps to 6ql6.1 is an initiator ofthe death receptor pathway that activates apoptotic substrates including PARP and DNA-activated
  • the calibrated ratios indicate the expression levels of a given gene in the E- C32.d and E-C32.2 double transfected clones relative to that in the the parental 8A1 cells.
  • a single anay was used to measure expression in E-C32.d clone.
  • expression was measured in replicate microarray hybridizations, and the mean ⁇ standard deviation ofthe calibrated ratio is listed for genes that had acceptable measurement quality in both.
  • Specific gene annotations can be reviewed at LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/index.html). Each result represents a total of duplicate experiments.
  • Example 7 Assessment ofthe Mechanism underlying the Genomic Effect on Suppression of Aneuploidy
  • Applicants were able to show cleaved PARP [Poly(ADP-ribose)polymerase] expression in E-C32.d and E- C32.2 double transfectants (Fig. 6b) by Western blot analysis.
  • Applicants detected upregulation of tankyrase in E-C32.d and E-C32.2 double transfectants by differential gene expression analysis (Table 2).
  • Tankyrase is a family member of the poly(ADP-ribosyl)ating proteins that has PARP activity 26 .
  • PARP cleavage by caspase(s) occurs early in apoptosis, earlier on or soon after the appearance of internucleosomal fragmentation of DNA 27 .
  • NIH:OVCAR-8 cell line was a gift by Dr. T.C. Hamilton (Fox Chase Center, Philadelphia, PA). Cells were cultured in DVIEM containing 10 % fetal bovine serum (FBS).
  • the antisense expressing vector for AS-c-erbB-2 (pRC/CMV-AS 5') and control vector, (pRC/CMV) was kindly provided by Dr. Shoshana Segal (NIH/NCI).
  • the antisense expressing vector for EGFR (pCDNA3.1/AS-EGFR) was constructed as previously described using pCDNA3.1 zeo (Invitrogen Corporation, San Diego, CA) 6 .
  • Parental 8A1 cells were stably transfected with vectors expressing AS-EGFR and/or AS-c-erbB-2 (pCDNA3.1/AS-EGFR and/or pRC/CMV/AS-c-erbB-2, respectively) and control vectors, Ev-Cv (pCDNA3.1 and/or pRC/CMV, respectively).
  • AS-EGFR and AS-c-erbB-2 inserts were sequenced using primers complementary to the vector sequences and ABI PRISM BigDyeTM Terminator Cycle Sequencing kit (PE- Applied Biosystems; Foster City, CA) according to the manufacturer's protocol. Sequencing products were separated on ABI Prism 377 DNA Sequencer (PE- Applied Biosystems; Foster City, CA).
  • Apoptosis was detected by Annexin V-FITC and propidium iodide staining using AnnexinV-FITC Apoptosis kit (Pharmingen, San Diego, CA). Twenty thousand cells were analyzed on FACSort (Becton Dickinson, San Diego CA).
  • Results were analyzed utilizing FCS Express (De Novo Software, Ontario, Canada). Cells were stained with propidium iodide and analyzed in duplicate with and without human peripheral blood lymphocytes as an internal standard. One hundred thousand cells were analyzed on FACSort. DNA index and ploidy were determined utilizing ModFitLT software (Verity Software, Topsham, ME).
  • Blotting was performed with anti-EGFR antibody (Ab-12, NeoMarkers, Fremont, CA) and anti-c-erbB-2 antibody (Ab-18, Santa Cruz Biotechnology, CA).
  • the expression of PARP and actin was analyzed by western blot using 20-40 ⁇ g protein/lane. Blotting was performed using anti-PARP mouse monoclonal antibody (MoAb) (Ab2, Oncogene Research, Boston, MA) according to manufacturer's instructions and anti-actin MoAb (119, Santa Cruz Biotechnology, CA). Bands were visualized with secondary HRP-co ⁇ jugated antibodies and the ECL system (Amersham Pharmacia).
  • MMP-9 activity was measured by zymography as previously described 29 . Briefly, cells were grown to 80 % confluence in complete medium and then incubated for 72 hours in serum-free medium. Aliquots of serum-free media were analyzed by gel electrophoresis. Dried gels were scanned by use of Chemihnager TM 5500 (Alpha Innotech Corporation, San Leandro, CA).
  • Matrigel (10 mg/ml; kindly provided by Dr. Hynda Kleinman, National Institute of Dental and Craniofacial Research, Bethesda, MD). The plates were incubated at 37°C in EVIEM medium (Gibco) containing 10 % FBS for various time periods. After the nonadherent cells were removed by washing gently with PBS, adherent cells were fixed with 70 % methanol for 5 min at room temperature and stained with Giemsa (Sigma, St. Louis, MO). Fibronectin expression was detected by staining the cells with anti-fibronectin MoAb (Transduction Laboratories, Lexington, KY) and FITC-labelled secondary MoAb.
  • the images were analyzed using an Olympus IX- 70 Laser Confocal Scanning Microscope equipped with a Olympus 60x/1.4 N.A. objective lens. Paraffin sections for control and transfected cells were stained with anti-progesterone Receptor MoAb (A6, Coulter Corporation, Miami, FL) using DAKO Envision Kit (Dako Corporation, CA). The images were analyzed using an Olympus Vanox Microscope equipped with a Zeiss 25x/0.8 N.A. objective lens.
  • CGH and SKY Comparative genomic hybridization and spectral karyotyping were done as described previously 8 .
  • Tumour cell lines after one passage in culture following stable transfection were processed and analyzed by CGH, spectral karyotyping (SKY), and specific chromosome paints (FISH).
  • Cells were anested at mitosis by treatment with colcemid (Gibco/BRL) at 0.1 ⁇ g /ml for 1 hour. Mitotic chromosome spreads were prepared, and SKY analysis was performed as described 30 .
  • FISH analysis metaphases were hybridized using directly labelled locus specific probes for chromosomes 16 and 20 (Vysis, Downers Grove, Illinois).
  • CyDye-labeled cDNA was prepared from RNA and hybridized to microanays as described at http://research.nhgri.nih.gov/microanay/index.html. Arrays were scanned using an Agilent microanay scanner (Agilent Technologies, Palo Alto, CA), and expression ratios were determined using the Anaysuite software package,
  • Salomon DS Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumourigenicity in ovarian cancer cells. Int.J. Cancer. 88,566-574 (2000).
  • IGF-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-betal on programmed cell death through a p53- and IGF-independent mechanism. JBiol.Chem. 272,12181-12188 (1997).

Abstract

The instant invention provides methods and reagents for treating cancer, through inducing apoptosis and/or inducing aneuploidy regression and/or differentiation in cancer cells. The methods of the invention comprise administering one or more inhibitors to simultaneously block signaling through the EGFR and the c-erbB2 receptor, such as simultaneously suppress phosphorylated EGFR and erbB2. The inhibitors may include anti-sense RNA of the receptors, short fragments of dsRNA corresponding to segments of the EGFR or c-erbB2 mRNA, DNA encoding such RNAs (preferably on a vector that can be delivered to target cancer cells), function-antagonizing antibodies against the receptors, or small peptide inhibitors that block receptor activation.

Description

METHOD FOR INDUCING APOPTOSIS AND ANEUPLOIDY REGRESSION IN CANCER CELLS
GOVERNMENT SUPPORT
The invention described herein was supported, in whole or in part, by Grant No. 2P30-CA-51008 (Cancer Center Support Grant, to Lombardi Cancer Center). The Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of death in the United States. Half of all men and one-third of all women in the US will develop cancer during their lifetimes. Today, millions of people are living with cancer or have had cancer. The sooner a cancer is found and treatment begins, the better are the chances for survival.
To illustrate, ovarian cancer is cancer that begins in the cells that constitute the ovaries, including surface epithelial cells, germ cells, and the sex cord-stromal cells. Cancer cells that metastasize from other organ sites to the ovary (most commonly breast or colon cancers) are not then considered ovarian cancer.
According to the American Cancer Society, ovarian cancer accounts for 4 percent of all cancers among women and ranks fifth as a cause of their deaths from cancer. The American Cancer Society statistics for ovarian cancer estimate that there will be 25,400 new cases and 14,300 deaths in 2003. The death rate for this disease has not changed much in the last 50 years.
Unfortunately, almost 70 percent of women with the common epithelial ovarian cancer are not diagnosed until the disease is in an advanced stage - i.e., has spread to the upper abdomen (stage III) or beyond (stage IV). The 5-year survival rate for these women is only 15 to 20 percent, whereas the 5-year survival rate for stage I disease patients approaches 90 percent and for stage II disease patients approaches 70 percent.
Breast cancer is the most common form of cancer in women in the United States, after skin cancer. Both its cause and its cure remain undiscovered. Based on the current life expectancy for women in the United States, one out of nine women will develop breast cancer in her lifetime - a risk that was one out of 14 in 1960.
In 2003, 211,300 new cases of female invasive breast cancer - (cancer that has spread to nearby tissue, lymph nodes under the arm, or other parts ofthe body) and 55,700 cases of female in situ breast cancer (noninvasive cancer) will be diagnosed. Of these noninvasive breast cancers, approximately 85 percent will be DCIS - ductal carcinoma in situ (abnormal cells that are found only in the lining of a milk duct and have not spread outside the duct). Breast cancer alone is expected to account for 32 percent of all new cancer cases among women in 2003 and a new case will be diagnosed every two and a half minutes.
Although its incidence is low, men can develop breast cancer too. In 2003, 1,300 cases of male breast cancer are expected to be diagnosed, and 400 men will die from this disease. Breast cancer incidence increases with age, rising sharply after age 40. About 77 percent of invasive breast cancers occur in women over age 50. The average age at diagnosis is 62.
When combined, breast cancer and ovarian cancer account for one-third of all cancers in women and approximately one-quarter of cancer-related deaths in females.
Cancer is an abnormal state in which uncontrolled proliferation of one or more cell populations interferes with normal biological functioning. The proliferative changes are usually accompanied by other changes in cellular properties, including reversion to a less differentiated, more developmentally primitive state.
The development of cancer consists of multiple, sequential, and interrelated steps that lead to the generation of an autonomous clone with aggressive growth potential. These steps include sustained growth and unlimited self-renewal through a process of growth signal self-sufficiency, decreased sensitivity to growth suppressive signals, and resistance to apoptosis. Genetic or cytogenetic events that initiate aberrant growth sustain cells in a prolonged "ready" state by preventing apoptosis. In addition to the loss of regulated growth, another feature of many tumors is the remodeling ofthe surrounding tissues. Local invasive infiltration and growth are key features in many tumors, which are accompanied by remodeling ofthe vasculature and the destruction ofthe surrounding normal tissues. The invasive character of certain tumors appears to depend partly on remodeling of the surrounding extracellular matrix, such as by the proteolytic destruction ofthe extracellular matrix components. One class of enzymes that is upregulated in certain tumors are the matrix metalloproteinases (MMPs), zinc dependent proteolytic enzymes, that cleave extracellular matrix (collagen, laminin, firbronectin, etc) as well as non-matrix substrates (growth factors, cell surface receptors, etc). The deregulation of MMPs is involved in many diseases, such as tumor metastasis, rheumatoid arthritis, and periodontal disease.
There are many established cancer treatment methods. Surgery reduces local tumor masses. However, surgery is invasive and not suitable for all types of cancers. Radiation therapy is less invasive, and advances in technology (such as "conformal radiation therapy") are making it possible to aim radiation more precisely than in the past. Chemotherapy generally works by killing rapidly proliferating cells (including normal cells) by damaging their DNA. Both of these methods may have undesirable side-effects, such as excessive killing of normal cells. There is a need to develop new cancer therapy methods, particularly those which are more specific or directed to abnormal cells and less damaging to normal cells.
SUMMARY OF THE INVENTION
The present invention relates to methods of inducing apoptosis and aneuploidy regression in cancer cells overexpressing EGFR and/or erbB2 receptor, methods of therapy useful to treat an individual who suffers from cancer or a precancerous condition, pharmaceutical and therapeutic agents and compositions useful in the methods. In one embodiment, the invention is a method of inducing apoptosis and aneuploidy regression in aneuploid cancer cells comprising contacting the cells with an agent that blocks signaling through the EGFR and erbB2 pathway(s) in sufficient quantity to block the signaling. In a further embodiment, the invention is a method of inducing apoptosis and aneuploidy regression in an individual, in aneuploid cells that overexpress EGFR and erbB2, comprising administering to the individual a therapeutically effective amount of an agent that blocks signaling through the EGFR and erbB2 pathways(s). As used herein, the term therapeutic agent or anti-tumor therapeutic agent refers to agents (compounds, molecules) that induce apoptosis or aneuploidy regression, or both, in cancer cells. A therapeutically effective amount of an agent is one which induces apoptosis in aneuploid cancer cells and/or aneuploidy regression in such cells, particularly in near-diploid cancer cells and survival of diploid cells.
A variety of therapeutic agents can be used in a method ofthe present invention, such an agent may inhibit the interaction of EGFR and/or erbB2 with their respective ligands; inhibit expression of EGFR and/or erbB2; or inhibit EGFR and/or erbB2 activity (e.g., by inhibiting clustering or phosphorylation of EGFR and/or erbB2, or inhibiting a downstream signaling event that occurs upon binding of EGFR/erbB2 to their ligand(s)). Therapeutic agents can be, for example, an antibody (monoclonal or polyclonal) or a fragment thereof, such as an antibody that is specifically immunoreactive with EGFR or erbB2; a protein, such as a mutated form or a mimic of EGFR or erbB2, a fragment of EGFR or erbB2, an extracellular domain of EGFR/erbB2 or a fusion protein; a nucleic acid, such as an antisense nucleic acid of EGFR/erbB2 or an RNAi construct of EGFR/erbB2; or a small organic molecule, such as a peptidomimetic.
One or more therapeutic agents (e.g, an antibody and a small organic molecule) that induce apoptosis or regression of aneuploidy in cancer cells overexpressing EGFR and erbB2 can be administered, simultaneously or sequentially, in a method ofthe present invention. Pharmaceutical compositions, which comprise at least one therapeutic agent that induces apoptosis and/or regression of aneuploidy in cancer cells that overexpress EGFR and erbB2 and a suitable carrier, are also the subject of this invention and are useful in the present methods. In specific embodiments, pharmaceutical compositions comprise at least one therapeutic agent that induces apoptosis in aneuploid cancer cells that overexpress EGFR and erbB2 and at least one therapeutic agent that induces aneuploidy regression in aneuploid cancer cells, particularly near-diploid cancer cells, that overexpress EGFR and erbB2.
In further embodiments of this invention, the method comprises administering at least one additional type of agent, referred to as a chemotherapeutic agent, in combination with a therapeutic agent. For example one or more chemotherapeutic agent(s) that affects growth of metastasis tumor cells in an additive or synergistic manner with the therapeutic agent can be administered. The chemotherapeutic agent can be, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, or vinorelbine. hi further embodiments, ionizing radiation can also be administered and/or surgical treatment provided. The method ofthe present invention is useful to treat a variety of types of cancers, such as breast cancer, ovarian cancer, skin cancer (for example, melanoma), cancer ofthe GI mucosa (such as colon cancer); neuro/glioblastoma, leukemia, such as acute promyelocytic leukemia (APL), adenocarcinoma, including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer. The present invention also relates to packaged pharmaceutical compositions comprising a pharmaceutical composition comprising at least one therapeutic agent that induces apoptosis and/or regression of aneuploidy in cancer cells that overexpress EGFR and erbB2 and a suitable carrier and instructions and/or a label for administration ofthe pharmaceutical composition for the treatment of patients.
A further embodiment ofthe invention is a method of treating a patient suffering from cancer in which the cancerous cells overexpress EGFR and erbB2 by administering to the patient a therapeutically effective amount of a therapeutic agent that induces apoptosis and/or aneuploidy regression in the cancer cells. In this embodiment, any ofthe therapeutic agents, or a pharmaceutical composition comprising such a therapeutic agent or agents can be admimstered, alone or in conjunction with a chemotherapeutic agent(s), radiation therapy, and/or surgery. One or more therapeutic agent can be admimstered, simultaneously or sequentially.
The present invention also encompasses use of therapeutic agent or agents in the manufacture of medicament for the treatment of cancer in which cancer cells overexpressEGFR and erbB2. The therapeutic agent(s) used in the manufacture of the medicament can be any ofthe therapeutic agents described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A shows status of EGFR and c-erbB-2 phosphorylation in selected parental 8A1 cells; control vector-transfected Ev-Cv cells containing vector for EGFR and vector for c-erbB-2; single AS-EGFR-transfected E2 cells; single AS-c- erbB-2-transfected C7 cells; and double AS-EGFR/AS-cerbB-2-transfected E-C32.d and E-C32.2 cells. Phosphorylation of EGFR and c-erbB-2 is detected by immunoprecipitation (IP) using the phospho-tyrosine (PTY)-specific 4G10 monoclonal antibody, followed by Western blot (WB) analysis with the EGFR-specific Ab-12 monoclonal antibody, or the c-erbB-2-sρecific Ab-18 monoclonal antibody, respectively. Equal protein-adjusted (normalized) cell lysates were used for all experiments. The data represent replicate analysis.
