1CZ8

VASCULAR ENDOTHELIAL GROWTH FACTOR IN COMPLEX WITH AN AFFINITY MATURED ANTIBODY

Summary for 1CZ8

• Entry DOI: 10.2210/pdb1cz8/pdb
• Related: 1BJ1 1FLT 1VPF 1VPP
• Descriptor: Vascular endothelial growth factor A, LIGHT CHAIN OF NEUTRALIZING ANTIBODY, HEAVY CHAIN OF NEUTRALIZING ANTIBODY, ... (5 entities in total)
• Functional Keywords: complex (antibody-antigen), cystine knot, fab fragment, immune system
• Biological source: Homo sapiens (human); More
• Cellular location: Secreted : P15692
• Total number of polymer chains: 6
• Total formula weight: 119059.19

Authors

Chen, Y.,Wiesmann, C.,Fuh, G.,Li, B.,Christinger, H.W.,McKay, P.,de Vos, A.M.,Lowman, H.B. (deposition date: 1999-09-01, release date: 2000-03-20, Last modification date: 2024-10-09)

Primary citation

Chen, Y.,Wiesmann, C.,Fuh, G.,Li, B.,Christinger, H.W.,McKay, P.,de Vos, A.M.,Lowman, H.B.
Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen.
J.Mol.Biol., 293:865-881, 1999

PubMed 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.

PubMed: 10543973
DOI: 10.1006/jmbi.1999.3192

Experimental method

X-RAY DIFFRACTION (2.4 Å)