Structure of a VEGF-VEGF receptor complex determined by electron microscopy.

Receptor tyrosine kinases are activated upon ligand-induced dimerization. Here we show that the monomeric extracellular domain of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) has a flexible structure. Binding of VEGF to membrane-distal immunoglobulin-like domains causes receptor dimerization and promotes further interaction between receptor monomers through the membrane-proximal immunoglobulin-like domain 7. By this mechanism, ligand-induced dimerization of VEGFR-2 can be communicated across the membrane, activating the intracellular tyrosine kinase domains.

Crystal structure of the Orf virus NZ2 variant of vascular endothelial growth factor-E. Implications for receptor specificity.

Mammalian vascular endothelial growth factors constitute a family of polypeptides, vascular endothelial growth factor (VEGF)-A, -B, -C, -D and placenta growth factor (PlGF), that regulate blood and lymphatic vessel development. VEGFs bind to three types of receptor tyrosine kinases, VEGF receptors 1, 2, and 3, that are predominantly expressed on endothelial and some hematopoietic cells. Pox viruses of the Orf family encode highly related proteins called VEGF-E that show only 25-35% amino acid identity with VEGF-A but bind with comparable affinity to VEGFR-2. The crystal structure of VEGF-E NZ2 described here reveals high similarity to the known structural homologs VEGF-A, PlGF, and the snake venoms Vammin and VR-1, which are all homodimers and contain the characteristic cysteine knot motif. Distinct conformational differences are observed in loop L1 and particularly in L3, which contains a highly flexible GS-rich motif that differs from all other structural homologs. Based on our structure, we created chimeric proteins by exchanging selected segments in L1 and L3 with the corresponding sequences from PlGF. Single loop mutants did not bind to either receptor, whereas a VEGF-E mutant in which both L1 and L3 were replaced gained affinity for VEGFR-1, illustrating the possibility to engineer receptor-specific chimeric VEGF molecules. In addition, changing arginine 46 to isoleucine in L1 significantly increased the affinity of VEGF-E for both VEGF receptors.

Targeting human cancer cells with VEGF receptor-2-directed liposomes.

Antibodies are among the most versatile tools used today to characterize and target molecules in cells and in biological tissues. The development of phage display libraries encoding a large repertoire of single chain antibodies, scFv, allows the rapid and efficient isolation of antibodies specific for almost any type of molecule. A great advantage of such recombinant antibodies is the possibility to functionalize them by introducing new amino acid sequences. This leads to new features that would be difficult to introduce into naturally occurring antibody molecules. This approach has been successfully applied to create molecules with new biological activities, e.g. by generating chimeric scFv antibodies carrying sequences derived from other biomolecules such as blood clotting factors or enzymes. Here, we describe a new antibody isolated from an M13 phage library that recognizes vascular endothelial growth factor receptor 2, VEGFR-2. This antibody, scFvVR-2H9 was coupled to liposomes and used to specifically target VEGFR-2-expressing human cancer cells in culture.