title: Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen.
abstract: 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.
authors.affiliation: Department of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: C
authors.initials: C
authors.lastname: Wiesmann
authors.forename: G
authors.initials: G
authors.lastname: Fuh
authors.forename: B
authors.initials: B
authors.lastname: Li
authors.forename: H W
authors.initials: HW
authors.lastname: Christinger
authors.forename: P
authors.initials: P
authors.lastname: McKay
authors.forename: A M
authors.initials: AM
authors.lastname: de Vos
authors.forename: H B
authors.initials: HB
authors.lastname: Lowman
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity / Q000276:immunology*; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D018360:Crystallography, X-Ray; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D006860:Hydrogen Bonding; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D008222:Lymphokines / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D011487:Protein Conformation; D013816:Thermodynamics; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D016228:Endothelial Growth Factors; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D008222:Lymphokines; D019151:Peptide Library; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000409:Alanine