Figure IB (Supplementary CGH Analysis, panels a-c) show chromosomal imbalances detected by CGH in a) 8A1 cells; b) E-C32.d transfectants compared to 8A1 cells that was used as a reference DNA , and c) E-C32.2 transfectants versus 8A1 cells. Losses are shown to the left (red vertical lines), gains are shown to the right (green vertical lines) ofthe chromosome ideograms. Figure 2 (panels 2a-2c) shows biological consequences of simultaneous suppression of EGFR and c-erbB-2 in ovarian cancer cells, a, Matrigel outgrowth of parental 8A1 cells, and b, Matrigel outgrowth ofthe E-C32.2 double transfectant simultaneously transfected with AS-EGFR and AS-c-erbB-2. The images are the results of three independent experiments, c, MMP-9 activity in parental 8A1 cells compared to single and double transfectants by zymogram analysis when seeding an equal number of cells. The experiment was repeated three times. Similar result was also obtained with E-C32.d double transfected clone.
Figure 3 (panels 3a-3g) shows cytogenetic analysis of parental 8A1 cells and double transfectant clones E-C32.d and E-C32.2. Metaphase spreads of a, 8A1 cells (58 chromosomes) after FISH with chromosome 16 (green) and 20 (red) specific painting probes; and b, (61 chromosomes) with chromosome 16q (green) and 20q (red) locus specific probes; c, d, e, multiple figures of apoptosis seen in the clone E- C32.d showing: c, chromosomal pulverization (note the presence of 3 copies of 20q probe). Arrows indicate 20q FISH signals; d, endoreduplication (octaploid cell with 12 copies of chromosome 20q probe); and e, nuclear fragmentation after endoreduplication (again, multiple copies of 20q probe can be seen); f, near-diploid metaphase spread (47 chromosomes) of E-C32.d cells with 3 copies of 20q locus - specific probe (Scale bar: 100 μm). Results similar to Figures 3c, 3d, and 3e ofthe double transfectant E-C32.d clone were also obtained with the E-C32.2 double transfectant clone, g, summary of CGH analysis ofthe E-C32.d and the E-C32.2 double transfected clones compared to the parental 8A1 cells. Blue and red lines represent the E-C32.d and the E-C32.2 double-transfected clones respectively. Lines to the left ofthe chromosomes represent losses, as lines to the right denote gains. Figure 4 (panels 4a-4f) shows fibronectin (FN) and progesterone receptor
(PR) expression. Immunofluorescent images of fibronectin distribution of a, parental 8A1 cells, and b, double transfectant E-C32.2 cells when cultured on Matrigel™. Scale bar is 50 μM. Progesterone receptor expression by immunohistochemical analysis in paraffin embedded cell pellets is shown for: c, the parental 8A1 cells, and d, the E-C32.2 double transfectant clones. Similar results were also obtained for the E-C32.d double-transfectant clone, and are shown in Figures 4b and 4d. Figure 5 shows the proposed model ofthe "bottle-neck" genomic rescue through apoptosis and regression of aneuploidy in ovarian cancer cells following simultaneous suppression of EGFR and c-erbB-2.
Figure 6 (panels 6a & 6b) shows apoptosis detected by AnnexinV/PI staining and PARP cleavage, a, AnnexinV/ PI staining was determined in the parental 8A1 cells, the single (E2, C7) and double transfectant (E-C32.d and E-C32.2) clones by FACS analysis. This analysis allowed flow cytometric quantitation of apoptosis occurring in the parental 8A1 cells, and cells transfected individually (E2 (AS- EGFR) and C7 (AS-c-erbB-2) cells) or simultaneously (EC32.d and EC32.2 cells (AS-EGFR/AS-c-erB-2)) with the AS-EGFR and/or the AS-c-erbB-2 constructs. Data for the vector control is not shown, (n = 2 in all groups), b, PARP cleavage analysis showed two bands that were specific for the full-length PARP (115 kDa) and the cleaved PARP fragment (85-90 kDa), in either the E-C32.2 double transfectant clone or the parental 8A1 cells. Similar result was also obtained in the E-C32.d double transfectant. Each result is representative of a total of three experiments.
Table 1. Summary of genomic analysis in parental 8A1, Ev-Cv control, E-
C32.d and E-C32.2 double transfectants. Chromosome number from metaphase spreads and DNA index by FACS analysis for each clone are compared with gains and losses from CGH analysis. Each result represents a duplicate experiment.
Table 2. Differential gene expression profile in single and double transfectants compared to the parental 8A1 cells were analyzed by cDNA microarray analysis. The calibrated ratios indicate the expression levels of a given gene in the E-C32.d and the E-C32.2 double- transfected clones relative to that in the parental 8A1 cells. A single array was used to measure expression in the E-C32.d clone. For the E-C32.d cells, expression was measured in replicate microarray hybridizations, and the mean ± standard deviation ofthe calibrated ratio is listed for genes that had acceptable measurement quality in both. Specific gene annotations can be reviewed at LocusLink (National Center for Biotechnology Information, or "NCBI" of NIH). Each result represents duplicate experiments.
Figure imgf000010_0001
DETAILED DESCRIPTION OF THE INVENTION
The ErbB family of receptor tyrosine kinases (RTKs) or subclass I RTKs so far comprises four members: epidermal growth factor receptor (EGFR /ErbB-l (Ullrich et al., 1984, Nature 309: 418), ErbB-2 (Yamamoto et al., 1986, Nature, 319: 230-234), ErbB-3 (Kraus et al., 1989, Proc. Natl Acad. Sci. USA, 86: 9193-9197; Plowman et al, 1990, Proc. Natl Acad. Sci. USA, 87: 4905-4909) and ErbB-4 (Plowman et al., 1993, Proc. Natl Acad. Sci. USA, 90: 1746-1750). The four proteins are widely expressed in epithelial, mesenchymal and neuronal tissues and play fundamental roles during development (Gassmann et al., 1995, Nature, 378: 390- 394; Lee et al., 1995, Nature, 378: 394-398; Sibilia and Wagner, 1995, J. Biol.
Chem., 269: 14661-14665; Threadgill et al, 1995, Science, 269: 230-234). Interest in the ErbB family of RTKs is high due also to the involvement of some of its members in human cancers (Hynes and Stern, 1994, J. Cell. Biochem., 39: 167-173; Salomon et al, 1995, Crit. Rev. Oncol. Hematol, 19: 183-232). Subclass I RTKs have an extracellular domain (ECD) which bears two cysteine-rich clusters and is responsible for interaction with polypeptide ligands. A direct consequence of ligand binding to the ECD is the formation of receptor dimers and stimulation ofthe intrinsic kinase activity, which leads to the phosphorylation of tyrosine residues in the intracellular domain ofthe receptors (van der Geer et al, 1994, Annu. Rev. Cell Biol, 10: 251-337). These serve as docking sites for a number of SH2- and PTB-domain containing proteins (Kavanaugh and Williams, 1994, Science, 266: 1862-1865; Cohen, GB. et al., 1995, Cell, 80: 237-248) including the adaptor proteins SHC (Pelicci et al., 1992, Cell, 70: 93-104) and Grb2 (Lowenstein et al, 1992, Cell, 70: 431-442) and the p85 subunit of phosphatidylinositol (Ptdlns) 3-kinase (Fedi et al., 1994, Mol. Cell. Biol, 14: 492-500; Prigent and Gullick, 1994, EMBO J, 13: 2831-2841), which link RTKs to intracellular signaling pathways such as the mitogen-activated protein kinase (MAPK) pathway (Egan and Weinberg, 1993, Nαtwre, 365: 781-783) or the S6 kinase cascade (Ming et al, 1994, Nature, 371: 426-429). Regulation of ErbB receptor function is complex. A large number of ligands, the EGF-related peptides, have been described. ErbB ligands can be classified into three groups and include: EGF and heparin binding EGF-like growth factor (HB- EGF), which bind ErbB-1 (Savage et al., 1972, J Biol Chem., 241: 7612-7621; Higashiyama et al., 1991, Science, 251: 936-939); betacellulin (BTC), which is a ligand of ErbB-1 and ErbB-4 (Shing et al., 1993, Science, 259: 1604-1607; Beerli and Hynes, 1996, J Biol Chem., 271: 6071-6076; Riese et al. 1996, Oncogene, 12: 345-353); neu differentiation factors (NDFs)/heregulins (Peles and Yarden, 1993, BioEssays, 15: 815-824), which are ligands of ErbB-3 and ErbB-4 (Plowman et al., 1993, Nature, 366: 473-475; Carraway et al.; 1994, J Biol. Chem., 269: 14303- 14306), the respective low and high affinity receptors (Tzahar et al., 1994, J Biol. Chem., 269: 25226-25233). By binding to the ECD of their respective receptors, EGF-related peptides induce not only receptor homodimers, but also heterodimers. Consequently, although none of these peptides directly bind ErbB-2, all of them induce its tyrosine phosphorylation by triggering heterodimerization and cross-phosphorylation (King et al., 1988, EMBO J., 7: 1647-1651; Plowman et al., 1993, Nature, 366: 473-475; Sliwkowski et al., 1994, J. Biol. Chem., 269: 14661-14665; Beerli and Hynes, 1996, J. Biol Chem., 271: 6071-6076). Cooperation of ErbB-2 with the other ErbB receptors has been reported (Alimandi et al., 1995, Oncogene, 10: 1813-1821; Wallasch et al., 1995, EMBOJ., 14: 4267-4275; Pinkas-Kramarski et al, 1996, EMBO J., 15: 2452-24; Zhang et al, 1996, J. Biol. Chem., 271: 3884-3890). Moreover, EGF and ΝDF receptors have been shown to compete for dimerization with ErbB-2 (Karunagaran et al, 1995, J. Biol. Chem., 270: 9982-9990; Chen et al., 1996, J. Biol. Chem., 271: 7620-7629). By means of intracellular expression of an endoplasmic reticulum (ER)-targeted single chain antibody (scFv) that leads to the specific and stable loss of cell surface ErbB-2 (Beerli et al., 1994, J Biol. Chem., 269 : 23931 -23936), it was previously shown that: (i) ErbB-2 enhances EGF-induced tyrosine phosphorylation of ErbB-1 and ΝDF-induced tyrosine phosphorylation of ErbB-3 and ErbB-4 (Beerli et al. 1995, Mol Cell Biol, 15: 6496-6505; Graus-Porta et al., 1995, Mol. Cell. Biol, 15: 1182-1191); (ii) ErbB-2 potentiates and prolongs the signal transduction pathways elicited by EGF and ΝDF (Beerli et al., 1995, Mol Cell Biol, 15: 6496-6505; Graus-Porta et al; 1995, Mol. Cell. Biol, 15: 1182-1191; Karunagaran et al., 1996, EMBOJ, 15: 254-264). In addition, it have been shown that ErbB-2 increases the affinity of both EGF and ΝDF for their receptors (Wada et al., 1990, Cell, 61: 1339-1347; Sliwkosky et al, 1994, J. Biol. Chem., 269: 14661- 14665; Karunagaran et al., 1996, EMBO J, 15: 254-264). Together, these results suggest that ErbB-2 acts as a common receptor subunit of all the other ErbB proteins and that the physiological receptors for the EGF-related peptides are ErbB-2- containing heterodimers.
Signal transduction in the erbB family of receptors is clearly complicated and several recent observations appear to argue against this model. First, it has been suggested that ErbB-1 -ErbB-3 heterodimers occur in cell lines overexpressing ErbB- 1, where EGF can efficiently elevate tyrosine phosphorylation of ErbB-3 (Kim et al., 1994, J. Biol. Chem., 269: 24747-24755; Soltoff et al, 1994, Mol Cell. Biol, 14: 3550-3558). Second, in cells that express moderate levels ofthe four receptor proteins, EGF, HB-EGF and BTC not only activate their respective receptors and ErbB-2, but also ErbB-3 (Beerli and Hynes, 1996, J. Biol. Chem., 271: 6071-6076). Third, cooperative signaling of ErbB-3 and ErbB-4 with not only ErbB-2 but also ErbB-1 has been demonstrated when the receptors were expressed in cells devoid of ErbB proteins (Cohen, B.D. et al., 1996, J. Biol. Chem., 271: 4813-4818; Pinkas- Kramarski et al., 1996, EMBO J., 15: 2452-2467; Zhang et al., 1996, J. Biol Chem., 271: 3884-3890). Finally, expression of different combinations of ErbB receptors in Ba/F3 hematopoietic cells has revealed that all heterodimers can be formed in response to the appropriate ligand (Riese et al, 1995, Mol. Cell. Biol, 15: 5770- 5776; Riese et al., 1996, Oncogene, 12: 345-353).
Expression of recombinant ErbB receptors in pair- wise combinations is a viable approach to study their function. However, the four ErbB proteins are often co-expressed and this approach does not allow delineation of which ErbB receptor interactions are biologically relevant. Different heterodimers have been shown to elicit very different biological responses (Riese et al., 1995, 1996, supra), implying the recruitment and activation of distinct signaling molecules. Thus, it appears to be rather difficult to predict the phenotypic outcome if the function of two or more erbB family receptors are negated in a particular cell, even if information regarding blocking the function of individual erbB receptors are known. In each particular cellular context, different heterodimers, and thus different signaling pathways may be activated. To understand in more detail ligand-induced ErbB receptor heterodimerization and transactivation, as well as the consequent diversification of intracellular cascades, Graus-Porta et al. (EMBO J, 16: 1647-1655, 1997) have down-regulated the cell surface expression of ErbB-1 and ErbB-2 in a number of cell lines that co-express various amounts ofthe four ErbB proteins. Specifically, ErbB-2-specifιc scFv-5R (Beerli et al, 1994, J. Biol. Chem., 269: 23931-23936) has been expressed in T47D (Graus-Porta et al., 1995, Mol Cell Biol, 15: 1182-1191) and A431 cells, leading to a complete loss of cell surface ErbB-2 due to its functional inactivation. ErbB-1 -specific scFv-RlR has been expressed in T47D cells (Jannot et al., 1996, Oncogene, 13: 275-282), leading to a dramatic reduction in cell surface ErbB-1. This approach has enabled them to analyze in more detail EGF-, HB-EGF-, BTC- and NDF-induced signaling and to unravel a hierarchy guiding the ligand-induced coordinated action of ErbB receptors.
Specifically, it was found that ligand-induced ErbB receptor heterodimerization follows a strict hierarchy and ErbB-2 is the preferred heterodimerization partner of all ErbB proteins. NDF-activated ErbB-3 or ErbB-4 heterodimerizewith ErbB-1 only when no ErbB-2 is available. If all ErbB receptors are present, NDF receptors preferentially dimerize with ErbB-2. Furthermore, EGF- and BTC-induced activation of ErbB-3 is impaired in the absence of ErbB-2, suggesting that ErbB-2 has a role in the lateral transmission of signals between other ErbB receptors. Finally, ErbB-1 activated by all EGF-related peptides (EGF, HB- EGF, BTC and NDF) couples to SHC, whereas only ErbB-1 activated by its own ligands associates with and phosphorylates Cbl. These results provide biochemical evidence that a given ErbB receptor has distinct signaling properties depending on its dimerization.
The current invention is based in part on the discovery that simultaneous targeting of EGFR and erbB2 signaling pathway contributes to increased apoptosis in aneuploid cancer cells and regression of aneuploidy in near-diploid cancer cells, resulting in death of aneuploid cells and rescue of near-diploid cancer cells. The terms "apoptosis" or "programmed cell death," refers to the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Apoptosis, is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise ("cellular suicide"). Cells undergoing apoptosis show characteristic morphological and biochemical features. These features include chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria and nuclear material. In vivo, these apoptotic bodies are rapidly recognized and phagocytized by either macrophages or adjacent epithelial cells. Due to this efficient mechanism for the removal of apoptotic cells in vivo, no inflammatory response is elicited. In vitro, the apoptotic bodies as well as the remaining cell fragments ultimately swell and finally lyse. This terminal phase of in vitro cell death has been termed "secondary necrosis."
Generally, "aneuploid" refers to the characteristic of having a chromosome complement that is not an exact multiple ofthe haploid number, with either fewer or more than the normal number of chromosomes in the cell. In an aneuploid cell, chromosomes may be present in multiple copies (e.g. Trisomy) or one of a homologous pair may be missing in a diploid cell. However, "aneuploid" or all grammatical variations thereof refers to a severe form of chromosomal complement change, such that an aneuploid cell contains on average 5 or more, preferably 8, 10, 20, 40, 50, or even 60 more chromosomes (either in whole or part) than a normal diploid cell.
Similarly, "near diploid" or all its grammatical variations refers to a less severe form of chromosomal complement change, such that a near diploid cell contains on average no more or no less than 3, preferably 2, or even 1 chromosome(s) (either in whole or part) when compared to a normal diploid cell.
"c-erb-B2," "erbB2," or "erbB-2" or "c-erbB-2" or any grammatical variants are used interchangeably. Similarly, "EGFR," "erbBl," "erb Bl," or any grammatical variants are used interchangeably.
As described herein, Applicant has shown that simultaneous suppression of phosphorylated EGFR and c-erbB2 in human tumor cells that overexpress EGFR and c-erbB2 induces apoptosis of aneuploid cell population and results in survival of near-diploid cells in the population. Further characterization of all genomic and chromosomal imbalances pre-existing in the parental cell line and those following double transfection revealed a trend toward normalization of the karyotype along with the activation of apoptotic pathways favoring a mechanism that involved cell selection. Inhibition of the invasive phenotype and increase in expression of progesterone receptor and fibronectin in double transfected clones indicated the presence of benign phenotype. Thus, Applicant has unexpectedly shown genomic rescue of near-diploid cancer.cells and apoptosis of aneuploid cancer cells as a result of simultaneous suppression of phosphorylated EGFR and c-erbB2 in human tumor cells. Simultaneous inhibition of EGFR and erbB2 provides a means of overcoming functional cooperation of these receptors and, surprisingly, inducing apoptosis of aneuploid cells and genomic rescue (regression toward normal/diploid genotype with the result that highly aneuploid cells die and more nearly diploid cells survive. As shown herein, simultaneous targeting of EGFR and erbB2 is a method that results in a trend toward normalization of the karyotype and activation of apoptotic pathways favoring a mechanism involving cell selection.
