One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals into intracellular signals that in turn modulate biochemical pathways. Examples of such extracellular signaling molecules include growth factors, cytokines, and chemokines. The cell surface receptors of these molecules and their associated signal transduction pathways are therefore one of the principal means by which cellular behavior is regulated. Because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in the molecular components of signal transduction pathways. Consequently, considerable attention has been devoted to the characterization of these receptor proteins.
HER4 (also known as ErbB4 and p180/erbB4), a member of the EGF family of receptor/tyrosine kinases, is a protein that has been shown to play a complex role in several signal transduction pathways by forming homo- and heterodimers with other members of the EGF family depending on their concentrations and the concentration of particular ligands. The two groups of ligands specific to HER3 and HER4 are collectively termed neuregulins (NRGs) because of their demonstrated role in the nervous system. HER3 and HER4 function as the ligand binding receptors and HER2 along with EGFR are considered co-receptors and are recruited as partners to HER3 and HER4 upon ligand binding. (Burden and Yarden, Neuron, 1997, 18, 847-855).
HER4, first cloned in 1993, is predominantly expressed in breast carcinoma cell lines, and in normal skeletal muscle, heart, pituitary, brain, and the cerebellum (Plowman et al., Proc. Natl. Acad. Sci. U. S. A., 1993, 90, 1746-1750). Disclosed in the European Patent Application EP 0 599 274 A1 are the recombinant polynucleotide encoding HER-4, antibodies to HER-4, plasmids encoding HER-4, host cells containing said plasmids and a HER-4 ligand (Plowman et al., 1994). Further disclosed in the PCT Publication WO 99/19488 are the HER-4 gene, antibodies to HER-4, vectors encoding HER-4, host cells expressing said vectors and bioassays for the detection of HER-4 (Klagsbrun et al., 1999).
HER4 has subsequently been shown to regulate the development of heart and neural tissues of the CNS (Carraway, BioEssays, 1996, 18, 263-266) and manifestations of altered HER4 regulation appear in both injury and disease states the most important being in the development of cancer. Cellular transformation and acquisition of the metastatic phenotype are the two main changes normal cells undergo during the progression to cancer and many studies have implicated HER4 in the development of the cancer phenotype. Overexpression of HER4 is correlated with decreased survival time in some cases of squamous cell carcinoma of the head and neck and Furger et al. showed that HER4 is overexpressed in granulosa cell tumors and that HRG/PE40, a toxic fusion protein, reduced tumor cell proliferation (Furger et al., Cancer Res., 1998, 58, 1773-1778). Recently, it has been demonstrated that HER-3 and HER-4 are expressed at high levels in gastric cancers with three out of six gastric cancers expression HER-3 and four out of six gastric cancers overexpressing HER-4 (Kataoka et al., Life Sci., 1998, 63, 553-564).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of HER4. Consequently, there remains a long felt need for agents capable of effectively inhibiting HER4 function. To date, strategies aimed at inhibiting HER4 function have involved the use of antibodies to HER2, fusion protein ligand toxins, and gene knockouts in mice and ribozymes.
Disclosed in the PCT Publication WO 99/23209 are enzymatic RNA molecules which cleave HER-4 mRNA and methods of using these ribozymes (Tang and Lippman, 1999).
Antibodies to HER2 have been used to disrupt the dimerization of HER4 with HER2 thereby inhibiting HER4 function (Fitzpatrick et al., FEBS Lett., 1998, 431, 102-106). The fusion protein, HAR-TX beta 2 was found to be cytotoxic toward several carcinoma cell lines expressing the HER4 receptor while it did not induce cytotoxicity in cells expressing HER2 or HER3 (Siegall et al., J. Biol. Chem., 1995, 270, 7625-7630). Finnaly, mice defective in HER4 showed impaired heart trabeculae resulting in embryonic death (Gassmann et al., Nature, 1995, 378, 390-394).
It is evident that these targeting strategies are indirect and often inhibit multiple pathways or result in lethal modifications to the cell or organism. Therefore, antisense oligonucleotides provide a promising new pharmaceutical tool for the effective modification of the expression of specific genes.