Selection and analysis of an optimized anti-VEGF antibody: Crystal structure of an affinity-matured Fab in complex with antigen

The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb V EGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by aff inity selection using monovalent phage display. After stringent binding sel ections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding v ariants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutat ions yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equi librium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The over all features of the binding interface seen previously with wild-type are pr eserved, and many contact residues are maintained in precise alignment in t he superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increase d van der Waals contact and improved hydrogen bonding. Site-directed mutant s confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several V EGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab bind ing. The results show that small changes even in a large protein-protein bi nding interface can have significant effects on the energetics of interacti on. (C) 1999 Academic Press.