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Abstract
Angiogenesis is the process that leads to the production of new blood vessels from pre-existing ones and is vital in the progression of a number of diseases, including cancer. The receptor tyrosine kinases KDR and Flt1, and their ligand vascular endothelial cell growth factor (VEGF), have been shown to be vitally important in this process and as such have become the focus of research by a large number of groups. This present study was centred about the generation of novel antibodies against the intracellular kinase insert domain of each receptor with the ultimate goal of blocking receptor signalling. During the course of this study, a number of novel anti-KDR and anti-Flt1 antibodies were produced by peptide vaccination, one of which (D11.0.6, an anti-KDR antibody) displayed excellent binding properties. It successfully detected denatured KDR in western blotting and also native KDR in immunoprecipitations. In both cases it was unique in recognising all three forms of the KDR receptor. D11.0.6 was used to investigate KDR turnover under a number of conditions and successfully detected changes in the total protein. It was then used to identify KDR in tissue sections by immunoperoxidase staining and later successfully used to intracellularly block KDR function. Injecting D11.0.6 into human umbilical vein endothelial cells reduced VEGF-induced calcium mobilisation by 77% thus showing VEGF-induced calcium mobilisation is due to KDR signalling. While Flt1 vaccinations did not lead to the production of a useful antibody, they did lead to useful insights into the requirements for peptide design, which will be important for future peptide vaccinations. D11.0.6 provides a novel reagent for blocking KDR signalling. This not only permits in vitro studies to investigate receptor signalling, but also could lead to a mechanism of blocking KDR function, and hence angiogenesis, in vivo. Thus DNA encoding this antibody could be targeted to tumour endothelial cells in vivo as a novel therapeutic.