Thus, the instant invention provides a method of cancer treatment by simultaneously inhibiting the function of a selected pair of erbB family receptors in cancer cells. The method selectively eliminates highly aneuploid tumor cells by apoptosis (or "programmed cell death"), while rescuing normal or diploid cells and less-severely damaged, near diploid tumor cells, which, as a result, exhibit more benign phenotypes.
Specifically, one aspect ofthe present invention provides a method for treating tumors expressing EGFR and erbB2 receptor, hi one embodiment, the treatment induces apoptosis in aneuploid cells and regression of aneuploidy in near- diploid tumor cells. Such method comprises administering an amount of an EGFR therapeutic agent (at least one agent and an erbB2 therapeutic agent and at least one agent that blocks signaling through the erbB2 pathway) that blocks signaling through the EGFR pathway, sufficient to reduce the aneuploidy and increase apoptosis of cancer cells (e.g., tumor cells). For example, a therapeutic agent may exert its inhibitory effect by inhibiting the interaction between EGFR and erbB2 with each other and/or their ligands, inhibiting gene expression of EGFR and erbB2, inhibiting activity of EGFR and erbB2, inhibiting clustering of EGFR and erbB2, inhibiting phosphorylation of EGFR and erbB2, or inhibiting any downstream signaling event upon binding of EGFR and erbB2. The therapeutic agents useful in this method are described in greater detail herein. A therapeutic agent can affect EGFR, erbB2 or both, hi the instances in which a therapeutic agent has both anti- EFGR and anti-erbB2 activity, only a single agent need be administered to induce apoptosis and aneuploid cells and aneuploidy regression in near-diploid cells and, as a result, treat cancer by killing highly aneuploid cells and rescuing non-diploid cells. Two or more therapeutic agents (e.g., an anti-EGFR agent and an anti-erbB2 agent; two or more anti-EGFR agents and one anti-erbB2 agent; one anti-erbB2 agent; and two or more of each type of agents can be administered according to the method of present invention.
As used herein, the term "EGFR therapeutic agent" or "erbB2 therapeutic agent" is a generic term which include any compound or molecule (agent) which blocks signaling through the EGFR and/or erbB2 pathway. For example, such anti- tumor therapeutic agents can inhibit functions (activity or expression) of erbB2 or EGFR, inhibit the interaction of erbB2 or EGFR with each other and/or with their ligands, inhibit the phosphorylation of erbB2 or EGFR, or inhibit any of the downstream signaling events upon binding of ligands for erbB2 or EGFR. Generally, substances that act as antagonists of erbB2 or EGFR may be used as anti- tumor therapeutic agents. Such anti-tumor therapeutic agents include, but are not limited to, a protein, a peptide, a small organic molecule, a peptidomimetic, an antibody, and a nucleic acid.
The term "expression" with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein coding sequence results from transcription and translation ofthe coding sequence.
"Inhibition of gene expression" refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene.
"Specificity" refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination ofthe outward properties ofthe cell or organism (as presented below in the examples) or by biochemical techniques, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
EGFR and or erbB2 has been implicated in a number of cancers, including: breast cancer (see van de Vijver et al., New Eng. J. Med. 319: 1239-1245, 1988, also see Slamon et al, Science 244: 707-712, 1989; Yu et al., Molec. Cell 2: 581-591, 1998); ovarian cancer (Slamon et al., Science 244: 707-712, 1989); skin cancer (for example, melanoma), see Sibilia et al., Cell 102: 211-220, 2000; cancer ofthe GI mucosa (such as colon cancer, see Pai et al, Nature Med. 8: 289-293, 2002); neuro/glioblastoma (Yang-Feng et al., Cytogenet. Cell Genet. 40: 784 only, 1985); leukemia, such as acute promyelocytic leukemia (APL) (Kaneko et al., Jpn. J. Cancer Res. 78: 16-19, 1987); adenocarcinoma (Akiyama et al., Science 232: 1644- 1646, 1986), including adenocarcinoma ofthe salivary gland (Semba et al., Proc. Nat. Acad. Sci. 82: 6497-6501, 1985); gastric cancer (Fukushige et al., Biochem. Biophys. Res. Commun. 134: 477-483, 1986); and prostate cancer (Qiu et al., Nature 393: 83-85, 1998). Thus, the instant invention can be used to treat a variety of different cancers, especially cancers that express at least one ofthe EGFR or erbB2 receptors as listed above.
In certain embodiments, the therapeutic agent is formulated with a pharmaceutically acceptable carrier. In other embodiments ofthe methods ofthe present invention, at least one additional chemotherapeutic agent that affects growth ofthe tumor cells in an additive or synergistic manner with a therapeutic agent is administered with the therapeutic agent(s). Optionally, methods ofthe present invention may further include ionizing radiation or any other cancer therapy (e.g., surgery) known in the art.
Another aspect ofthe present invention provides a packaged pharmaceutical. Such packaged pharmaceutical comprises: (i) an EGFR therapeutic agent and an erbB2 therapeutic agent, preferably in therapeutically effective amounts; and (ii) instructions and/or a label for administration ofthe therapeutic agents for the treatment of patients having tumors that express EGFR and erbB2. As used herein the term "patient" or "subject" to be treated by the method of the invention can mean either a human or non-human animal. "Animal" refers to mammals, preferably mammals such as humans.
As described above, the therapeutic agent blocks signaling through the EGFR / erbB2 pathways, for example, by inhibiting the interaction between EGFR and erbB2 with each other and/or their ligands, inhibiting gene expression of EGFR and erbB2, inhibiting activity of EGFR and erbB2, inhibiting clustering of EGFR and erbB2, inhibiting phosphorylation of EGFR and erbB2, or inhibiting any downstream signaling event upon binding of EGFR and erbB2. Another aspect of the present invention provides a method for treating a patient (individual) suffering from a cancer, especially in patient suffering from a cancer in which EGFR and erbB2 are overexpressed. In one embodiment, the method comprises treating the patient with therapeutically effective amount of an EGFR therapeutic agent and an erbB2 therapeutic agent. The method may further include assessing the EGFR and erbB2 status of a sample of tumor cells from a patient before said treatment, so that only patients overexpressing both receptors are treated. As described herein, the EGFR and/or erbB2 status includes, but is not limited to, gene expression of EGFR and/or erbB2, activity of EGFR and/or erbB2 (e.g., protein-protein interaction, clustering, phosphorylation, etc.), and any downstream signaling through the EGFR and/or erbB2 pathway.
The present invention also relates to methods of treating an individual (patient) in whom cells overexpress EGFR and erbB2 and, therefore, is in what can be seen to be a precancerous state or condition. By administering a therapeutic agent(s) that inhibits EGFR and a therapeutic agent(s) that inhibits cerbB2, the progression ofthe precancerous cell to become a cancer cell can be prevented or limited (the progression will occur to a lesser extent that would occur without the treatment). This approach is useful, for example, in individuals at risk for developing a cancer in which EGFR and erbB2 are overexpressed, such as a woman whose family history puts her at risk for developing ovarian or breast cancer. It is possible using available methods, to monitor or assess individuals for overexpression of EGFR and erbB2. Those individuals in whom expression of EGFR and/or erbB2 exceeds a certain level (e.g., a level above which the likelihood of developing cancer in which the two are overexpressed is increased) can be treated with a therapeutically effect amount of therapeutic agent that induces apoptosis of aneuploid cells and a therapeutic agent that induces aneuploidy regression in near- diploid cells. As a result, abnormal cells (especially highly aneuploid cells) are induced to undergo apoptosis and more normal (nearly diploid) cells revert or differentiate to diploidy and an essentially normal karyotype.
"Overexpression" generally means having a higher expression level than the "normal" level in "normal" cells. For example, "overexpression of EGFR" in a cancer cells means expression of the EGFR protein and/or mRNA is at least 10% more, preferably 20%, 50%, 100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold or more when compared to normal EGFR protein and/or mRNA levels in comparable normal cells. Preferably, cancer cells are compared to the same types of normal cells. For example, breast carcinoma cancer cells are compared to normal breast epithelial cells, preferably normal breast epithelial cells from the same patient. Some times, EGFR and/or erbB2 DNA may be amplified in cancer cells, leading to overexpression of receptor mRNA and/or protein. The quantitation of expression level of mRNA and/or protein can be done using routine methods well-known in the art, such as Northern and Western blots, or commercial kits. Yet another aspect of the present invention provides use of an EGFR therapeutic agent and an erbB2 therapeutic agent in the manufacture of medicament for the treatment of cancer that expresses EGFR and erbB2. As described above, the EGFR/erbB2 therapeutic agent(s) block signaling through the EGFR and erbB2 pathway, for example, by inhibiting the interaction between EGFR and or erbB2 with their ligands or with each other, by inhibiting gene expression of EGFR and/or erbB2, by inhibiting activity of EGFR and/or erbB2, by inhibiting clustering of EGFR and/or erbB2, by inhibiting phosphorylation of EGFR and/or erbB2, or by inhibiting any downstream signaling event upon binding of EGFR and/or erbB2 to their ligands. Exemplary cancers include, but are not limited to, breast cancer, ovarian cancer, skin cancer (for example, melanoma), cancer ofthe GI mucosa (such as colon cancer); neuro/glioblastoma, leukemia, such as acute promyelocytic leukemia (APL), adenocarcinoma, including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer.
Exemplery EGFR and/or erbB2 Therapeutic Agents
As used herein, the EGFR/erbB2 therapeutic agents ofthe present invention include any compound, (agent) which blocks the EGFR receptor and erbB2 receptor signaling. For example, such therapeutic agents can inhibit functions (activity or expression), interactions between EGFR/erbB2 and their ligands, interactions between the EGFR and the erbB2 receptors, or phosphorylation ofthe EGFR/erbB2 receptors, or inhibit any ofthe downstream signaling events upon binding of EGFR/ erbB2 with their ligands. Generally, substances that act as antagonists of EGFR and/or erbB2 may be used as anti-tumor therapeutic agents. Such EGFR/erbB2 therapeutic agents include, but are not limited to, a protein, a peptide, a small organic molecule, a peptidomimetic, an antibody, and a nucleic acid (e.g., antisense, nucleic acid, siRNA or vectors expressing siRNA). As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. In certain aspects, the EGFR therapeutic agents and erbB2 therapeutic agents include a polypeptide which is a mutated form, a mimic or a fragment of EGFR and/or erbB2 protein. Such therapeutic agents can bind to natural ligands of EGFR and/or erbB2, and prevent the interaction between EGFR and/or erbB2 with their ligands or with each other. For example, the EGFR therapeutic agents and/or the erbB2 therapeutic agents may include a soluble polypeptide having the amino acid sequence ofthe extracellular domain of EGFR and/or erbB2, or a sufficient portion ofthe extracellular domain, or a polypeptide having an amino acid sequence conferring a similar enough conformation to allow specific binding to ligands of EGFR and/or erbB2. Since the ligand binding region of EGFR and erbB2 are known or can be readily determined using art-recognized techniques (such as in vitro binding assay using fragments ofthe receptors and the natural ligands), such dominant negative mutated form, mimic or fragments can be readily made without undue experimentation. hi another example, antagonists which comprise the extracellular domain of EGFR and/or erbB2 but do not possess clustering activity, can be produced as EGFR and/or erbB2 therapeutic agents. Optionally, such EGFR and/or erbB2 therapeutic agents may be a fusion (hybrid) protein. For example, the extracellular domain of EGFR and/or erbB2 may be fused to the Fc domain of human IgG. Non-clustered forms of these hybrid proteins act as antagonists and thus can be used for anti-tumor purposes. These non-clustered forms ofthe hybrid proteins may bind the ligand and/or a wild-type receptor, leading to the formation of a non-functional receptor- ligand complex, thus antagonizing EGFR and/or erbB2 signaling. The extracellular domain that do not possess clustering activity can be identified using a number of routine art-recognized technologies. For example, progressive deletion analysis of smaller and smaller portions ofthe extracellular domain can be used to identify regions ofthe ECD that are required for receptor multimerization. Similarly, Ala scanning mutagenesis may be used to identify point mutations that are detrimental to ligand binding or receptor multimerization (which can be assessed using, for example, yeast two-hybrid assays). hi certain aspects, the EGFR therapeutic agents and the erbB2 therapeutic agents can be antibodies, such as antibodies that are specifically reactive with EGFR, erbB2, or both. Antibodies may be polyclonal or monoclonal; intact or truncated, e.g., F(ab')2, Fab, Fv; xenogeneic, allogeneic, syngeneic, or modified forms thereof, such as humanized or chimeric antibodies. Alternatively, these antibodies may be encoded by polynucleotides, and expressed upon transfection of such polynucleotides into the target cancer cell. While not wishing to be bound by any particular theory, these antibodies or fragments thereof may bind the surface receptors, and antagonize receptor function by receptor intemahzation. Alternatively, immune system ofthe host may recognize and eliminate such Ab- engaged cancer cells, through, for example, natural killer cells (NK cells). The term "encodes," unless evident from its context, will be meant to include DNA sequences that encode a polypeptide, as the term is typically used, as well as DNA sequences that are transcribed into inhibitory antisense molecules.
For example, by using immunogens derived from an EGFR, an erbB2 polypeptide, or from both, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (see, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, a hamster or rabbit can be immunized with an immunogenic form ofthe peptide (e.g., a polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. A full-length or an immunogenic portion of an EGFR polypeptide an erbB2 polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies. In one embodiment, antibodies ofthe invention are specific for the extracellular portion of the EGFR and/or erbB2 protein, hi another embodiment, antibodies ofthe invention are specific for the intracellular portion or the transmembrane portion ofthe EGFR and/or erbB2 protein.
Following immunization of an animal with an antigenic preparation of an EGFR polypeptide an erbB2 polypeptide or both, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells. Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, (1975) Nature, 256: 495- 497), the human B cell hybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an EGFR and/or erbB2 polypeptide and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
The term antibody as used herein is intended to include fragments thereof which are also specifically reactive with an EGFR polypeptide an erbB2 polypeptide or both. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments. An antibody ofthe present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an EGFR polypeptide or erbB2 polypeptide conferred by at least one CDR region ofthe antibody. Techniques for the production of single chain antibodies (US Patent No. 4,946,778) can also be adapted to produce single chain antibodies. Also, transgenic mice or other organisms including other mammalian species, may be used to express humanized antibodies. In certain embodiments, the antibodies further comprises a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co- factor). In certain specific embodiments, an antibody ofthe invention is a monoclonal antibody, and in certain embodiments the invention makes available methods for generating novel antibodies. For example, a method for generating a monoclonal antibody that binds specifically to an EGFR polypeptide or erbB2 polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the EGFR polypeptide or erbB2 polypeptide effective to stimulate a detectable immune response, obtaining antibody-producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody- producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the EGFR and/or erbB2 polypeptide. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to the EGFR and/or erbB2 polypeptide. The monoclonal antibody may be purified from the cell culture.
In addition, the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties ofthe antibody obtained. For example, an antibody to be used for certain therapeutic purposes will preferably be able to target an antigen on a particular cell type, as opposed to antigen in solution. Accordingly, to obtain antibodies of this type, it maybe desirable to screen for antibodies that bind to cells that express the antigen of interest (e.g., by fluorescence activated cell sorting), or at least confirm that the antibody can bind to EGFR or erbB2 receptor on cell surface. A variety of different techniques are available for testing antibody: antigen interactions to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g. the Biacore binding assay, Bia-core AB, Uppsala, Sweden), sandwich assays (e.g. the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), Western blots, immunoprecipitation assays and immunohistochemistry.
In certain aspects, the EGFR therapeutic agents and erbB2 therapeutic agents ofthe present invention comprise a nucleic acid. In one embodiment, the invention relates to the use of antisense nucleic acid complementary to polynucleotides encoding EGFR and/or erbB2 polypeptides or variants thereof, to decrease expression of EGFR and/or erbB2. Such an antisense nucleic acid can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion ofthe cellular mRNA which encodes an EGFR polypeptide or erbB2 polypeptide. Alternatively, the construct is an oligonucleotide (DNA or RNA) which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding an EGFR and/or erbB2 polypeptide. Such oligonucleotides are optionally modified oligonucleotide which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and is therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in nucleic acid therapy have been reviewed, for example, by van der Krol et al., (1988) Biotechniques 6:958-976; and Stein et al, (1988) Cancer Res 48:2659-2668.
In another embodiment, the invention relates to the use of RNA interference (RNAi) to effect knockdown of EGFR encoding nucleic acid (gene) erbB2 encoding nucleic acid (gene). RNAi constructs comprise double stranded RNA that can specifically block expression of a target gene. RNAi provides a useful method of inhibiting gene expression in vitro or in vivo. RNAi constructs can comprise either long stretches of dsRNA identical or substantially identical to the target nucleic acid sequence or short stretches of dsRNA identical or substantially identical to only a region of the target nucleic acid sequence.
As used herein, the term "RNAi construct" is a generic term including small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form siRNAs. RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts which can produce siRNAs in vivo.
