VEGF AND THE FAB FRAGMENT OF A HUMANIZED NEUTRALIZING ANTIBODY - CRYSTAL-STRUCTURE OF THE COMPLEX AT 2.4 ANGSTROM RESOLUTION AND MUTATIONALANALYSIS OF THE INTERFACE

Background: Vascular endothelial growth factor (VEGF) is a highly spec ific angiogenic growth factor; anti-angiogenic treatment through inhib ition of receptor activation by VEGF might have important therapeutic applications in diseases such as diabetic retinopathy and cancer. A ne utralizing anti-VEGF antibody shown to suppress tumor growth in an in vivo murine model has been used as the basis for production of a human ized version. Results: We present the crystal structure of the complex between VEGF and the Fab fragment of this humanized antibody, as well as a comprehensive alanine-scanning analysis of the contact residues on both sides of the interface. Although the VEGF residues critical fo r antibody binding are distinct from those important for high-affinity receptor binding, they occupy a common region an VEGF, demonstrating that the neutralizing effect of antibody binding results from steric b locking of VEGF-receptor interactions. Of the residues buried in the V EGF-Fab interface, only a small number are critical for high-affinity binding; the essential VEGF residues interact with those of the Fab fr agment, generating a remarkable functional complementarity at the inte rface. Conclusions: Our findings suggest that the character of antigen -antibody interfaces is similar to that of other protein-protein inter faces, such as ligand-receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptid e chain, whereas those essential for binding to the receptor are distr ibuted over four different segments and span across the dimer interfac e.