Characterization of a new potent, in vivo neutralizing monoclonal antibodyto human vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an important mediator of tumor -induced angiogenesis and represents a potential target for anticancer ther apy. Therefore, we prepared a panel of monoclonal antibodies (mAb) against both the VEGF(121) and VEGF(165) isoforms. Three of them completely neutral ized the mitogenic stimulation by VEGF of human umbilical vein endothelial cells at mAb concentrations below 0.1 mu g/ml. The most potent one, with a dissociation constant (K-d) of 8 pM, inhibited, in a dose-dependent manner, VEGF-induced angiogenesis in a growth factor implant model in mice. A comp lete inhibition of the angiogenic response was obtained by daily intraperit oneal injections of 10 mu g mAb/mouse. Angiogenesis induced by basic fibrob last growth factor was not inhibited by the mAb. Epitope mapping of the mAb , performed by competitive enzyme-linked immunosorbent assay and Western bl ot analysis, showed that it did not bind to the reduced and denatured monom er of VEGF. Substitutions of three residues (Q87R, G88K, Q89K), located on the major surface loop beta 5 to beta 6 of VEGF, resulted in the complete l oss of binding (more than 400-fold reduction). The results suggest that the mAb binds primarily to a conformation-dependent epitope on the VEGF dimeri c form, encompassing one of the loop regions involved in KDR receptor bindi ng. The mAb with its strong neutralizing properties represents a useful age nt for effective blocking of VEGF-mediated tumor neovascularization.