Optionally, the RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions ofthe cell to the nucleotide sequence of at least a portion ofthe mRNA transcript for the gene to be inhibited (i.e., the "target" gene). The double-stranded RNA need only be sufficiently similar to natural RNA that it mediate RNAi under the conditions used (such as physiological condition). Thus, the RNAi construct can include sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center ofthe siRNA duplex are most critical and may essentially abolish cleavage ofthe target RNA. In contrast, nucleotides at the 3' end ofthe siRNA strand that is complementary to the target RNA do not significantly contribute to specificity ofthe target recognition. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90%) sequence identity, or even 100% sequence identity, between the inhibitory
RNA and the portion ofthe target gene is preferred. Alternatively, the duplex region ofthe RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion ofthe target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C hybridization for 12-16 hours; followed by washing).
A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm (melting temperature) of 55°C, can be used, e.g., 5χSSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5xSSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5χ or 6χSSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5χ or 6χSSC. SSC is a 0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). A minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
Unless specified, the term "standard hybridization conditions" refers to a Tm of about 55°C, and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60°C; in a more preferred embodiment, the Tm is 65°C. In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68°C in 0.2xSSC, at 42°C in 50% formamide, 4χSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions. Suitable hybridization conditions for oligonucleotides (e.g., for oligonucleotide probes or primers) are typically somewhat different than for full- length nucleic acids (e.g., full-length cDNA), because of the oligonucleotides' lower melting temperature. Because the melting temperature of oligonucleotides will depend on the length of the oligonucleotide sequences involved, suitable hybridization temperatures will vary depending upon the oligoncucleotide molecules used. Exemplary temperatures may be 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides) and 60°C (for 23 -base oligonucleotides). Exemplary suitable hybridization conditions for oligonucleotides include washing in 6χSSC/0.05% sodium pyrophosphate, or other conditions that afford equivalent levels of hybridization.
The double-stranded structure may be formed by a single self- complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region ofthe RNA are targeted for genetic inhibition.
The subject RNAi constructs can be "small interfering RNAs" or "siRNAs." These nucleic acids are from about 19 to about 30 nucleotides in length, and in specific embodiments 19-23 or 21-23 nucleotides in length. The siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In a particular embodiment, the 21-23 nucleotides siRNA molecules comprise a 3' hydroxyl group, hi certain embodiments, the siRNA constructs can be generated by processing of longer double-stranded RNAs, for example, in the presence ofthe enzyme dicer. In one embodiment, the Drosophila in vitro system is used, hi this embodiment, dsRNA is combined with a soluble extract derived from Drosophila embryo, thereby producing a combination. The combination is maintained under conditions in which the dsRNA is processed to RNA molecules of from about 19 to about 23 nucleotides or from about 19 to about 23 nucleotides or from about 21 to about 23 nucleotides. The siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs.
Production of RNAi constructs can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase ofthe treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro. The RNAi constructs may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of an nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase. The RNAi construct may be produced enzymatically or by partial total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. Methods of chemically modifying RNA molecules can be adapted for modifying RNAi constructs (see, e.g., Heidenreich et al. (1997) Nucleic Acids Res. 25:776- 780; Wilson et al. (1994) J Mol Recog 7:89-98; Chen et al. (1995) Nucleic Acids Res 23:2661-2668; Hirschbein et al. (1997) Antisense Nucleic Acid Drug Dev 7:55- 61). Merely to illustrate, the backbone of an RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2'-substituted ribonucleosides, a- configuration). hi some cases, at least one strand ofthe siRNA molecules has a 3' overhang of from about 1 to about 6 nucleotides in length, although may be from 2 to 4 nucleotides in length. More preferably, the 3' overhangs are 1-3 nucleotides in length. In certain embodiments, one strand has a 3' overhang and the other strand is blunt-ended or also has an overhang. The length ofthe overhangs may be the same or different for each strand. In order to further enhance the stability ofthe siRNA, the 3' overhangs can be stabilized against degradation, hi one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotide 3' overhangs by 2l-deoxythyinidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2' hydroxyl significantly enhances the nuclease resistance ofthe overhang in tissue culture medium and may be beneficial in vivo.
The RNAi construct can also be in the form of a long double-stranded RNA. In certain embodiments, the RNAi construct is at least 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, the RNAi construct is 400-800 bases in length. The double-stranded RNAs are digested intracellularly, e.g., to produce siRNA sequences in the cell. However, use of long double-stranded RNAs in vivo is not always practical, presumably because of deleterious effects which may be caused by the sequence-independent dsRNA response. In such embodiments, the use of local delivery systems and/or agents which reduce the effects of interferon or PKR are preferred.
Alternatively, the RNAi construct is in the form of a hairpin structure (named as hairpin RNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al, Genes Dev, 2002, 16:948-58; McCaffrey et al., Nature. 2002, 418:38-9; McManus et al., RNA. 2002, 8:842-50; Yu et al., Proc Natl Acad Sci U S A, 2002, 99:6047-52). Preferably, such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.
PCT application WO 01/77350 describes an exemplary vector for bidirectional transcription of a transgene to yield both sense and antisense RNA transcripts ofthe same transgene in a eukaryotic cell. Accordingly, in certain embodiments, the present invention provides a recombinant vector having the following unique characteristics: it comprises a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene for an RNAi construct of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene f agment in a host cell.
The following exemplery RNAi sequences for EGFR and erbB2 are for illustration purposes only. Other similar sequences may be used to achieve similar results. The same approach may be employed to design and produce RNAi sequences for all pairs of target genes ofthe instant invention described above. siRNA sequences for c-erbB-2 (HER2)
Original Sequence of HER2:
1 aattctcgag ctcgtcgacc ggtcgacgag ctcgagggtc gacgagctcg agggcgcgcg 61 cccggccccc acccctcgca gcaccccgcg ccccgcgccc tcccagccgg gtccagccgg 121 agccatgggg ccggagccgc agtgagcacc atggagctgg cggccttgtg ccgctggggg
A nts 1-21 aattctcgag ctcgtcgacc g
B nts 21-41 accggtcgac gagctcgagg g
C nts 33-53 agggtcgacg agctcgaggg c
D nts 52-72 gggcgcgcgc ccggccccca c
E nts 69-89 ccacccctcg cagcaccccg c
F nts 86-106 ccgcgccccg cgccctccca g
G nts 103-123 ccagccgggt ccagccggag c
H nts 120-140 gagccatggg gccggagccg c
I nts 137-157 ccgcagtgag caccatggag c
More specifically: A nts 1 -21 aattctcgag ctcgtcgacc g
AntiSense 5 ' -P04 uuaagagcuc gagcagcuggc tt 3' -OH
Sense -P04 aauucucgag cucgucgaccg tt
B nts 21-41 ggtcgacgag ctcgagggtc g AntiSense 5' -P04 ccagcugcuc gagcucccag ctt 3' -OH Sense 5'-P04 ggucgacgag cucgaggguc gtt 3' -OH
C nts 33-53 cgagggtcga cgagctcgag g AntiSense 5'-P04 gcucccagcu gcucgagcuc ctt 3' -OH Sense 5'-P04 cgagggucga cgagcucgag gtt 3' -OH D nts 52-72 gggcgcgcgc ccggccccca c .
AntiSense 5' -P04 cccgcgcgcg ggccgggggu gtt 3' -OH
I sense 5'-P04 gggcgcgcgc ccggccccca ctt 3' -OH
E nts 69-89 ccacccctcg cagcaccccg c
AntiSense 5' -P04 gg ggggagc gucguggggc gtt 3' -OH sense 5'-P04 ccaccccucg cagcaccccg ctt 3' -OH
F nts 86-106 ccgcgccccg cgccctccca g AntiSense 5'-P04 ggcgcggggc gcgggagggu ctt 3' -OH sense 5' -P04 ccgcgccccg cgcccuccca gtt 3' -OH
G nts 103-123 ccagccgggt ccagccggag c AntiSense 5' -P04 ggucggccca ggucggccuc gtt 3' -OH sense 5'-P04 ccagccgggu ccagccggag ctt 3' -OH
H nts 120-140 gagccatggg gccggagccg c
AntiSense 5'-P04 cucgguaccc cggccucggc gtt 3' -OH sense 5'-P04 gagccauggg gccggagccg ctt 3' -OH
I nts 137-157 ccgcagtgag caccatggag c
AntiSense 5' -P04 ggcgucacuc gugguaccuc gtt 3' -OH
Sense 5' -P04 ccgcagugag caccauggag ctt 3' -OH
N nts 135-155 agccgcagtg agcaccatgg a AntiSense 5' -P04 ucggcgucac ucgugguacc utt 3' -OH Sense 5'-P04 agccgcagug agcaccaugg att 3' -OH
SiRNA for EGFR Original sequence of EGF:
1441 gcctttgaga acctagaaat catacgcggc aggaccaagc aacatggtca gttttctctt
1501 gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga gataagtgat 1561 ggagatgtga taatttcagg aaacaaaaat ttgtgctatg caaatacaat aaactggaaa
1621 aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc
1681 tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg
1741 gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag
1801 tgcaagcttc tggagggtga gccaagggag tttgtggaga actctgagtg catacagtgc 1861 cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg accagacaac
1921 tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga
1981 gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca tgtgtgccac
2041 ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg ctgtccaacg
2101 aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct cttgctgctg 2161 gtggtggccc tggggatcgg cctcttcatg cgaaggcgcc acatcgttcg gaagcgcacg nts 1491-1511 gttttctctt gcagtcgtca g
AntSense 5' -P04 caaaagagaa cgucagcagu ctt 3' -OH sense 5' -P04 guuuucucuu gcagucguca gtt 3' -OH
nts 1581-1601 aaacaaaaat ttgtgctatg c
AntiSense 5' -P04 uuuguuuuua aacacgauac gtt 3' -OH
Sense 5'-P04 aaacaaaaau uugugcuaug ctt 3' -OH
nts 1630-1650 gggacctccg gtcagaaaac c AntiSense 5' -P04 cccuggaggc cagucuuuug gtt 3' -OH Sense 5'-P04 gggaccuccg gucagaaaac ctt 3' -OH
nts 1720-1740 cccgagggct gctggggccc g AntiSense 5' -P04 gggcucccga cgaccccggg ctt 3' -OH Sense 5' -P04 cccgagggcu gcuggggccc gtt 3' -OH
nts 1741-1761 gagcccaggg actgcgtctc t
AntiSense 5' -P04 cucggguccc ugacgcagag att 3' -OH
Sense 5' -P04 gagcccaggg acugcgucuc utt 3' -OH
nts 1808-1828 ttctggaggg tgagccaagg g
AntiSense 5' -P04 aagaccuccc acucgguucc ctt 3' -OH
Sense 5' -P04 uucuggaggg ugagccaagg gtt 3' -OH
nts 1840-1860 aactctgagt gcatacagtg c AntiSense 5'-P04 uugagacuca cguaugucac gtt 3' -OH
Sense 5' -P04 aacucugagu gcauacagug ctt 3' -OH
nts 1876-1896 cctcaggcca tgaacatcac c
AntiSense 5' -P04 ggaguccggu acuuguagug gtt 3' -OH
Sense 5' -P04 ccucaggcca ugaacaucac ctt 3' -OH
nts 1951-1971 ccccactgcg tcaagacctg c AntiSense 5'-P04 ggggugacgc aguucuggac gtt 3' -OH Sense 5'-P04 ccccacugcg ucaagaccug ctt 3' -OH
nts 1991-2001 aaaacaacac cctggtctgg a
AntiSense 5' -P04 uuuuguugug ggaccagacc utt 3' -OH
Sense 5' -P04 aaaacaacac ccuggucugg att 3' -OH
nts 2109-2119 taagatcccg tccatcgcca c
AntiSense 5' -P04 auucuagggc agguagcggu gtt 3' -OH
Antisense 5' -P04 uaagaucccg uccaucgcca ctt 3' -OH
nts 2200-2220 cacatcgttc ggaagcgcac g AntiSense 5'-P04 guguagcaag ccuucgcgug ctt 3' -OH Sense 5' -P04 cacaucguuc ggaagcgcac gtt 3' -OH
These siRNAs or expression constructs can be constructed by PCR, and the PCR products can then be directly transfected into mammalian cells, resulting in functional expression of siRNAs. This approach should prove useful for identification of other optimal siRNA-target combinations and for multiplexing siRNA expression in mammalian cells.
"Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. Alternatively, "Host cell" means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell. For example, a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme. Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal). In another embodiment, the invention relates to the use of ribozyme molecules designed to catalytically cleave an mRNA transcript to prevent translation of mRNA (see, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225; and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591. The ribozymes ofthe present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and which has been extensively described (see, e.g., Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al, 1986,
Nature, 324:429-433; published International patent application No. WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). In a further embodiment, the invention relates to the use of DNA enzymes to inhibit expression of EGFR and/or erbB2 gene. DNA enzymes incorporate some of the mechanistic features of both antisense and ribozyme technologies. DNA enzymes are designed so that they recognize a particular target nucleic acid sequence, much like an antisense oligonucleotide. However much like ribozymes, they are catalytic and specifically cleave the target nucleic acid. Briefly, to design DNA enzyme that specifically recognizes and cleaves a target nucleic acid, one of skill in the art must first identify the unique target sequence. Preferably, the unique or substantially unique target sequence is a G/C rich sequence of approximately 18 to 22 nucleotides. High G/C content helps insure a stronger interaction between the DNA enzyme and the target sequence. When synthesizing the DNA enzyme, the specific antisense recognition sequence that will target the enzyme to the message is divided so that it comprises the two arms ofthe DNA enzyme, and the DNA enzyme loop is placed between the two specific arms. Methods of making and administering DNA enzymes can be found, for example, in U.S. Patent No. 6,110,462.
In certain aspects, the EGFR and erbB2 therapeutic agents ofthe present invention include a small molecule (e.g., a peptidomimetic). Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules (e.g., a peptidomimetic). As used herein, the term "peptidomimetic" includes chemically modified peptides and peptide-like molecules that contain non-naturally occurring amino acids, peptoids, and the like. Peptidomimetics provide various advantages over a peptide, including enhanced stability when administered to a subject. Methods for identifying a peptidomimetic are well known in the art and include the screening of databases that contain libraries of potential peptidomimetics. For example, the Cambridge Structural Database contains a collection of greater than 300,000 compounds that have known crystal structures (Allen et al., Acta Crystallogr. Section B, 35:2331 (1979)). Where no crystal structure of a target molecule is available, a structure can be generated using, for example, the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the Available Chemicals Directory (Molecular Design Limited, Informations Systems; San Leandro Calif.), contains about 100,000 compounds that are commercially available and also can be searched to identify potential peptidomimetics of CCL21 or a chemokine receptor.
As described herein, small molecule compounds may encompass numerous chemical classes, although typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, sulfhydryl or carboxyl group. Candidate small molecule compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds can be modified through conventional chemical, physical, and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, and amidification, to produce structural analogs.
The present invention also contemplates anti-tumor therapeutic agents obtainable from the screening methods described below.
Drug Screening Assays
There are numerous approaches to screening for EGFR and erbB2 therapeutic agents in tumor therapy. For example, high-throughput screening of compounds or molecules can be carried out to identify agents or drugs which inhibit tumor growth. Test agents to be assessed for their anti-tumor effects can be any chemical (element, molecule, compound, drug), made synthetically, made by recombinant techniques or isolated from a natural source. For example, test agents can be peptides, polypeptides, peptoids, sugars, hormones, or nucleic acid molecules (such as antisense or RNAi nucleic acid molecules). In addition, test agents can be small molecules or molecules of greater complexity made by combinatorial chemistry, for example, and compiled into libraries. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Test agents can also be natural or genetically engineered products isolated from lysates or growth media of cells — bacterial, animal or plant ~ or can be the cell lysates or growth media themselves. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps.
For example, an assay can be carried out to screen for compounds that specifically inhibit binding of EGFR and/or erbB2 to their ligand, or vice- versa, such as by inhibition of binding of labeled ligand- or receptor-Fc fusion proteins to immortalized cells. Compounds identified through this screening can then be tested in animal models of cancer (e.g., tumor xenografts implanted in nude mice) to assess their anti-tumor activity in vivo.
In one embodiment of an assay to identify a substance that interferes with interaction of EGFR and/or erbB2 with their ligand, samples of cells expressing EGFR and/or erbB2 are contacted with either labeled ligand (e.g., full-length, or a soluble portion thereof, or a fusion protein such as a fusion ofthe extracellular domain and the Fc domain of IgG) or labeled ligand plus a test compound (or group of test compounds). The amount of labeled ligand which has bound to the cells is determined. A lesser amount of label (where the label can be, for example, a radioactive isotope, a fluorescent or colormetric label) in the sample contacted with the test compound(s) is an indication that the test compound(s) interferes with binding. The reciprocal assay using cells expressing a ligand (e.g., an EGFR and/or erbB2 ligand or a soluble form thereof) can be used to test for a substance that interferes with the binding of an EGFR and/or erbB2 receptor or soluble portion thereof.
An assay to identify a substance which interferes with interaction between an EGFR and or erbB2 with their ligand can be performed with the component (e.g., cells, purified protein, including fusion proteins and portions having binding activity) which is not to be in competition with a test compound, linked to a solid support. The solid support can be any suitable solid phase or matrix, such as a bead, the wall of a plate or other suitable surface (e.g., a well of a microtiter plate), column pore glass (CPG) or a pin that can be submerged into a solution, such as in a well. Linkage of cells or purified protein to the solid support can be either direct or through one or more linker molecules.
