Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of Shiga-like toxin-VEGF fusion proteins

Abstract

Growing endothelial cells at the sites of angiogenesis express high numbers of VEGF receptors and therefore may be particularly sensitive to VEGF-mediated drug delivery. To test this hypothesis we have constructed a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF121 (SLT-VEGF/L). Wild-type A-subunit is a site-specific N-glycosidase of 28S rRNA that inhibits protein synthesis after being delivered into cells by separate cell-binding B-subunits. SLT-VEGF/L retains functional activities of both SLT and VEGF121 moieties, since it inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2.5 x 10(5) VEGFR-2/cell with an IC50 of 0.2 nM and rapidly induces apoptosis at concentrations >1 nM. We found that sensitivity of VEGFR-2 transfected PAE cells to SLT-VEGF/L declined as the cellular VEGFR-2 density decreased; PAE cells expressing 25000 VEGFR-2/cell were as sensitive as parental cells lacking the receptor. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but take place without significant inhibition of protein synthesis. Selective cytotoxicity of SLT-VEGF/L against growing endothelial cells overexpressing VEGFR-2 suggests that it may be useful in targeting similar cells at the sites of angiogenesis.