Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3

Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activat es VEGF receptor-2 (VEGFR-2/Flkl/KDR) and VEGFR-3 (Flt4). These receptor ty rosine kinases, localized on vascular and lymphatic endothelial cells, sign al for angiogenesis and lymphangiogenesis. VEGF-D consists of a central rec eptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal prop eptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs ra ised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. Th e mAbs bind the fully processed VHD with high affinity and also bind unproc essed VEGF-D. We demonstrate, using bioassays for the binding and cross-lin king of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized recepto rs, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two r eceptors may be in close proximity. Furthermore, VD1 blocks the mitogenic r esponse of human microvascular endothelial cells to VEGF-D. The anti-(VEGF- D) mAbs raised to the bioactive region of this growth factor will be powerf ul tools for analysis of the biological functions of VEGF-D.