In one embodiment, an isolated or purified protein (e.g., an EGFR and/or erbB2 receptor) can be immobilized on a suitable affinity matrix by standard techniques, such as chemical cross-linking, or via an antibody raised against the isolated or purified protein, and bound to a solid support. The matrix can be packed in a column or other suitable container and is contacted with one or more compounds (e.g., a mixture) to be tested under conditions suitable for binding ofthe compound to the protein. For example, a solution containing compounds can be made to flow through the matrix. The matrix can be washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds. Compounds which remain bound can be released by a suitable elution buffer. For example, a change in the ionic strength or pH ofthe elution buffer can lead to a release of compounds. Alternatively, the elution buffer can comprise a release component or components designed to disrupt binding of compounds (e.g., one or more ligands or receptors, as appropriate, or analogs thereof which can disrupt binding or competitively inhibit binding of test compound to the protein).
Fusion proteins comprising all of, or a portion of, a protein (e.g., an EGFR and/or erbB2 receptor) linked to a second moiety not occurring in that protein as found in nature can be prepared for use in another embodiment ofthe method. Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope). The fusion proteins can be produced by inserting the protein (e.g., an EGFR and/or erbB2 receptor) or a portion thereof into a suitable expression vector which encodes an affinity ligand.
The expression vector can be introduced into a suitable host cell for expression. Host cells are disrupted and the cell material, containing fusion protein, can be bound to a suitable affinity matrix by contacting the cell material with an affinity matrix under conditions sufficient for binding ofthe affinity ligand portion ofthe fusion protein to the affinity matrix. In one aspect of this embodiment, a fusion protein can be immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion ofthe fusion protein to the matrix, and is contacted with one or more compounds (e.g., a mixture) to be tested, under conditions suitable for binding of compounds to the receptor or ligand protein portion ofthe bound fusion protein. Next, the affinity matrix with bound fusion protein can be washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds without significantly disrupting binding of specifically bound compounds. Compounds which remain bound can be released by contacting the affinity matrix having fusion protein bound thereto with a suitable elution buffer (a compound elution buffer). In this aspect, compound elution buffer can be formulated to permit retention ofthe fusion protein by the affinity matrix, but can be formulated to interfere with binding ofthe compound(s) tested to the receptor or ligand protein portion ofthe fusion protein. For example, a change in the ionic strength or pH ofthe elution buffer can lead to release of compounds, or the elution buffer can comprise a release component or components designed to disrupt binding of compounds to the receptor or ligand protein portion ofthe fusion protein (e.g., one or more ligands or receptors or analogs thereof which can disrupt binding of compounds to the receptor or ligand protein portion ofthe fusion protein). Immobilization can be performed prior to, simultaneous with, or after contacting the fusion protein with compound, as appropriate. Various permutations ofthe method are possible, depending upon factors such as the compounds tested, the affinity matrix selected, and elution buffer formulation. For example, after the wash step, fusion protein with compound bound thereto can be eluted from the affinity matrix with a suitable elution buffer (a matrix elution buffer). Where the fusion protein comprises a cleavable linker, such as a thrombin cleavage site, cleavage from the affinity ligand can release a portion ofthe fusion with compound bound thereto. Bound compound can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction. In other embodiments, other assays can be used for screening for compounds that decrease the expression level (protein or nucleic acid) of EGFR and/or erbB2. Methods of detecting and optionally quantitating proteins can be achieved by techniques such as antibody-based detection assays, hi these cases, antibodies may be used in a variety of detection techniques, including enzyme-linked immunosorbent assays (ELISAs), immunoprecipitations, and Western blots. On the other hand, methods of detecting and optionally quantitating nucleic acids generally involve preparing purified nucleic acids and subjecting the nucleic acids to a direct detection assay or an amplification process followed by a detection assay. Amplification may be achieved, for example, by polymerase chain reaction (PCR), reverse transcriptase (RT), and coupled RT-PCR. Detection of nucleic acids is generally accomplished by probing the purified nucleic acids with a probe that hybridizes to the nucleic acids of interest, and in many instances, detection involves an amplification as well. Northern blots, dot blots, microarrays, quantitative PCR, and quantitative RT-PCR are all well known methods for detecting nucleic acids.
In some cases, one or more compounds can be tested simultaneously. Where a mixture of compounds is tested, the compounds selected by the foregoing processes can be separated (as appropriate) and identified by suitable methods (e.g., PCR, sequencing, chromatography). Large combinatorial libraries of compounds (e.g., organic compounds, peptides, nucleic acids) produced by combinatorial chemical synthesis or other methods can be tested (see e.g., Ohlmeyer, M.H.J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S.H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to tagged compounds; see also, Rutter, WJ. et al., U.S. Patent No. 5,010,175; Huebner, V.D. et al., U.S. Patent No. 5,182,366; and Geysen, H.M., U.S. Patent No. 4,833,092). Where compounds selected from a combinatorial library by the present method carry unique tags, identification of individual compounds by chromatographic methods is possible. Where compounds do not carry tags, chromatographic separation, followed by mass spectrophotometry to ascertain structure, can be used to identify individual compounds selected by the method, for example.
The instant invention also provides a method to identify agents that inhibits both EGFR and erbB2 receptors. In this embodiment, a plurality of agents are first identified as EGFR antagonists using any ofthe above described methods. Then these EGFR antagonists may be further tested, using any ofthe above methods, for their ability to inhibit erbB2. Alternatively, the method can be carried out in reverse (first identify erbB2 antagonists, then identify among these antagonists EGFR inhibitors).
Methods of Treatment
In certain embodiments, the present invention provides methods of treating an individual suffering from cancer through administering to the individual a therapeutically effective amount of an EGFR and/or erbB2 therapeutic agent as described above. In other embodiments, the invention provides methods of preventing or reducing the onset of cancer in an individual through administering to the individual a therapeutically effective amount of an EGFR therapeutic agent and a therapeutically effective amount of an erbB2 therapeutic agent. These methods are particularly aimed at therapeutic and prophylactic treatments of animals, and more particularly, humans.
In certain embodiments of such methods, one or more EGFR and/or erbB2 therapeutic agents can be administered, together (simultaneously) or at different times (sequentially), hi addition, EGFR and/or erbB2 therapeutic agents can be administered with another type of compounds for treating cancer. The two types of compounds may be administered simultaneously or sequentially.
In certain embodiments, gene therapy may be applicable with the use of nucleic acids encoding EGFR and/or erbB2 therapeutic polypeptides (for example, fragments of EGFR and/or erbB2, see below). Alternatively, an antisense nucleic acid or an RNAi construct can be used for reducing or inhibiting expression of EGFR and/or erbB2. Preferably, such gene therapy is specific for tumor tissues.
Methods ofthe present invention can be used to treat a variety of cancer types, including but not limited to: breast cancer, ovarian cancer, skin cancer (for example, melanoma), cancer ofthe GI mucosa (such as colon cancer); neuro/glioblastoma, leukemia, such as acute promyelocytic leukemia (APL), adenocarcinoma, including adenocarcinoma ofthe salivary gland, gastric cancer, and prostate cancer. In addition, EGFR and or erbB2 therapeutic agents can be also combined with VEGF inhibitors.
Pharmaceutical Compositions In certain embodiments, EGFR and/or erbB2 therapeutic agents of the present invention are formulated with a pharmaceutically acceptable carrier or salt. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention, e.g., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
Such therapeutic agents can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the compound included in the pharmaceutical preparation may itself be active, or may be a prodrug. The term "prodrug" refers to compounds which, under physiological conditions, are converted into therapeutically active agents.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Pharmaceutical compositions ofthe EGFR and/or erbB2 therapeutic agents include those suitable for oral/ nasal, topical, parenteral and/or intravaginal administration. The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount ofthe compound which produces a therapeutic effect.
Methods of preparing these pharmaceutical compositions or compositions include combining an anti-tumor therapeutic agent and a carrier and, optionally, one or more accessory ingredients. In general, the pharmaceutical compositions can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product. Pharmaceutical compositions for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of an artemisinin-related compound as an active ingredient. An artemisinin-related compound may also be administered as a bolus, electuary or paste. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), one or more EGFR and/or erbB2 therapeutic agents ofthe present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tefrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. hi particular, therapeutic agents or pharmaceutical compositions can be topically, either to skin or to mucosal membranes, such as those ofthe cervix and vagina. The topical pharmaceutical compositions may further include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2- pynolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur.
Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an artemisinin-related compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to an EGFR and/or erbB2 therapeutic agent, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Pharmaceutical compositions suitable for parenteral administration may comprise one or more anti-tumor therapeutic agents in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood ofthe intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions ofthe invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance ofthe required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, hi addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.
Injectable depot forms are made by forming microencapsule matrices of one or more anti-tumor therapeutic agents in biodegradable polymers such as polylactide-polyglycohde. Depending on the ratio of drug to polymer, and the nature ofthe particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable pharmaceutical compositions are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. Pharmaceutical compositions for intravaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds ofthe invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Optionally, such pharmaceutical compositions suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray pharmaceutical compositions containing such carriers as are known in the art to be appropriate.
Methods of Administration hi certain embodiments, the subject methods ofthe invention can be used alone. Alternatively, the subject methods may be used in combination with other conventional anti-cancer therapeutic approaches directed to treatment or prevention of proliferative disorders (e.g., tumor). For example, such methods can be used in prophylactic cancer prevention, prevention of cancer recunence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present invention recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of an EGFR / erbB2 therapeutic agent which blocks signaling through the EGFR and/or erbB2 receptor (as described above).
A wide variety of conventional compounds have been shown to have anti- neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each ofthe treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.
When an EGFR and/or erbB2 therapeutic agent ofthe present invention is administered in combination with another conventional anti-neoplastic agent, either concomitantly or sequentially, such therapeutic agent may enhance the therapeutic effect ofthe anti-neoplastic agent or overcome cellular resistance to such anti- neoplastic agent. This may allow decrease of dosage of an anti-neoplastic agent, thereby reducing the undesirable side effects, or restores the effectiveness of an anti- neoplastic agent in resistant cells.
Pharmaceutical compounds that may be used for such combination chemotherapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxahplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.
These chemotherapeutic compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5 -fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes - dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.
Depending on the nature ofthe cancer and the therapy, administration ofthe
EGFR and/or erbB2 therapeutic agents ofthe invention may be continued while the other therapy is being administered and/or thereafter. Administration ofthe EGFR and/or erbB2 therapeutic agents may be made in a single dose, or in multiple doses, hi some instances, administration ofthe EGFR and/or erbB2 therapeutic agents is commenced at least several days prior to the conventional therapy, while in other instances, administration is begun either immediately before or at the time ofthe administration ofthe conventional therapy.
Delivery means
The instant invention also provides expression vectors comprising a nucleotide sequence encoding a subject EGFR and/or erbB2 therapeutic agent
(nucleotide) operably linked to at least one regulatory sequence. "Operably linked" when describing the relationship between two DNA regions simply means that they are functionally related to each other. For example, a promoter or other transcriptional regulatory sequence is operably linked to a coding sequence if it controls the transcription ofthe coding sequence. Alternatively, "operably linked" is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the encoded polypeptide.
Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in
Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For instance, any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the chimeric polypeptides of this invention. Such useful expression control sequences, include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAG or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of inhibitors desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
As used herein, "transformed cells" refers to cells that have spontaneously converted to a state of unrestrained growth, e.g., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. For purposes of this invention, the terms "transformed phenotype of malignant mammalian cells" and "transformed phenotype" are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals. As will be apparent, the subject gene constructs can be used to cause expression ofthe subject therapeutic agents in target cells, e.g., to produce inhibitors that inhibits the function of EGFR and c-erbB2 receptors. In certain therapeutic applications, the ex vtvo-derived inhibitors are utilized in a manner appropriate for therapy in general. For such therapy, the inhibitors or vectors encoding inhibitors ofthe invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration, hi such embodiments, a polypeptide inhibitor may by combined with a pharmaceutically acceptable excipient, e.g., a non-pyrogenic excipient. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection being prefened, including intramuscular, intravenous, intraperitoneal, and subcutaneous injection, the inhibitors ofthe invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the inhibitors may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the peptides are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, particularly cosmetic pharmaceutical compositions, the oligomers ofthe invention are formulated into ointments, salves, gels, or creams as generally known in the art. Alternative means of administration of peptides have been developed.
Sustained-release pharmaceutical compositions (Putney, et al. Nature Biotechnology 1998, 16, 153-157) are advantageous, requiring fewer administrations and, often, lower dosages. Techniques for oral delivery of peptides have been reviewed (Fasano, A. Trends in Biotechnology 1998, 16, 152-157), as have several site- specific means of peptide delivery (Pettit, D.K. et al. Trends in Biotechnology 1998, 16, 343-349). Additional techniques for therapeutic administration of peptides are known to those of skill in the art. Genetic material inhibitors ofthe present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces the desired inhibitor (e.g. RNA).
In one embodiment, the genetic material is provided by use of an "expression" construct, which can be transcribed in a cell to produce the inhibitor. Such expression constructs may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively transfecting cells either ex vivo or in vivo with genetic material encoding an inhibitor. Approaches may include insertion ofthe antisense nucleic acid in viral vectors including recombinant retro viruses, adenoviruses, adeno-associated viruses, human immunodeficiency viruses, and herpes simplex viruses- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection ofthe gene construct or calcium phosphate precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice ofthe particular gene delivery system will depend on such factors as the phenotype ofthe intended target and the route of administration, e.g., locally or systemically.
As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked. One type of vector is a genomic integrated vector, or "integrated vector," which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to that they are operatively linked are refened to herein as "expression vectors." In the present specification, "plasmid" and "vector" are used interchangeably unless otherwise clear from the context. In the expression vectors, regulatory elements controlling transcription can be generally derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually confened by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as retroviruses, adenoviruses, and the like, may be employed.
One approach to in vivo introduction of genetic material encoding one ofthe subject inhibitors into a cell is by use of a viral vector containing said genetic material. Infection of cells with a viral vector has the advantage that a large proportion ofthe targeted cells can receive the nucleic acid. Additionally, inhibitors encoded by genetic material in the viral vector, e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid. Such a strategy may be particularly effective when skeletal muscle cells are the targets ofthe vector (Fisher, KJ. et al. Nature Medicine 1997, 3, 306- 312).
Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transfened nucleic acids are stably integrated into the chromosomal DNA ofthe host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility ofthe spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication- defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part ofthe retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one ofthe antisense E6AP constructs, rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZTP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone manow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395- 1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641- 647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345 ; and PCT Application WO 92/07573).
In choosing retroviral vectors as a gene delivery system for genetic material encoding the subject chimeric polypeptides, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction ofthe genetic material, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors. In fact, such limitation on infection can be particularly beneficial in the instant circumstances wherein the normal tissue (e.g., nontransformed cells) sunounding the target cancer cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors. Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retro viral-based vectors, by modifying the viral packaging proteins on the surface ofthe viral particle (see, for example, PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification ofthe infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Man et al. (1992) J Gen Virol 73:3251-3255; and Goud et al. (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda et al. (1991) JBiol Chem 266:14143-14146). Coupling can be in the form ofthe chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating chimeric proteins (e.g., single-chain antibody/e«v chimeric proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.
Moreover, use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences (such as breast or ovary-specific regulatory sequences) which control expression ofthe genetic material of the retroviral vector.
Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting non- dividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al.
(1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard
(1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and, as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity ofthe adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts ofthe viral El and E3 genes but retain as much as 80% ofthe adenoviral genetic material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology, E. J. Munay, Ed.
(Humana, Clifton, NJ, 1991) vol. 7. pp. 109-127). Expression ofthe inserted genetic material can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences. Yet another viral vector system useful for delivery of genetic material encoding the subject chimeric polypeptides is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is also one ofthe few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell Biol 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol Cell. Biol. 4:2072-2081 ; Wondisford et al. (1988) Mol. Endocrinol 2:32-39; Tratschin et al. (1984) J. Virol 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).
Other viral vector systems that may have application in gene therapy have been derived from herpes virus, vaccinia virus, and several RNA viruses. In addition to viral transfer methods, such as those illustrated above, non- viral methods can also be employed to cause expression of genetic material encoding the subject inhibitors in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In prefened embodiments, non- viral gene delivery systems ofthe present invention rely on endocytic pathways for the uptake of genetic material by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, polylysine conjugates, and artificial viral envelopes.
In a representative embodiment, genetic material can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and, optionally, which are tagged with antibodies against cell surface antigens ofthe target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075). For example, lipofection of papilloma-infected cells can be carried out using liposomes tagged with monoclonal antibodies against PV- associated antigen (see Viac et al. (1978) J Invest Dermatol 70:263-266; see also Mizuno et al. (1992) Neurol Med. Chir. 32:873-876). hi yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For example, genetic material encoding the subject chimeric polypeptides can be used to fransfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g., polylysine (see U.S. Patent 5,166,320). It will also be appreciated that effective delivery ofthe subject nucleic acid constructs via mediated endocytosis can be improved using agents which enhance escape ofthe gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides ofthe influenza HA gene product can be used as part ofthe delivery system to induce efficient disruption of DΝA-containing endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) PNAS 89:7934; and Christiano et al. (1993) PΝAS 90:2122). In clinical settings, the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation ofthe gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction ofthe target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression ofthe gene, or a combination thereof. In other embodiments, initial delivery ofthe recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3054-3057).
