Impact of intrinsic affinity on functional binding and biological activity of EGFR antibodies

Abstract

Aberrant expression and activation of EGF receptor (EGFR) has been implicated in the development and progression of many human cancers. As such, targeted therapeutic inhibition of EGFR, for example by antibodies, is a promising anticancer strategy. The overall efficacy of antibody therapies results from the complex interplay between affinity, valence, tumor penetration and retention, and signaling inhibition. To gain better insight into this relationship, we studied a panel of EGFR single-chain Fv (scFv) antibodies that recognize an identical epitope on EGFR but bind with intrinsic monovalent affinities varying by 280-fold. The scFv were converted to Fab and IgG formats, and investigated for their ability to bind EGFR, compete with EGF binding, and inhibit EGF-mediated downstream signaling and proliferation. We observed that the apparent EGFR-binding affinity for bivalent IgG plateaus at intermediate values of intrinsic affinity of the cognate Fab, leading to a biphasic curve describing the ratio of IgG to Fab affinity. Mathematical modeling of antibody-receptor binding indicated that the biphasic effect results from nonequilibrium assay limitations. This was confirmed by further observation that the potency of EGF competition for antibody binding to EGFR improved with both intrinsic affinity and antibody valence. Similarly, both higher intrinsic affinity and bivalent binding improved the potency of antibodies in blocking cellular signaling and proliferation. Overall, our work indicates that higher intrinsic affinity combined with bivalent binding can achieve avidity that leads to greater in vitro potency of antibodies, which may translate into greater therapeutic efficacy.

Figure 1

Impact of EGFR density on the apparent binding affinities of IgG to EGFR-overexpressing cells. A, low and high intrinsic affinity IgG (C10 and 2224) had their functional affinities measured on cells of varying EGFR density (B).

Figure 2

Impact of intrinsic and apparent antibody affinity and concentration on the ability of EGF to inhibit antibody binding to EGFR-overexpressing cells. A, at 1 nM IgG, EGF inhibited the binding of IgG in proportion to their intrinsic affinities; At 10 nM (B) and 100 nM (C) IgG concentration, there was less effect of EGF on IgG binding, especially of the higher intrinsic affinity 2224 and to a lesser extent P2/4. (D) EGF inhibited the binding of Fab in proportion to their intrinsic affinities. Comparing (C) to (D), it is clear that an increase in apparent affinity due to IgG avidity significantly reduces the reduction of antibody binding by EGF.

Figure 3

Impact of intrinsic and apparent affinity on EGF induced EGFR phosphorylation. A, increasing scFv intrinsic affinity reduced EGF induced EGFR phosphorylation; this effect was greatly increased by increasing functional affinity using an IgG constructed from the scFv V-genes. B, at lower antibody concentrations, no effect of scFv on EGF induced EGFR phosphorylation was observed and the inhibitory effect of the IgG was significantly reduced. Anti-ErbB2 IgG C6.5 was used as the control antibody.

Figure 4

Inhibition of A431 cell proliferation by anti-EGFR scFv, IgG and Fab. A, inhibition of proliferation of A431 cells increased with increasing scFv intrinsic affinity, with an IC50 of 35 nM for the highest affinity 2224 scFv; (B) Inhibition of proliferation of A431 cells increased with increasing Fab intrinsic affinity, but this effect was less than that observed for the scFv. (C) Inhibition of proliferation of A431 cells increased with increasing IgG intrinsic affinity. 3D12 = control anti-botulinum neurotoxin antibody.