In vitro and in vivo studies of a VEGF₁₂₁/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors

Vascular endothelial growth factor (VEGF) plays a key role in the growth and metastasis of solid tumors. We generated a fusion protein containing VEGF₁₂₁ linked by a flexible G₄S tether to the toxin gelonin (rGel) and expressed this as a soluble protein in bacteria. Purified VEGF₁₂₁/rGel migrated as an 84-kDa homodimer under nonreducing conditions. VEGF₁₂₁/rGel bound to purified, immobilized Flk-1, and the binding was competed by VEGF₁₂₁. Both VEGF₁₂₁/rGel and VEGF₁₂₁ stimulated cellular kinase insert domain receptor (KDR) phosphorylation. The VEGF₁₂₁/rGel fusion construct was highly cytotoxic to endothelial cells overexpressing the KDR/Flk-1 receptor. The IC₅₀ of the construct on dividing endothelial cells expressing 105 or more KDR/Flk-1 receptors per cell was 0.5-1 nM, as compared with 300 nM for rGel itself. Dividing endothelial cells overexpressing KDR were approximately 60-fold more sensitive to VEGF₁₂₁/rGel than were nondividing cells. Endothelial cells overexpressing FLT-1 were not sensitive to the fusion protein. Human melanoma (A-375) or human prostate (PC-3) xenografts treated with the fusion construct demonstrated a reduction in tumor volume to 16% of untreated controls. The fusion construct localized selectively to PC-3 tumor vessels and caused thrombotic damage to tumor vessels with extravasation of red blood cells into the tumor bed. These studies demonstrate the successful use of VEGF₁₂₁/rGel fusion construct for the targeted destruction of tumor vasculature in vivo.