Moreover, the pharmaceutical preparation can consist essentially ofthe gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral packages, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system. In the latter case, methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals, and can be adapted for release of viral particles through the manipulation ofthe polymer composition and form. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of an the viral particles by cells implanted at a particular target site. Such embodiments ofthe present invention can be used for the delivery of an exogenously purified virus, which has been incorporated in the polymeric device, or for the delivery of viral particles produced by a cell encapsulated in the polymeric device.
By choice of monomer composition or polymerization technique, the amount of water, porosity and consequent permeability characteristics can be controlled. The selection ofthe shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response. The generation of such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and the Sabel et al. U.S. Patent No. 4,883,666. hi another embodiment of an implant, a source of cells producing a the recombinant virus is encapsulated in implantable hollow fibers. By "recombinant virus" is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle. Such fibers can be pre-spun and subsequently loaded with the viral source
(Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S. Patent No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178- 183), or can be co-extruded with a polymer which acts to form a polymeric coat about the viral packaging cells (Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent No. 4,353,888; Sugamori et al. (1989) Trans. Am. Artifi Intern. Organs 35:791-799; Sefton et al. (1987) Biotechnol Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials 12:50-55). Again, manipulation ofthe polymer can be carried out to provide for optimal release of viral particles. Inhibitors of the present invention can be designed by using molecular modeling. A computer model of EGFR or c-erbB2 receptor may be used to identify any compounds that might bind EGFR or c-erbB2 receptor in the ligand binding sites. Alternatively, antagonistic compounds mimicking the natural ligands of these receptors might be designed in silica. Alternatively, the nature ofthe inhibitory sequence can be determined by calculation, based on knowledge of a receptor or binding pocket. Other calculational strategies will be known to those skilled in the art. Calculations such as these can be useful for directing the synthesis of inhibitors ofthe present invention in a time- and material-efficient manner, before actual synthesis and screening techniques begin. Methods for screening inhibitors ofthe present invention are well known in the art, independent ofthe use of computer modeling. The use of peptide libraries is one way of screening large numbers of polypeptides at once. In one screening assay, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind a target molecule, such as a receptor protein via this gene product is detected in a "panning assay". For instance, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting chimeric polypeptide detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). hi an alternate embodiment, the peptide library is expressed as chimeric polypeptides on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at very high concentrations, a large number of phage can be screened at one time. Second, since each infectious phage displays the combinatorial gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd, and fl are most often used in phage display libraries, as either ofthe phage gill or gVIII coat proteins can be used to generate chimeric polypeptides without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO 90/02809; Ganard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBOJ 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
The field of combinatorial peptide libraries has been reviewed (Gallop et al. J. Med. Chem. 1994, 37, 1233-1251), and additional techniques are known in the art (Gustin, K Virology 1993, 193, 653-660; Goeddel et al. U.S. Patent 5,223,408; Markland et al. PCT publication WO92/15679; Bass et al. Proteins: Structure, Function and Genetics 1990, 8, 309-314;Cunningham, B.C. Science 1990, 247, 1461-1465; Lowman, H.B. Biochemistry 1991, 30, 10832-10838; Fowlkes et al. U.S. Patent No. 5,789,184; Houghton, Proc. Natl Acad. Sci. U.S.A. 1985, 82, 5131-5135) for generating and screening peptide libraries.
U.S. Pat. No. 6,420,110 (incorporated herein by reference) discloses a method for isolating biologically active peptides. Using the techniques disclosed therein, a polypeptide inhibitor ofthe present invention may be developed which interacts with a chosen receptor, and inhibits the signaling of said receptor. The inhibition can be readily tested in many suitable in vitro or in vivo models, using such art-recognized techniques as reporter genes and/or selectable markers under the control of promoters responsive to ΕGFR and/or c-erbB2 receptors. In a representative example, this method is utilized to identify polypeptide inhibitors which have antagonistic activity with respect to one or more types of cells expressing at least one ofthe EGFR or erbB2 receptors. One of skill in the art will readily be able to modify the procedures outlined below to find polypeptides with any desired activity. In the example, in the display mode, the chimeric polypeptide library can be panned with the target cells or immobilized EGFR or erbB2 receptor in order to enrich for polypeptides which bind to that cell or receptor. At that stage, the polypeptide library can also be panned against one or more control cell lines (that does not express any ofthe two EGFR / erbB2 receptors) in order to remove polypeptides which bind the control cells. In this manner, the polypeptide library which is then tested in the secretion mode can be enriched for polypeptides which selectively bind target cells (relative to the control cells). Thus, for example, the display mode can produce a polypeptide library enriched for polypeptides which preferentially bind EGFR+ and or erbB2+ tumor cells relative to normal cells, which preferentially bind p53- cells relative to p53+ cells, which preferentially bind hair follicle cells relative to other epithelial cells, or any other differential binding characteristic. hi the secretion mode, the polypeptides are tested for antiproliferative activity against the target cell using any of a number of techniques known in the art. For instance, BrdU or other nucleotide uptake can be measured as an indicator of proliferation. As above, the secretion mode can include negative controls in order to select for polypeptides with specific antiproliferative activity.
In similar fashion, polypeptides can be isolated from the library based on their ability to induce apoptosis or cell lysis, for example, in a cell-selective manner. Also, this method can be used to identify polypeptides with angiogenic or antiangiogenic activity. For instance, the polypeptide library can be enriched for polypeptides that bind to endothelial cells but which do not bind to fibroblasts. The resulting sub-library can be screened for polypeptides which inhibit capillary endothelial cell proliferation and/or endothelial cell migration. Polypeptides scoring positive for one or both of these activities can also be tested for activity against other cell types, such as smooth muscle cells or fibroblasts, in order to select polypeptides active only against endothelial cells. Furthermore, this method can be used to identify anti-infective polypeptides, for example, which are active as anti-fungal or antibacterial agents.
In addition, this assay can be used for identifying effectors of a receptor protein or complex thereof. In general, the assay is characterized by the use of a test cell which includes a target receptor or ion channel protein whose signal transduction activity can be modulated by interaction with an extracellular signal, the transduction activity being able to generate a detectable signal.
In general, such assays are characterized by the use of a mixture of cells expressing a target receptor protein or ion channel capable of transducing a detectable signal in the reagent cell. The receptor/channel protein can be either endogenous or heterologous. In combination with the disclosed detection means, a culture ofthe instant reagent cells will provide means for detecting agonists or antagonists of receptor function.
The ability of particular polypeptides to modulate a signal transduction activity of the target receptor or channel can be scored for by detecting up or down- regulation ofthe detection signal. For example, second messenger generation (e.g., GTPase activity, phospholipid hydrolysis, or protein phosphorylation patterns as examples) can be measured directly. Alternatively, the use of an indicator gene can provide a convenient readout. In other embodiments a detection means consists of an indicator gene. In any event, a statistically significant change in the detection signal can be used to facilitate identification of compounds which modulate receptor or ion channel activities.
By this method, polypeptides which induce a signal pathway from a particular receptor or channel can be identified. If a test polypeptide does not appear to induce the activity ofthe receptor/channel protein, the assay may be repeated as described above, and modified by the introduction of a step in which the reagent cell is first contacted with a known activator ofthe target receptor/channel to induce signal transduction, and the test peptide can be assayed for its ability to inhibit the activated receptor/channel, for example, to identify antagonists. In yet other embodiments, peptides can be screened for those which potentiate the response to a known activator ofthe receptor. EXAMPLES
The following examples are for illustrative purpose only, and should in no way be construed to be limiting in any respect ofthe claimed invention.
Example 1. Assessment of the effect of inhibiting EGFR and erbB2 in cancer cells
There have been previous reports of a functional interaction between cAMP- dependent protein kinase (PKA) and EGFR signaling due to reduced proliferation (decrease in PKA-Typel) and induction of differentiation (increase in PKA-Typell) in NLH:OVCAR-8 human ovarian cancer cells that are stably transfected with an EGFR-antisense expression vector (AS-EGFR)5. A morphological alteration observed in AS-EGFR cells was associated with a decrease in the expression of E- cadherin, a- and ^catenin6. Although AS-EGFR cells showed reduced proliferation and tumour formation in nude mice , they formed colonies in soft-agar with an incidence similar to parental cells (unpublished observation). A subsequent study showed that AS-EGFR cells have reduced α6-integrin subunit expression (a well characterized laminin- 1 receptor) and MMP-9 activity and decreased adhesion to laminin-1. On the other hand, transfection of NIH:OVCAR-8 cells with an inducible c-erbB-2-antisense expression vector (AS-c-erbB-2) resulted in inhibition of cell growth and colony formation in soft agar4. These data support the hypothesis that EGFR and c-erbB-2 overexpression in NIH:OVCAR-8 cells enhances an invasive phenotype and cell growth.
Applicant describes here use of antisense-EGFR and antisense-c-erbB-2 to study the combined effect of inhibiting both receptors in NIH:OVCAR-8 cells, paying particular attention to 1) the potential loss of invasive phenotype due to decreased EGFR expression, and 2) the potential inhibition of transforming ability due to decreased c-erbB-2 expression. Specifically, Applicants report that cells with both EGFR and erbB2 inhibition exhibit reduced outgrowth in Matrigel, reduced MMP-9 expression, and a decreased ability to form colonies in soft agar. Applicants cloned NIH:OVCAR-8 cells and analyzed the subtle changes in the clones by CGH analysis (data not shown). After stable transfection of parental 8A1 cells (a subclone of NTH:OVCAR-8 cells) with AS-EGFR and/or AS-c-erbB-2 and Ev-Cv vectors, Applicants selected several clones and verified the reduction of phosphorylated protein expression for EGFR and c-erbB-2 receptors by immunoprecipitation and Western blotting of cell lysate after loading equal amounts of total protein. The 8A1 parental cells, as well as the double- vector control transfectants Ev-Cv, expressed high amounts of active EGFR and c-erbB-2, whereas E2 cells (singly AS-EGFR transfected) expressed decreased level of phosphorylated EGFR. Similarly, the C7 cells (singly AS-c-erbB-2 transfected) expressed decreased level of phosphorylated c-erbB-2 (Fig.l). Double transfectants for AS-EGFR and AS-c-erbB-2, including clones E-C32.d and E-C32.2, expressed decreased levels of both phosphorylated EGFR and phosphorylated c-erbB-2 in comparison to the parental 8A1 cells (Fig.l). These observations indicate that simultaneous suppression of EGFR and c-erbB-2 phosphorylation by antisense-expressing constructs was highly effective.
Example 2. Assessment of cellular phenotypic changes
Applicants examined the cellular phenotypic changes in single and double AS-EGFR and/or AS-c-erbB-2 transfectants; and compared them to the parental 8A1 cells (Fig. 2). When grown on Matrigel™, a naturally occurring basement membrane-like extracellular matrix, the parental 8A1 cells and the Ev-Cv control transfectants were polygonal in shape with interconnected cell clusters (Fig. 2a). In contrast, in the E-C32.d and the E-C32.2 double transfectants, Applicants observed viable as isolated, spherical, single cells (Fig. 2b). Zymography analysis showed a more than 3 fold decrease in secreted MMP-9 compared to the parental 8A1 cells (30% in E-C32.2 cells versus 100% in 8A1 cells (Fig. 2c).
Example 3. Induction of Apoptosis and Aneuploidy Regression in Cancer cells
Genomic instability is thought to be a driving force of carcinogenesis, producing many genetic changes that confer growth advantage, invasive and metastatic ability to tumor cells7. Reduced malignancy, therefore, may be accompanied by a more stable genetic phenotype. Applicants have compared the chromosomal profiles ofthe single and double antisense-transfected clones to the parental clone by molecular cytogenetic techniques, including fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). A direct chromosomal enumeration (after Giemsa-staining) coupled with CGH data revealed a striking tendency for reduction of chromosomal aneuploidy in the double transfectants. The parental 8A1 cells had a ploidy range from 42-113, with the major clone having 58 chromosomes indicative of significant chromosomal instability (Fig. 3a and b). In the E-C32.d and the E-C32.2 double transfected clones, however, Applicants observed a reduction in the chromosomal number, with clonal ploidy of 46-52 and 46-50, respectively (Fig. 3f) (Table 1). Consistent with the decrease in chromosome copy number, Applicants observed a decrease in ploidy of E-C32.d and E-C32.2 double transfectants by fluorescence activated cell sorting (FACS) analysis (Table 1). Chromosomal analysis revealed that a high percentage of cells (40%) were undergoing apoptosis in the E-C32.d clone, as evidenced by chromosomal pulverization (Fig. 3c) and nuclear fragmentation (Fig. 3d). Chromosomal endoreduplication and non-disjunction (Fig. 3e) were indicated by giant metaphase plates that were either blocked in mitosis or at the point of mitotic exit. In contrast, only 5 % ofthe parental 8 Al cells were apoptotic. Taken together, Applicants concluded that a subpopulation of E-C32.d and E-C32.2 double transfectants underwent apoptosis, while the majority of cells that were rescued from apoptosis had a near- normal diploidy (46-50 in the case ofthe E-C32.2 clone).
Table 1 Summary of genomic analysis in parental 8A1, Ev-Cv control, E-C32.d and E-C32.2 double transfectants
Cell Line DNA Modal Losses Gains
Chromosome Index Number (range)
Parental 1.2 58 (42-113) lpter-p32, lq23-32, 2p, 2ql4-q21, 2q33- lp31-cen, 2cen-q21, 4cen-4q21, 4q24-
Subcloneδ qter, 3q22-q28, 4p,4q32-qter, 6q22-qter, q28, 5, 6pter-p21.3, 6ql2-q21,
Al 7pl3, 7qll.2, 7q22-31, 8p, 9p, 10q25, lip, 7q31-qter, 8pll.2-ql l.2, 8q22-qter, llq23.3-qter, 13cen-ql4, 14q24-qter, 15q, 1 Opter-p 13, 10cen-q24, 11 cen- 16pll.2, 16q22-qter, 17p-13-cen, 17ql2, q23,13q21-qter, 17q22-q24, 20ql3.2- 18q, 19ql3.1-qter, 21q22, 22q, Xpter-p22.1 qter
Ev-Cv 1.2 No difference with parental subclone No difference from parental subclone
E-C32.d 1.14 49 (46-52) 5, 7q32-qter, 10, llq23.3-qter, 12p 4p, 15q22-qter, X
E-C32.2 1.14 48 (46-50) 5, 7q32-qter, 8q22-qter, 10, llq23.3- 12ql5-q21, 15q22-qter, 18q23.1- qter, 12p, 20q qter, X
Chromosome number from metaphase spreads and DNA index by FACS analysis for each clone is compared with gains and losses from CGH analysis. Each result represents a duplicate experiment.
Example 4. Different Degrees of Chromosomal Imbalance in the Parental Cancer Cells and the Double-Tranfectants
Applicants expanded our analysis by CGH to view a global picture of all chromosomal imbalances in the parental cell line and second, in the single or double transfectants, in order to pin-point the differences among the parental 8A1 cells, the double vector control cells (Ev-Cv), the single transfectants (E2, C7), and the double transfectants (E-C32.d, E-C32.2).
DNA from the parental 8A1 cells (rather than DNA from normal donors) was used as a reference for comparison with the E-C32.d and the E-C32.2 double transfectants8. CGH analysis of chromosomal imbalances in the parental 8A1 cells revealed a high level of genomic imbalances (Fig. 3g and Fig. IB - Supplementa CGH analysis) as summarized in Table 1. Applicants observed losses of chromosomes 5 and chromosomal region 1 lq23.3-qter in both double transfectants, while the parental 8A1 cells had a gain of chromosome 5 (Table 1). Gain of chromosome 5 is one ofthe earliest genetic events for different human ovarian surface epithelial cells immortalized by HPV16-E6E7 viral oncogenes9. Applicants also observed loss of chromosome 8q22-qter in E-C32.2 double transfected cells, while the same region was a gain in the parental 8A1 cells (Table 1). The amplification of c-myc on chromosomal region 8q24 (~30 %) is a common finding in ovarian and other cancers10'11. CGH data analysis showed a loss of chromosome 8q22-qter in the E-C32.2 double transfectant (Table 1). A loss of chromosome 20q was also observed (Table 1). Interestingly, forty percent of ovarian primary carcinomas also have a gain in copy number of chromosome region 20q12. This region includes MMP-9 (20ql2.2-13.1), a putative oncogene, EEF1A2 (20ql3), and a recently identified testis-cancer gene BORIS (Brother ofthe Regulator of
Imprinted Sites) 13'14. BORIS is the first gene from the testis-cancer family that has oncogenic properties and maps to 20ql3.2, a hot spot of amplification in many cancers including ovarian cancer1 . A gain of chromosome region 18q23.1-qter in the E-C32.2 double transfectant was seen. This region is frequently lost (-33%) in ovarian carcinomas and was also lost in parental 8A1 cells and Ev-Cv control transfectants16.
Example 5. Assessment of Gene Regulation in Double-Transfected Closes
Recent results revealed a major direct role of DNA copy number alteration in the transcriptional program of human cancer cells17. Therefore a more normal karyotype is expected to be associated with a more normal gene expression pattern. Applicants investigated the genes that were up- and down-regulated in double transfected clones in comparison to the parental 8A1 and the Ev-Cv control cells using cDNA microanay analysis. Applicants observed a consistent and marked up- regulation of IGFBP-3 expression (7pl3-pl2) in both the E-C32.d and the E-C32.2 double transfectants (Table 2). Interestingly, this finding conelated with the loss of 7pl3 in parental 8A1 cells by CGH analysis (Table 1). Normal ovarian epithelial cells have been shown to express high levels of IGFBP-3 which also induces apoptosis in p53 negative cells12'18. The finding might also suggest an IGFBP-3 induced apoptosis that would eliminate highly aneuploid cells in the model. Applicants have observed that Caspase-8 and DNA-activated kinase mRNA were also upregulated in E-C32.d and E-C32.2 double transfectants (Table 2).
