Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen
| Autor: | Y, Chen, C, Wiesmann, G, Fuh, B, Li, H W, Christinger, P, McKay, A M, de Vos, H B, Lowman |
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| Rok vydání: | 1999 |
| Předmět: |
Models
Molecular Vascular Endothelial Growth Factor A Umbilical Veins Protein Conformation Molecular Sequence Data Antibody Affinity Endothelial Growth Factors Crystallography X-Ray Epitopes Immunoglobulin Fab Fragments Mice Peptide Library Animals Humans Amino Acid Sequence Antigens Lymphokines Alanine Binding Sites Vascular Endothelial Growth Factors Antibodies Monoclonal Hydrogen Bonding Mutation Thermodynamics Endothelium Vascular Cell Division |
| Zdroj: | Journal of molecular biology. 293(4) |
| ISSN: | 0022-2836 |
| Popis: | The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections 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 variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium 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 overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants 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 VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction. |
| Databáze: | OpenAIRE |
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