VEGF RNA Aptamer Binding Specificity and Inhibition of Ligand binding to VEGF Receptors in vitro

Abstract

Abstract: : Purpose:Macugen^TM (pegaptanib sodium) is a pegylated RNA aptamer that binds to and blocks VEGF165 function with high specificity and potency. The aim of this study was to determine the binding specificity of Macugen^TM with regard to the different VEGF isoforms, and to members of the VEGF family that are homologous to VEGF165. We also examined aptamer inhibition of VEGF165 binding to its three high affinity receptors: KDR, Flt–1, and neuropilin–1 (Npn–1) in vitro. The inhibitory profile (IC₅₀) of Macugen^TM in a VEGF bioassay in HUVECs was examined. Methods:The target ligand binding affinities (K_D) of the VEGF RNA aptamer were determined using a modified filter binding assay using serially diluted growth factors and a constant amount of radio–labeled aptamer. Binding of the aptamer to the VEGF188 isoform was examined using a Far–Western method where radio–labeled aptamer was used to detect re–natured VEGF188 protein immobilized on a membrane. Inhibition of I¹²⁵–VEGF165 binding to VEGF receptor/Fc fusion proteins in the presence Macugen^TM was examined using a plate–binding assay. Data from the binding assays were analyzed by non–linear regression. The IC₅₀ of Macugen^TM in an assay measuring VEGF–induced tissue factor (TF) expression in HUVEC was determined using real–time quantitative RT–PCR (TaqMan). Results: The VEGF aptamer exhibited high–affinity specific binding (K_D of 70 to 100 pM) for VEGF165, however, did not bind VEGF121. Far–Western analysis suggests the aptamer binds less efficiently to VEGF188. The VEGF aptamer did not bind to the other VEGF family members tested (i.e., VEGF–B₁₆₇, VEGF–C, PDGF–BB, PlGF). Macugen^TM (pegylated VEGF aptamer) potently inhibited the binding of VEGF165 to all three of its receptors (i.e., VEGF–R1, VEGF –R2, Npn–1) in vitro with low nM potency. Macugen^TM also inhibited VEGF–induced tissue factor expression with an IC₅₀ in the low nM range. Conclusions: The VEGF RNA aptamer is a high affinity, highly–specific inhibitor of the heparin–binding VEGF isoform, VEGF165. Macugen^TM ’s specificity, and potent inhibition of VEGF receptor binding and bioactivity strongly support its use as a therapeutic in the treatment of ocular neovascular conditions.

Keywords: age–related macular degeneration • pharmacology • neovascularization