Interestingly, Caspase-8, which maps to 6ql6.1 is an initiator ofthe death receptor pathway that activates apoptotic substrates including PARP and DNA-activated
10 90 1 kinase ' ' . Differential gene expression analysis also showed upregulation of fibronectin (FN) mRNA in E-C32.d and E-C32.2 double transfectants in contrast to parental 8A1 cells (Table 2). In addition, confocal laser scanning microscopic images indicated a significant difference in FN expression and distribution by immunofluorescence staining analysis. Parental 8A1 cells deposited relatively less FN that localized mainly to the cell periphery (Fig. 4a). hi contrast, E-C32.2 double transfectants had an increase in FN expression and fibrillogenesis occuned over the entire ventral cell surface (Fig. 4b). The data conelates with studies showing the presence of FN throughout the stroma of normal ovarian tissue and the loss of FN in advanced ovarian tumours22. Thus, these findings supported a positive conelation between CGH profiles of regional gains and losses and levels of differential gene expression in our model system (Fig. 5).
Table 2 Differential gene expression profile in single and double transfectants compared to parental 8A1 cells were analyzed by cDNA microarray analysis
Figure imgf000072_0001
The calibrated ratios indicate the expression levels of a given gene in the E- C32.d and E-C32.2 double transfected clones relative to that in the the parental 8A1 cells. A single anay was used to measure expression in E-C32.d clone. For the E- C32.d cells, expression was measured in replicate microarray hybridizations, and the mean ± standard deviation ofthe calibrated ratio is listed for genes that had acceptable measurement quality in both. Specific gene annotations can be reviewed at LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/index.html). Each result represents a total of duplicate experiments.
Example 6. Progesterone receptor expression
A decrease in progesterone receptor (PR) levels in ovarian adenocarcinomas and an increase in EGFR expression in comparison to benign, borderline and normal ovarian tissues has been shown earlier23. Therefore, Applicants investigated PR expression in parental and double transfected clones by immunohistochemical analysis. Double transfectants showed a significant increase in PR number
(p<0.0001) and intensity of staining (/ θ.0001) in E-C32.2 double transfectant compared to parental 8A1 cells (Fig. 4c, and 4d). The significant increase in PR expression and induction of apoptosis by simultaneous inhibition of EGFR and c- erbB-2 is consistent with the study showing a direct interaction of PARP with the DNA binding domain of human PR providing an insight into the cellular mechanisms of regulation of receptor tyrosine-kinase signaling pathway24. PR positive tumours are associated with an improved patient survival25. Collectively, the phenotypic characterization suggest that simultaneous targeting of EGFR and c- erbB-2 induces apoptosis in cells that are aneuploid and overexpress EGFR and c- erbB-2 while cells that are near-diploid or diploid and have a more normal phenotype survive (Fig. 5).
Example 7. Assessment ofthe Mechanism underlying the Genomic Effect on Suppression of Aneuploidy To investigate the mechanisms underlying the genomic effect on suppression of aneuploidy, Applicants studied apoptosis by AnnexinV/ FITC cell surface (early apoptosis) and Propidium Iodide (PI) nuclear (late apoptosis) staining. Conespondingly, Applicants found a significant conelation of "genomic recovery" with the induction of apoptosis. A significant increase in apoptosis when measured by AnnexinV/PI staining was observed in both single and double transfected clones when compared to parental 8A1 cells (Fig. 6a). In addition, Applicants were able to show cleaved PARP [Poly(ADP-ribose)polymerase] expression in E-C32.d and E- C32.2 double transfectants (Fig. 6b) by Western blot analysis. Interestingly, Applicants detected upregulation of tankyrase in E-C32.d and E-C32.2 double transfectants by differential gene expression analysis (Table 2). Tankyrase is a family member of the poly(ADP-ribosyl)ating proteins that has PARP activity 26. PARP cleavage by caspase(s) occurs early in apoptosis, earlier on or soon after the appearance of internucleosomal fragmentation of DNA27. Although the underlying molecular events of these interactions remain unclear, it is speculated that PARP takes part mainly in intenelated events in the nucleus including DNA repair, cell cycle regulation and apoptosis contributing to the maintenance of stability of the genome 21 ' 28.
This study is the first to show karyotype reversion to a near-diploid state following dual suppression of EGFR and c-erbB-2 receptor expression in a carcinoma cell line. Intrinsic mechanisms of cellular response to double antisense transfection for these receptors in a clone of cancer cells included reversal ofthe malignant phenotype, reduction of aneuploidy and genomic imbalances, normalization of gene expression profiles, and reduction of invasiveness (MMP-9 activity and Matrigel outgrowth).
In summary, Applicants conclude that simultaneous inhibition of EGFR and c-erbB-2 expression by antisense transfection of a subclone of aneuploid cancer cells selects for a near-diploid cell population with a benign phenotype. Similar experiments employing a dual siRNA-induced inhibition of these erbB receptors are expected to yield the same results.
Methods
The following methods and materials were used in the examples. Transfection
NIH:OVCAR-8 cell line was a gift by Dr. T.C. Hamilton (Fox Chase Center, Philadelphia, PA). Cells were cultured in DVIEM containing 10 % fetal bovine serum (FBS). The antisense expressing vector for AS-c-erbB-2 (pRC/CMV-AS 5') and control vector, (pRC/CMV) was kindly provided by Dr. Shoshana Segal (NIH/NCI). The antisense expressing vector for EGFR (pCDNA3.1/AS-EGFR) was constructed as previously described using pCDNA3.1 zeo (Invitrogen Corporation, San Diego, CA)6. Parental 8A1 cells were stably transfected with vectors expressing AS-EGFR and/or AS-c-erbB-2 (pCDNA3.1/AS-EGFR and/or pRC/CMV/AS-c-erbB-2, respectively) and control vectors, Ev-Cv (pCDNA3.1 and/or pRC/CMV, respectively). To verify the identity ofthe plasmids used in transfections, AS-EGFR and AS-c-erbB-2 inserts were sequenced using primers complementary to the vector sequences and ABI PRISM BigDye™ Terminator Cycle Sequencing kit (PE- Applied Biosystems; Foster City, CA) according to the manufacturer's protocol. Sequencing products were separated on ABI Prism 377 DNA Sequencer (PE- Applied Biosystems; Foster City, CA).
Analysis of Apoptosis and Ploidy
Apoptosis was detected by Annexin V-FITC and propidium iodide staining using AnnexinV-FITC Apoptosis kit (Pharmingen, San Diego, CA). Twenty thousand cells were analyzed on FACSort (Becton Dickinson, San Diego CA).
Results were analyzed utilizing FCS Express (De Novo Software, Ontario, Canada). Cells were stained with propidium iodide and analyzed in duplicate with and without human peripheral blood lymphocytes as an internal standard. One hundred thousand cells were analyzed on FACSort. DNA index and ploidy were determined utilizing ModFitLT software (Verity Software, Topsham, ME).
Biochemistry
Cells (lxlO6 cells) in tissue culture were lysed in RJLPA buffer; 250mM NaCl, 20mM Na2HPO4, 1 % Triton, 1 % deoxycholic acid, 0.1 % sodium dodecyl sulfate, protease inhibitor (Complete Mini, Roche), ImM NaVO4. Total protein (800μg/ml) was immunoprecipitated with the following mouse monoclonal antibodies specific to phosphotyrosine (4G10, Upstate, Lake Placid, NY), resolved by 4-12 % SDS- PAGE gels and transfened to nitrocellulose sheets. Blotting was performed with anti-EGFR antibody (Ab-12, NeoMarkers, Fremont, CA) and anti-c-erbB-2 antibody (Ab-18, Santa Cruz Biotechnology, CA). The expression of PARP and actin was analyzed by western blot using 20-40μg protein/lane. Blotting was performed using anti-PARP mouse monoclonal antibody (MoAb) (Ab2, Oncogene Research, Boston, MA) according to manufacturer's instructions and anti-actin MoAb (119, Santa Cruz Biotechnology, CA). Bands were visualized with secondary HRP-coηjugated antibodies and the ECL system (Amersham Pharmacia).
MMP-9 activity was measured by zymography as previously described29. Briefly, cells were grown to 80 % confluence in complete medium and then incubated for 72 hours in serum-free medium. Aliquots of serum-free media were analyzed by gel electrophoresis. Dried gels were scanned by use of Chemihnager ™ 5500 (Alpha Innotech Corporation, San Leandro, CA).
Matrigel Outgrowth and hnmuno-cvto and -histochemistry Cells were seeded on glass coverslips in 12-well plates coated with 0.5 ml of
Matrigel (10 mg/ml; kindly provided by Dr. Hynda Kleinman, National Institute of Dental and Craniofacial Research, Bethesda, MD). The plates were incubated at 37°C in EVIEM medium (Gibco) containing 10 % FBS for various time periods. After the nonadherent cells were removed by washing gently with PBS, adherent cells were fixed with 70 % methanol for 5 min at room temperature and stained with Giemsa (Sigma, St. Louis, MO). Fibronectin expression was detected by staining the cells with anti-fibronectin MoAb (Transduction Laboratories, Lexington, KY) and FITC-labelled secondary MoAb. The images were analyzed using an Olympus IX- 70 Laser Confocal Scanning Microscope equipped with a Olympus 60x/1.4 N.A. objective lens. Paraffin sections for control and transfected cells were stained with anti-progesterone Receptor MoAb (A6, Coulter Corporation, Miami, FL) using DAKO Envision Kit (Dako Corporation, CA). The images were analyzed using an Olympus Vanox Microscope equipped with a Zeiss 25x/0.8 N.A. objective lens.
CGH and SKY Comparative genomic hybridization and spectral karyotyping were done as described previously8. Tumour cell lines after one passage in culture following stable transfection, were processed and analyzed by CGH, spectral karyotyping (SKY), and specific chromosome paints (FISH). Cells were anested at mitosis by treatment with colcemid (Gibco/BRL) at 0.1 μg /ml for 1 hour. Mitotic chromosome spreads were prepared, and SKY analysis was performed as described30. For FISH analysis, metaphases were hybridized using directly labelled locus specific probes for chromosomes 16 and 20 (Vysis, Downers Grove, Illinois).
Differential Gene Expression
Cells were grown in duplicate in 150-mm dishes (Corning, Corning, NY) and RNA was isolated using TRIzol LS (Invitrogen, Carlsbad, CA), and then further purified using an RNeasy spin column (Qiagen, Valencia, CA). RNA eluted from the columns was immediately concentrated to approximately 3 μg/μl using a Microcon 30 centrifugal filter device (Millipore, Bedford, MA) and stored at -80°C until used for microanay analysis. cDNA microanays containing approximately 6,400 clones were obtained through a collaboration with the Cancer Genetics Branch at The National Human Genome Research Institute, NIH. CyDye-labeled cDNA was prepared from RNA and hybridized to microanays as described at http://research.nhgri.nih.gov/microanay/index.html. Arrays were scanned using an Agilent microanay scanner (Agilent Technologies, Palo Alto, CA), and expression ratios were determined using the Anaysuite software package,
(http://research.nhgri.nih.gov/microanay/image-analysis.html) which is a collection of software tools customized for analyzing cDNA microanays, written as extensions to the IPLab image analysis software package (Scanalytics, Fiarfax, VA) for the Apple Macintosh computer.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific method and reagents described herein, including alternatives, variants, additions, deletions, modifications and substitutions. Such equivalents are considered to be within the scope of this invention and are covered by the following claims. REFERENCES
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EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific method and reagents described herein, including alternatives, variants, additions, deletions, modifications and substitutions. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

CLAIMS:
1. A method of inducing apoptosis and aneuploidy regression, and/or differentiation towards a normal phenotype, in cancer cells overexpressing EGFR and erbB2 receptor, comprising administering (a) a therapeutically effective amount of at least one therapeutic agent that blocks signaling through EGFR in said cells, and (b) a therapeutically effective amount of at lest one therapeutic agent that blocks signaling through erbB2 in said cells, whereby apoptosis is induced in highly aneuploid cancer cells and aneuploidy regression is induced with the survival of near-diploid cancer cells and/or diploid normal cells.
2. The method of claim 1 , wherein the said therapeutic agent of (a) inhibits the interaction between EGFR and a ligand, and the therapeutic agent of (b) inhibits interaction between erbB2 and a ligand.
3. The method of claim 1 , wherein the therapeutic agent of (a) inhibits expression of EGFR, and the therapeutic agent of (b) inhibits expression of erbB2.
4. The method of claim 1 , wherein the therapeutic agent of (a) inhibits activity of EGFR, and the therapeutic agent of (b) inhibits activity of erbB2.
5. The method of claim 1 , wherein the therapeutic agent of (a) inhibits clustering of EGFR with an erbB family receptor, and the therapeutic agent of (b) inhibits clustering of erbB2 with an erbB2 family recedptor.
6. The method of claim 1, wherein the therapeutic agent of (a) inhibits phosphorylation of EGFR, and the therapeutic agent of (b) inhibits phosphorylation of erbB2.
7. The method of claim 1 , wherein the therapeutic agent of (a) inhibits a downstream signaling event upon binding of EGFR to a ligand, and the therapeutic agent of (b) inhibits a downstream signaling event upon binding of erbB2 to a ligand.
8. The method of claim 1 , wherein the therapeutic agent of (a) blocks signaling through EGFR by inhibiting: the interaction between EGFR and a ligand, expression of EGFR, activity of EGFR, clustering of EGFR with an erbB family receptor, phosphorylation of EGFR, or a downstream signaling event upon binding of EGFR to a ligand, and wherein the therapeutic agent of (b) blocks signaling through erbB2 by inhibiting: the interaction between erbB2 and a ligand, expression of erbB2, activity of erbB2, clustering of erbB2 with an erbB family receptor, phosphorylation of erbB2, or a downstream signaling event upon binding of erbB2 to a ligand.
9. The method of any one of claims 1-8, wherein the therapeutic agent of (a) is an antibody and the therapeutic agent of (b) is an antibody.
10. The method of claim 9, wherein the antibody of (a) is specifically immunoreactive with EGFR, and the antibody of (b) is specifically immunoreactive with erbB2.
11. The method of any one of claims 1 -8, wherein the therapeutic agent of (a) is a protein, and the therapeutic agent of (b) is a protein.
12. The method of claim 11 , wherein the protein of (a) is a mutated form or a mimic of EGFR, and the protein of (b) is a mutated form or a mimic of erbB2.
13. The method of claim 11 , wherein the protein of (a) is a fragment of EGFR, and the protein of (b) is a fragment of erbB2.
14. The method of claim 11 , wherein the protein of (a) comprises an extracellular domain of EGFR, and the protein of (b) comprises an extracellular domain of erbB2.
15. The method of any one of claims 12-14, wherein the protein of (a) and the protein of (b) are both fusion proteins.
16. The method of any one of claims 1 -8, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both nucleic acids.
17. The method of claim 16, wherein the nucleic acid of (a) is an antisense nucleic acid of EGFR, the nucleic acid of (b) is an antisense nucleic acid of erbB2.
18. The method of claim 16, wherein the nucleic acid of (a) is an RNAi construct of EGFR, and the nucleic acid of (b) is an RNAi construct of erbB2.
19. The method of any one of claims 1-8, wherein the therapeutic agent of (a) and the therapeutic agent of (b) are both small molecule compounds.
20. The method of claim 19, wherein the therapeutic agent of (a) and the therapeutic agent of (b) are both peptidomimetics.
21. The method of any one of claims 1-20, wherein the therapeutic agent of (a) and the therapeutic agent of (b) are both formulated with pharmaceutically acceptable carriers.
22. The method of any one of claims 1-8, further comprising administering at least one chemotherapeutic agent that affects growth ofthe tumor cells in an additive or synergistic manner with the therapeutic agent of (a) and/or (b).
23. The method of claim 22, wherein the chemotherapeutic agent is selected from the group consisting of: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, mefhotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxahplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.
24. The method of any one of claims 1-8, further comprising administering ionizing radiation.
25. The method of any one of claims 1 -24, wherein the cancer cells are selected from the group consisting of: breast cancer cells; ovarian cancer cells; skin cancer cells (for example, melanoma), cancer cells ofthe GI mucosa (such as colon cancer); neuro/glioblastoma cancer cells; leukemia cells, such as acute promyelocytic leukemia (APL), adenocarcinoma cells, including cells from adenocarcinoma ofthe salivary gland or gastric cancer, and prostate cancer cells.
26. A packaged pharmaceutical comprising (a) an EGFR therapeutic agent; (b) an erbB2 therapeutic agent; and (c) instructions and/or a label for administration of said therapeutic agent for the treatment of patients having tumors that overexpress EGFR and erbB2.
27. The packaged pharmaceutical of claim 26, wherein said EGFR therapeutic agent inhibits the interaction between EGFR and a ligand, and said erbB2 therapeutic agent inhibits the interaction between erbB2 and a ligand.
28. The packaged pharmaceutical of claim 26, wherein said EGFR therapeutic agent inhibits gene expression of EGFR, and said erbB2 therapeutic agent inhibits gene expression of erbB2.
29. The packaged pharmaceutical of claim 26, wherein said EGFR therapeutic agent inhibits activity of EGFR, and said erbB2 therapeutic agent inhibits activity of erbB2.
