Binding and Neutralization of Vascular Endothelial Growth Factor and Related Ligands by VEGF Trap, Ranibizumab and Bevacizumab

Abstract

Purpose: : To evaluate the in vitro activity and binding characteristics of VEGF Trap (aflibercept), bevacizumab (BEV) and ranibizumab (RAN) to vascular endothelial growth factor-A (VEGF-A₁₂₁ ,VEGF-A₁₆₅)_, VEGF-B_10-118 and placental growth factor (PlGF-1 and PlGF-2).

Methods: : Kinetic binding parameters were measured using Surface Plasmon Resonance (Biacore) and equilibrium binding constant in solution was determined using (Kinexa) technology. VEGF-A₁₂₁, VEGF-A₁₆₅ and PlGF-2 induced activation of VEGF receptors (VEGFR1, VEGFR2) were investigated using HEK293 cells stably transfected with an NFΚB-luciferase plasmid and either human VEGFR1 or VEGFR2. VEGF-A₁₆₅ calcium mobilization and migration were investigated using human umbilical vein endothelial cells (HUVEC).

Results: : VEGF Trap bound with high affinity to human VEGF-A₁₂₁ (K_D=0.36pM) and VEGF-A₁₆₅ (K_D=0.49pM) and to the equivalent proteins from rabbit, rat and mouse. High affinity binding also was observed between VEGF Trap and human PlGF-1 (K_D=392pM) and PlGF-2 (K_D=38.8pM). BEV and RAN bound to VEGF-A₁₆₅ with 118- and 94-fold lower affinity than VEGF Trap, respectively as determined by Surface Plasmon Resonance. In the VEGFR1 cell line, VEGF Trap effectively blocked luciferase activity induced by 20 pM VEGF-A₁₂₁ (IC₅₀=15pM) or VEGF-A₁₆₅ (IC₅₀=16pM) and was ~50 to 90 times more potent than BEV or RAN. Only VEGF Trap was able to block PlGF-2-induced (40 pM) luciferase activity in this cell line (IC₅₀=2.89 nM). In the VEGFR2 cell line, VEGF Trap effectively blocked luciferase activity induced by 20 pM VEGF-A₁₂₁ (IC₅₀=16pM) or 20 pM VEGF-A₁₆₅ (IC₅₀=26pM) and was 30-50 times more potent than BEV or RAN.

Conclusions: : These data differentiate VEGF Trap from bevacizumab and ranibizumab in terms of binding affinity for VEGF-A isoforms, as well as its ability to bind PlGF and VEGF-B. In addition, VEGF Trap demonstrates increased potency relative to bevacizumab and ranibizumab in blocking VEGF induced receptor(s) activation and HUVEC migration.

Keywords: protein purification and characterization • vascular endothelial growth factor • oncology