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.

Submission Details

ID: kGTbL7D64

Submitter: Shu-Ching Ou

Submission Date: Feb. 22, 2019, 1:32 p.m.

Version: 1

Publication Details
Chen Y;Wiesmann C;Fuh G;Li B;Christinger HW;McKay P;de Vos AM;Lowman HB,J Mol Biol (1999) Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen. PMID:10543973
Additional Information

ΔΔG values were reported in AB-Bind article: (DOI: 10.1002/pro.2829)

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3UPA 2011-11-17T00:00:00+0000 1.8 A general strategy for the generation of human antibody variable domains with increased aggregation resistance
1F6L 2000-06-22T00:00:00+0000 2.8 VARIABLE LIGHT CHAIN DIMER OF ANTI-FERRITIN ANTIBODY
5W38 2017-06-07T00:00:00+0000 1.8 1.80A resolution structure of human IgG3 Fc (N392K)
6D58 2018-04-19T00:00:00+0000 2.39 Crystal structure of a Fc fragment of Human IgG3
4ZNC 2015-05-04T00:00:00+0000 2.28 Fc fragment of human IgG in complex with the C domain of staphylococcal protein A mutant - Q9W
4WWI 2014-11-11T00:00:00+0000 2.31 Crystal structure of the C domain of staphylococcal protein A in complex with the Fc fragment of human IgG at 2.3 Angstrom resolution
3EO1 2008-09-26T00:00:00+0000 3.1 Structure of the Fab Fragment of GC-1008 in Complex with Transforming Growth Factor-Beta 3
5LG1 2016-07-05T00:00:00+0000 2.7 Room temperature structure of human IgG4-Fc from crystals analysed in situ
4D2N 2014-05-12T00:00:00+0000 2.7 Crystal structure of deglycosylated serum-derived human IgG4 Fc
1ADQ 1997-02-18T00:00:00+0000 3.15 CRYSTAL STRUCTURE OF A HUMAN IGM RHEUMATOID FACTOR FAB IN COMPLEX WITH ITS AUTOANTIGEN IGG FC

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
193.4 V,W Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A Q9MYV3 VEGFA_CANLF
189.8 V,W Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A Q9GKR0 VEGFA_HORSE
185.2 V,W Fab fragment,Vascular endothelial growth factor A P50412 VEGFA_SHEEP
187.2 V,W Fab fragment,Vascular endothelial growth factor A P15691 VEGFA_BOVIN
195.8 V,W Fab fragment,Vascular endothelial growth factor A P49151 VEGFA_PIG
200.0 V,W Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A P15692 VEGFA_HUMAN
183.0 J,L Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A P04432 KVD39_HUMAN
183.0 J,L Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A P01597 KV139_HUMAN
200.0 J,L Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A P01834 IGKC_HUMAN
182.2 H,K Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A P01861 IGHG4_HUMAN
181.8 H,K Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A P01860 IGHG3_HUMAN
200.0 H,K Fab fragment, heavy chain,Fab fragment, light chain,Vascular endothelial growth factor A P01857 IGHG1_HUMAN
183.0 V,W Fab fragment,Vascular endothelial growth factor A P16612 VEGFA_RAT
183.0 V,W Fab fragment,Vascular endothelial growth factor A Q00731 VEGFA_MOUSE
180.8 V,W Fab fragment,Vascular endothelial growth factor A Q99PS1 VEGFA_MESAU
187.0 V,W Fab fragment,Vascular endothelial growth factor A P26617 VEGFA_CAVPO