30. The packaged pharmaceutical of claim 26, wherein said EGFR therapeutic agent inhibits clustering of EGFR with an erbB family receptors, and said erbB2 therapeutic agent inhibits clustering of erbB2 with an erbB family receptors.
31. The packaged pharmaceutical of claim 26, wherein said EGFR therapeutic agent inhibits phosphorylation of EGFR, and said erbB2 therapeutic agent inhibits phosphorylation of erbB2.
32. The packaged pharmaceutical of claim 26, wherein said EGFR therapeutic agent inhibits a downstream signaling event upon binding of EGFR with a ligand, and said erbB2 therapeutic agent inhibits a downstream signaling event upon binding of erbB2 with a ligand.
33. The packaged pharmaceutical of claim 26, wherein said EGFR therapeutic agent inhibits: the interaction between EGFR and a ligand, expression of EGFR, activity of EGFR, clustering of EGFR with an erbB family receptor, phosphorylation of EGFR, or a downstream signaling event upon binding of EGFR to its ligand, and said erbB2 therapeutic agent inhibits: the interaction between erbB2 and a ligand, expression of erbB2, activity of erbB2, clustering of erbB2 with an erbB family receptor, phosphorylation of erbB2, or a downstream signaling event upon binding of erbB2 to a ligand.
34. The packaged pharmaceutical of any one of claims 26-33, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both antibodies.
35. The packaged pharmaceutical of claim 34, wherein the antibody of (a) is specifically immunoreactive with EGFR, and the antibody of (b) is specifically immunoreactive with erbB2.
36. The packaged pharmaceutical of any one of claims 26-33, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both proteins.
37. The packaged pharmaceutical of claim 36, wherein the protein of (a) is a mutated form or a mimic of EGFR, and the protein of (b) is a mutated form or a mimic of erbB2.
38. The packaged pharmaceutical of claim 36, wherein the protein of (a) is a fragment of EGFR, and the protein of (b) is a fragment of erbB2.
39. The packaged pharmaceutical of claim 36, wherein the protein of (a) comprises an extracellular domain of EGFR, and the protein of (b) comprises an extracellular domain of erbB2.
40. The packaged pharmaceutical of any one of claims 36-39, wherein the protein of (a) and the protein of (b) are both fusion proteins.
41. The packaged pharmaceutical of any one of claims 26-33, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both nucleic acids.
42. The packaged pharmaceutical of claim 41, wherein the nucleic acid of (a) is an antisense nucleic acid of EGFR, and the nucleic acid of (b) is an antisense nucleic acid of erbB2.
43. The packaged pharmaceutical of claim 41 , wherein the nucleic acid of (a) is an RNAi construct of EGFR, and the nucleic acid of (b) is an RNAi construct of erbB2.
44. The packaged pharmaceutical of any one of claims 26-33, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both small molecule compounds.
45. The packaged pharmaceutical of claim 44, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both peptidomimetics.
46. The packaged pharmaceutical of any one of claims 26-33, wherein said therapeutic agent of (a) is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound; and said therapeutic agent of (b) is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound.
47. A method of treating a patient suffering from cancer in which cancer cells overexpress EFGR and erbB2, the method comprising administering to the patient (a) a therapeutically effective amount of at least one therapeutic agent that suppresses phosphorylated EGFR, and (b) a therapeutically effective amount of at least one therapeutic agent that suppresses phosphorylated erbB2.
48. The method of claim 47, wherein said therapeutic agent of (a) inhibits the interaction between EGFR with a ligand, and said therapeutic agent of (b) inhibits the interaction between erbB2 with a ligand.
49. The method of claim 47, wherein said therapeutic agent of (a) inhibits gene expression of EGFR, and said therapeutic agent of (b) inhibits gene expression of erbB2.
50. The method of claim 47, wherein said therapeutic agent of (a) inhibits activity of EGFR, and said therapeutic agent of (b) inhibits activity of erbB2.
51. The method of claim 47, wherein said therapeutic agent of (a) inhibits clustering of EGFR with an erbB family receptor, and said therapeutic agent of (b) inhibits clustering of erbB2 with an erbB family receptor
52. The method of claim 47, wherein said therapeutic agent of (a) inhibits phosphorylation of EGFR, and said therapeutic agent of (b) inhibits phosphorylation of erbB2.
53. The method of claim 47, wherein said therapeutic agent of (a) inhibits a downstream signaling event upon binding of EGFR to a ligand, and said therapeutic agent of (b) inhibits a downstream signaling event upon binding of erbB2 to a ligand.
54. The method of claim 47, wherein said therapeutic agent of (a) inhibits: the interaction between EGFR and a ligand, expression of EGFR, activity of EGFR, clustering of EGFR with an erbB family receptor, phosphorylation of EGFR, or a downstream signaling event upon binding of EGFR to its ligand, and said therapeutic agent of (b) inhibits: the interaction between erbB2 and a ligand, expression of erbB2, activity of erbB2, clustering of erbB2 with an erbB family receptor, phosphorylation of erbB2, or a downstream signaling event upon binding of erbB2 to a ligand.
55. The method of any one of claims 47-54, wherein the therapeutic agent of (a) is an antibody, and the therapeutic agent of (b) is an antibody.
56. The method of claim 55, wherein the antibody of (a) is specifically immunoreactive with EGFR, and the antibody of (b) is specifically immunoreactive with erbB2.
57. The method of any one of claims 47-54, wherein the therapeutic agent of (a) and the therapeutic agent of (b) are both proteins.
58. The method of claim 57, wherein the protein of (a) is a mutated form or a mimic of EGFR, and the protein of (b) is a mutated form or a mimic of erbB2.
59. The method of claim 57, wherein the protein of (a) is a fragment of EGFR, and the protein of (b) is a fragment of erbB2.
60. The method of claim 57, wherein the protein of (a) comprises an extracellular domain of EGFR, and the protein of (b) comprises an extracellular domain of erbB2.
61. The method of any one of claims 57-60, wherein the protein of (a) and the protein of (b) are both fusion proteins.
62. The method of any one of claims 47-54, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both nucleic acids.
63. The method of claim 62, wherein the nucleic acid of (a) is an antisense nucleic acid of EGFR, and the nucleic acid of (b) is an antisense nucleic acid oferbB2.
64. The method of claim 62, wherein the nucleic acid of (a) is an RNAi construct of EGFR, and the nucleic acid of (b) is an RNAi construct of erbB2.
65. The method of any one of claims 47-54, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both small molecule compounds.
66. The method of claim 65, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both peptidomimetics.
67. The method of any one of claims 47-54, wherein said therapeutic agent of (a) is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound; and said therapeutic agent of (b) is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound.
68. Use of (a) an EGFR therapeutic agent and (b) an erbB2 therapeutic agent in the manufacture of medicaments for the treatment of cancer that over expresses EGFR and erbB2.
69. The use of claim 68, wherein said EGFR therapeutic agent inhibits the interaction between EGFR and a ligand, and said erbB2 therapeutic agent inhibits the interaction between erbB2 and a ligand.
70. The use of claim 68, wherein said EGFR therapeutic agent inhibits gene expression of EGFR, and said erbB2 therapeutic agent inhibits gene expression of erbB2.
71. The use of claim 68, wherein said EGFR therapeutic agent inhibits activity of EGFR, and said erbB2 therapeutic agent inhibits activity of erbB2.
72. The use of claim 68, wherein said EGFR therapeutic agent inhibits clustering of EGFR with an erbB family receptor, and said erbB2 therapeutic agent inhibits clustering of erbB2 with an erbB family receptor.
73. The use of claim 68, wherein said EGFR therapeutic agent inhibits phosphorylation of EGFR, and said erbB2 therapeutic agent inhibits phosphorylation of erbB2.
74. The use of claim 68, wherein said EGFR therapeutic agent inhibits a downstream signaling event upon binding of EGFR to a ligand, and said erbB2 therapeutic agent inhibits a downstream signaling event upon binding of erbB2 to a ligand.
75. The use of claim 68, wherein said EGFR therapeutic agent inhibits: the interaction between EGFR and a ligand, expression of EGFR, activity of
EGFR, clustering of EGFR with an erbB family receptor, phosphorylation of EGFR, or a downstream signaling event upon binding of EGFR to a ligand, and wherein said erbB2 therapeutic agent inhibits: the interaction between erbB2 and a ligand, expression of erbB2, activity of erbB2, clustering of erbB2 with an erbB family receptor, phosphorylation of erbB2, or a downstream signaling event upon binding of erbB2 to a ligand.
76. The use of any one of claims 68-75, wherein said EGFR therapeutic agent and said erbB2 therapeutic agent are both antibodies.
77. The use of claim 76, wherein the antibody of said EGFR therapeutic agent is specifically immunoreactive with EGFR, and the antibody of said erbB2 therapeutic agent is specifically immunoreactive with erbB2.
78. The use of any one of claims 68-75, wherein said EGFR therapeutic agent and said erbB2 therapeutic agent are both proteins.
79. The use of claim 78, wherein the protein of said EGFR therapeutic agent is a mutated form or a mimic of EGFR, and the protein of said erbB2 therapeutic agent is a mutated form or a mimic of erbB2.
80. The use of claim 78, wherein the protein of said EGFR therapeutic agent is a fragment of EGFR, and the protein of said erbb2 therapeutic agent is a fragment of erbB2.
81. The use of claim 78, wherein the protein of said EGFR therapeutic agent comprises an extracellular domain of EGFR, and the protein of said erbB2 therapeutic agent comprises an extracellular domain of erbB2.
82. The use ofany one of claims 78-81, wherein the protein of said EGFR therapeutic agent and the protein of said erbB2 therapeutic agent are both fusion proteins.
83. The use of any one of claims 68-75, wherein said EGFR therapeutic agent and said erbB2 therapeutic agent are both nucleic acids.
84. The use of claim 83, wherein the nucleic acid of said EGFR therapeutic agent is an antisense nucleic acid of EGFR, and the nucleic acid of said erbB2 therapeutic agent is an antisense nucleic acid of erbB2.
85. The use of claim 84, wherein the nucleic acid of said EGFR therapeutic agent is an RNAi construct of EGFR, and the nucleic acid of said erbB2 therapeutic agent is erbB2.
86. The use of any one of claims 68-75, wherein said EGFR therapeutic agent and said erbB2 therapeutic agent are both small molecule compounds.
87. The use of claim 86, wherein said EGFR therapeutic agent and said erbB2 therapeutic agent are both peptidomimetics.
88. The use of any one of claims 68-75, wherein said EGFR therapeutic agent is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound; and said erbB2 therapeutic agent is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound.
89. A pharmaceutical composition for the treatment of patients having tumors that overexpress EGFR and erbB2, said pharmaceutical composition comprising (a) an EGFR therapeutic agent; and (b) an erbB2 therapeutic agent.
90. The pharmaceutical composition of claim 89, wherein said EGFR therapeutic agent inhibits the interaction between EGFR and a ligand, and said erbB2 therapeutic agent inhibits the interaction between erbB2 and a ligand.
91. The pharmaceutical composition of claim 89, wherein said EGFR therapeutic agent inhibits gene expression of EGFR, and said erbB2 therapeutic agent inhibits gene expression of erbB2.
92. The pharmaceutical composition of claim 89, wherein said EGFR therapeutic agent inhibits activity of EGFR, and said erbB2 therapeutic agent inhibits activity of erbB2.
93. The pharmaceutical composition of claim 89, wherein said EGFR therapeutic agent inhibits clustering of EGFR with an erbB family receptors, and said erbB2 therapeutic agent inhibits clustering of erbB2 with an erbB family receptors.
94. The pharmaceutical composition of claim 89, wherein said EGFR therapeutic agent inhibits phosphorylation of EGFR, and said erbB2 therapeutic agent inhibits phosphorylation of erbB2.
95. The pharmaceutical composition of claim 89, wherein said EGFR therapeutic agent inhibits a downstream signaling event upon binding of EGFR with a ligand, and said erbB2 therapeutic agent inhibits a downstream signaling event upon binding of erbB2 with a ligand.
96. The pharmaceutical composition of claim 89, wherein said EGFR therapeutic agent inhibits: the interaction between EGFR and a ligand, expression of EGFR, activity of EGFR, clustering of EGFR with an erbB family receptor, phosphorylation of EGFR, or a downstream signaling event upon binding of EGFR to its ligand, and said erbB2 therapeutic agent inhibits: the interaction between erbB2 and a ligand, expression of erbB2, activity of erbB2, clustering of erbB2 with an erbB family receptor, phosphorylation of erbB2, or a downstream signaling event upon binding of erbB2 to a ligand.
97. The pharmaceutical composition of any one of claims 89-96, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both antibodies.
98. The pharmaceutical composition of claim 97, wherein the antibody of (a) is specifically immunoreactive with EGFR, and the antibody of (b) is specifically immunoreactive with erbB2.
99. The pharmaceutical composition of any one of claims 89-96, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both proteins.
100. The pharmaceutical composition of claim 99, wherein the protein of (a) is a mutated form or a mimic of EGFR, and the protein of (b) is a mutated form or a mimic of erbB2.
101. The pharmaceutical composition of claim 99, wherein the protein of (a) is a fragment of EGFR, and the protein of (b) is a fragment of erbB2.
102. The pharmaceutical composition of claim 99, wherein the protein of (a) comprises an extracellular domain of EGFR, and the protein of (b) comprises an extracellular domain of erbB2.
103. The pharmaceutical composition of any one of claims 99- 102, wherein the protein of (a) and the protein of (b) are both fusion proteins.
104. The pharmaceutical composition of any one of claims 89-96, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both nucleic acids.
105. The pharmaceutical composition of claim 104, wherein the nucleic acid of (a) is an antisense nucleic acid of EGFR, and the nucleic acid of (b) is an antisense nucleic acid of erbB2.
106. The pharmaceutical composition of claim 104, wherein the nucleic acid of (a) is an RNAi construct of EGFR, and the nucleic acid of (b) is an RNAi construct of erbB2.
107. The pharmaceutical composition of any one of claims 89-96, wherein said therapeutic agent of (a) and said therapeutic agent of (b) are both small molecule compounds.
108. The pharmaceutical composition of claim 107, wherein said therapeutic agent i of (a) and said therapeutic agent of (b) are both peptidomimetics.
109. The pharmaceutical composition of any one of claims 89-96, wherein said therapeutic agent of (a) is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound; and said therapeutic agent of (b) is selected from: a protein, including an antibody; a nucleic acid; or a small molecule compound.
110. A method of inducing apoptosis and aneuploid regression in cancer cells overexpressing EGFR and erbb2 receptors, comprising administering a therapeutically effective amount of an inhibitor of EGFR and a therapeutically effective amount of an inhibitor of erbB2 receptor, wherein a therapeutically effective amount of an inhibitor of EGFR and a therapeutically effective amount of an inhibitor of erbB2 receptor are amounts sufficient to induce apoptosis in highly aneuploid cancer cells overexpressing EGFR and erbB2 receptor, and aneuploid regression in near diploid cancer cells overexpressing EGFR and erbB2 receptor.
111. A method of treating cancer in an individual, wherein cancer cells in the individual overexpresses EGFR and erbB2, comprising administering to the individual therapeutic agents which simultaneously suppress phosphorylated EGFR and erbB2, whereby apoptosis and/or differentiation is induced in highly aneuploid cancer cells and aneuploid regression is induced in near- diploid and/or diploid cancer cells.
112. The method of claim 111, wherein the therapeutic agents comprises antisense EGFR and antisense erbB2 in sufficient amounts to induce apoptosis in highly aneuploid cancer cells and aneuploid regression in near-diploid cancer cells.
113. The method of claim 111, wherein the cancer is ovarian cancer or breast cancer.
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US7696320B2 (en) 2004-08-24 2010-04-13 Domantis Limited Ligands that have binding specificity for VEGF and/or EGFR and methods of use therefor
EP3698807A1 (en) 2005-01-21 2020-08-26 Genentech, Inc. Fixed dosing of her antibodies
EP2399605A1 (en) 2005-02-23 2011-12-28 Genentech, Inc. Extending time to disease progression or survival in cancer patients
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US8940302B2 (en) 2007-03-02 2015-01-27 Genentech, Inc. Predicting response to a HER inhibitor
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EP2170404A2 (en) * 2007-06-22 2010-04-07 Intradigm Corporation Compositions comprising human egfr-sirna and methods of use
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WO2011014457A1 (en) 2009-07-27 2011-02-03 Genentech, Inc. Combination treatments
WO2011146568A1 (en) 2010-05-19 2011-11-24 Genentech, Inc. Predicting response to a her inhibitor
US9427458B2 (en) 2010-07-22 2016-08-30 Consejo Nacional De Investigaciones Cientificas Y Tecnicas, Conicet Methods for inhibiting cancer cell proliferation
WO2012031027A1 (en) 2010-08-31 2012-03-08 Genentech, Inc. Biomarkers and methods of treatment
EP3264089A1 (en) 2010-08-31 2018-01-03 Genentech, Inc. Biomarkers and methods of treatment
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WO2013148315A1 (en) 2012-03-27 2013-10-03 Genentech, Inc. Diagnosis and treatments relating to her3 inhibitors
US10240207B2 (en) 2014-03-24 2019-03-26 Genentech, Inc. Cancer treatment with c-met antagonists and correlation of the latter with HGF expression
WO2017194554A1 (en) 2016-05-10 2017-11-16 Inserm (Institut National De La Sante Et De La Recherche Medicale) Combinations therapies for the treatment of cancer
WO2018200505A1 (en) 2017-04-24 2018-11-01 Genentech, Inc. Erbb2/her2 mutations in the transmbrane or juxtamembrane domain

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