Gentherapeutische Modelle zur anti-angiogenen Therapie experimenteller Tumoren

The development of anti-angiogenic therapy approaches as well as the specific drug release at site of interest (drug targeting) presents a promising advance in conventional anti cancer therapies. In this study, the membrane bound fusion proteins Lodavin and Scavidin were used, consisting of parts of an endocytotic receptor and an extracellular avidin domain. Because of the physicochemical properties of biotin that allow coupling to chemotherapeutics or radionuclides and its exceptional high affinity to avidin, the fusion proteins could be implemented for gene therapeutical and clinical benefit. Due to the strong and transient expression of Flk-1 in endothelial cell during tumor angiogenesis, the regulatory elements of the flk-1 gene were used for the expression of the fusion proteins in transgenic mice. The generated Flk-1-Lodavin and Flk-1-Scavidin transgenic animals showed an endothelial specific expression of both fusion proteins in all organs throughout the embryonic development without any obvious phenotype. Adult transgenic mice showed no expression of the transgenes. Only exception has been vessels of the spleen in Flk-1-Lodavin transgenic mice. Analysis of experimental BFS-1 fibrosarcomas confirmed expression of both fusion proteins during the neovascularisation of tumors grown in transgenic mice. Furthermore, retrovirus-mediated gene transfer of the fusion proteins via co-injection of BFS-1 tumor cells with virus-producing GP+E86 cells resulted in a selective infection of tumor endothelial cells but not of tumor cells. In addition, a tumor cell specific model with an inducible expression of Scavidin was generated. Binding studies has been performed with the animal models. After systemic administration of a biotinylated agent, the ligand accumulated exclusively in the Lodavin-expressing tissue. These results confirm the functionality of the system as an in vivo targeting model. In future studies biotinylated conjugates for diagnostical or therapeutical purposes should be tested systemically in the presented animal models. The transcription factor Ets-1 plays an essential role in the transcriptional regulation of important angiogenic processes. It is expressed transiently in activated tumor endothelial cells and regulates the expression of proteins involved in angiogenesis like endothelial receptor tyrosin kinases, matrix metalloproteinases and adhesion molecules. Here, Ets-1-dependent pathways have been blocked by overexpression of a dominant negative transcription factor mutant of Ets-1 (dnEts-1). Initially, the dominant negative effect of the mutant was confirmed by inhibition of the Ets-responsive flk-1 promoter in vitro. In vivo, retroviral gene transfer of dnEts-1 was performed via co-injection of GP+E86 cells with B16 melanoma or BFS-1 fibrosarcoma cells in syngeneic mice. In both models, no alteration in tumor angiogenesis or tumor progression could be confirmed, although Flk-1 expression was inhibited. These results indicate that Ets-1 exerts pleiotropic functions in endothelia cells. Future studies should be done to block additional pathways, because Ets-1 is only one factor out of a complex that regulates the expression of genes involved in tumor angiogenesis. Possible targets are hypoxia-inducible factors (HIF). A positive influence on the activation of several endothelial promoters involved in angiogenesis has already been demonstrated especially for HIF2α. Together with Ets-1, HIF2α acts synergistically on stimulation of the flk-1 promoter activity. Here, the dominant negative effect of a mutant (dnHIF) was confirmed by inhibition of the flk-1 promoter in vitro. Retroviruses should be generated for the dnhif gene transfer in vivo to block HIF-dependent pathways during development of experimental tumors. Implications on the tumor vascularization also by co-expression with dnEts-1 should be tested in future studies and analyzed as a potential anti-angiogenic model.