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

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.

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

“…Novel paratopes arising from these procedures conserve the critical functional features, together with modifications aimed at incorporating/improving biological functions. Such approaches have led to affinity 48 and specificity optimization, 47 and even to the incorporation of dual specificities within the same paratope. 49 While a deeper mechanistic understanding of antibody biological effects would require knowing not only the identity of the epitope/paratope, but also the geometry of the Ag/Ab complex (which would only be revealed through experimental structural studies), in silico docking simulations were able to predict a plausible picture of nimotuzumab interactions.…”

High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

“…While lowlevel random diversification of phagemid-cloned Ag gene by error-prone PCR was initially performed, 25,26 resembling the usual yeast display-based mapping procedures, detailed information about the residues contributing to epitope formation has been recently obtained through comprehensive mutagenesis scanning of the role of solvent-exposed side chains within a discrete surface patch. [5][6][7][8] Once the target Ag is phage-displayed and a putative relevant antigenic region within it is identified through any available method, focused exploration of such region can result in a very high local sequence space coverage (Fig. 1).…”

“…While full-length interleukin-2 (IL-2), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) have been displayed on filamentous phages for mapping purposes, [5][6][7]9 restricting the scanning to the individual domain containing the epitope(s) under investigation is the alternative of choice for larger multi-domain Ags, as it has been done with phage-displayed EGF receptor (EGFR) domain III. 8 In any case, the original folding/antigenicity of the molecule must be preserved.…”

“…Beyond antigenicity, phage-displayed IL-2 and VEGF were shown to bind their natural receptors. 5,6 Phage-displayed IL-2 molecules successfully passed an even more stringent test: they were biologically active on IL-2-dependent cells. 7,27,28 VEGF deserves particular attention because the native Ag is a homodimer.…”

“…The current article summarizes mapping experiences accumulated during the last 2 y on the bases of phage display of native Ags and a comprehensive mutagenesis exploration of their surfaces to locate the target epitopes. [5][6][7][8][9] The usefulness of such an approach in the context of a plethora of available epitope discovery techniques is discussed. Despite the wide use of phage display to study protein interactions, including epitope mapping, 10 the particular strategy we review here had not been fully exploited until recently.…”