r84 Inhibits VEGFR2 Figure 1. r84 binds human and mouse VEGF-A and specifically blocks VEGF-A binding to VEGFR2, not VEGFR1. A, Recombinant human VEGF coated at 0.5 mg/mL was detected with a titration of fully human monoclonal antibody r84. r84 bound to VEGF was detected with an anti-human Fc HRP-conjugated antibody, demonstrating r84 binds both human and mouse VEGF-A (open squares and circles, respectively). B, Recombinant human and mouse VEGF-A, and human VEGF-B, -C, -D, and PlGF coated at 0.5 mg/mL was detected with r84 at 1 mg/mL. Binding of r84 to VEGF family member was detected as in A, demonstrating r84 binds only human and mouse VEGF-A and not other VEGF family members. VEGFR2 (C) and VEGFR1 (D, left panel) coated at 1 mg/mL were incubated with 4.76 nM or 2.38 nM biotinylated VEGF, respectively, +/2 the indicated fold excesses of antibody (Control IgG, 2C3, r84). r84, 2C3 specifically block biotinylated-VEGF binding to VEGFR2 (C), but not VEGFR1 (D, left panel). In contrast, a 500-fold molar excess bevacizumab (bev) reduces biotinylated-VEGF binding to VEGFR1, compared to biotinylated-VEGF alone or plus r84 (D, right panel). doi:10.1371/journal.pone.0012031.g001 effects of anti-VEGF antibodies on VEGF-induced endothelial cell migration. Three different endothelial cell lines, selected for their VEGF receptor expression, were used for the migration assays. Human dermal microvascular endothelial cells (HDMEC) express VEGFR1 and VEGFR2, porcine aortic endothelial cells (PAE)- KDR and PAE-Flt-1 express high levels of VEGFR2 or VEGFR1, respectively [16]. Human VEGF significantly induced migration of all three cell types compared to serum free media (SFM) alone (p,0.05 for HDMEC, p,0.001 for PAE-KDR, -Flt-1), and a non- specific control IgG did not affect VEGF-induced migration (Figure 2A). Both r84 and bevacizumab significantly inhibited VEGF-induced migration of VEGFR2-expressing endothelial cells (p,0.001, Figure 2A, HDMEC, PAE-KDR). However, only bevacizumab was able to decrease the migration of PAE-Flt-1 cells PLoS ONE | www.plosone.org 4 August 2010 | Volume 5 | Issue 8 | e12031