12^th Asian Biologics and Biosimilars Congress

Angiogenesis is a highly controlled process of growing new blood vessels under normal circumstances. However, in a large number of pathologies, such as solid tumor growth, angiogenesis is a crucial component of the disease process. Therefore, inhibitors of angiogenesis are being investigated as potential therapeutics for tumor growth. During angiogenesis endothelial cells of existing blood vessels undergo a complex process of reshaping, migration, growth, and organizing into new vessels. Vascular Endothelial Growth Factor (VEGF) is a central mediator of this process and acts via receptors whose

expression is restricted almost exclusively to endothelial cells. Because of its selectivity, VEGF represents a unique vehicle for delivery of inhibitors of angiogenesis to endothelial cells. Among potential inhibitors of angiogenesis, the shiga-like toxin-1 (SLT-I) produced by E. coli O157:H7 has the advantage that endothelial cells appear to be particularly sensitive to its action. The hypothesis that combining an SLT-I toxin with VEGF as a delivery vehicle would serve as a highly selective and active inhibitor of angiogenesis. To this end, fusion proteins containing VEGF121 and two forms of shiga-like toxin-I (SLT-I) were developed and tested in vitro for activities that have the potential to inhibit angiogenesis in vivo. Plasmids encoding the fusion proteins VEGF121/A1 containing the catalytically active fragment of the SLT-I A subunit and VEGF121/A containing the full length A subunit of SLT-I were constructed in pET-29a and pET-32a systems. Escherichia coli BL21 (DE3) pLysS bacteria were transformed with the plasmid constructs for the expression of these two fusion proteins. Both puri ed fusion proteins inhibited the translation of luciferase mRNA as a reporter gene in vitro translation system, indicating that both fusion proteins retain the N-glycosidase activity of SLT-I. However, only VEGF121/A1 fusion proteins displayed the ability to induce

auto-phosphorylation of the VEGF receptor KDR/FLK-1 and displayed a strong, selective growth inhibition of cultured cells expressing KDR/FLK-1 receptors. These results indicated that VEGF/SLT fusion proteins are promising therapeutic agents that can be developed into powerful and selective inhibitors of angiogenesis.