Jump to main content
Jump to site search

Issue 8, 2013
Previous Article Next Article

Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor

Author affiliations

Abstract

We have previously reported the isolation of a novel single-chain variable fragment (scFv) against vascular endothelial growth factor (VEGF), from a phage-displayed human antibody repertoire. This scFv, denominated 2H1, was shown to block the binding of VEGF to its receptor but exhibited a moderate binding affinity. Here, we describe the affinity maturation of the 2H1 scFv. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of the parental scFv as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was designed and constructed by combining recurrent replacements found among selected variants in a single molecule, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant obtained here, L3H6, showed an overall affinity improvement of 18-fold over the parental scFv and exhibited an enhanced potency to block the binding of VEGF to its receptor. Using phage display and extensive mutagenesis of VEGF, we determined the fine specificity of L3H6. This functional mapping revealed a novel neutralizing epitope on human VEGF defined by the residues Y25, T71, E72, N100, K101, E103 and R105. The conformational epitope recognized by L3H6 was recapitulated by grafting human VEGF residues into the mouse molecule, providing further confirmation of the nature of the identified epitope.

Graphical abstract: Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor

Back to tab navigation

Supplementary files

Publication details

The article was received on 04 Apr 2013, accepted on 16 May 2013 and first published on 16 May 2013


Article type: Paper
DOI: 10.1039/C3MB70136K
Citation: Mol. BioSyst., 2013,9, 2097-2106
  •   Request permissions

    Affinity maturation and fine functional mapping of an antibody fragment against a novel neutralizing epitope on human vascular endothelial growth factor

    H. Lamdan, J. V. Gavilondo, Y. Muñoz, A. Pupo, V. Huerta, A. Musacchio, L. Pérez, M. Ayala, G. Rojas, R. F. Balint and J. W. Larrick, Mol. BioSyst., 2013, 9, 2097
    DOI: 10.1039/C3MB70136K

Search articles by author

Spotlight

Advertisements