We report here the engineering of heterodimeric proteins that efficiently downregulate EGFR surface levels without activating the receptor. The wide variation in effects of the combinations of monovalent and homo- and hetero-bivalent constructs strongly suggests the existence of stereospecific constraints on binding-induced downregulation, as opposed to simple gross clustering. As expected, monovalent binding alone does not reduce EGFR levels. Homodimers, aside from weak downregulation by D-D, also are ineffective. In fact, strong reduction in EGFR levels is only observed for selected heterodimers of non-competitive clones. Constructs D-B, D-C, and D-E yield the strongest downregulation whereas A-D, B-D, C-D, and E-D exhibit modest efficacy. Non-competitive heterodimers including clone D, which binds to a novel epitope at the junction of domains III and IV, are generally effective except for D-A. Non-competitive heterodimers including clone A are less consistent. C-A and A-B are weakly effective against all three cell types; A-C and A-E are weakly effective against only two cell types; and B-A and E-A are ineffective. Thus, a combination of non-competitive clones is necessary but not sufficient for strong downregulation. Non-competitive heterobivalent constructs are theoretically topologically able to form receptor clusters because of the ability to bind two heterodimers to a single receptor thereby propagating receptor linkages whereas homobivalents or competitive heterodimers can only form two-receptor complexes. Meanwhile, the reduced efficacy of some non-competitive heterodimers may arise from the inability to simultaneously bind two receptors given the distance and steric constraints of the epitopes targeted and the length of the bivalent linker. pH-dependent binding effects may also play a role.
The heterodimers elicit a response that is qualitatively distinct from that resulting from EGF binding. This is perhaps most clearly demonstrated by the ability of heterodimers to downregulate EGFR overexpressed in HEK cells, which EGF fails to downregulate, perhaps due to a saturation of the endocytic machinery. Also, multiple receptor mutants, including kinase inactive K721R, are downregulated to the same extent as wild-type receptor. Mutation of neither T669 nor S1046, whose phosphorylations are implicated in receptor internalization (Countaway et al., 1992
; Winograd-Katz and Levitzki, 2006
), nor T654, whose phosphorylation either inhibits ubiquitination or accelerates recycling (Bao et al., 2000
), impacts downregulation. In addition, mutation of Y845, Y1068, Y1148 or Y1173, which are important in the ERK signaling pathway (Downward et al., 1985
; Yamauchi et al., 1997
; Biscardi et al., 1999
; Wu et al., 2002
; Amos et al., 2005
; Morandell et al., 2008
), has no effect on Fn3-heterodimer-induced downregulation. These results are corroborated by phosphorylation analyses. Seven of the eight key sites studied on EGFR, with the exception of T669, demonstrated significantly lower phosphorylation with heterobivalent D-C compared to EGF stimulation. Conversely, no phosphorylation is observed at T654, S1046 and Y1068. Y845, Y1086, Y1148 and Y1173 exhibit no agonism at multiple time points and weak phosphorylation at 1h. Moreover, western blot analysis demonstrates ERK phosphorylation upon treatment with EGF but not upon treatment with any of the heterodimers tested. Global phosphoproteomic analysis also exhibits substantially more phosphorylation from EGF than D-B, D-C, or a combination of B and D monomers. Thus, unlike EGF, Fn3-Fn3 constructs achieve receptor downregulation without significant receptor agonism.
A simple mathematical analysis of receptor trafficking indicates that steady-state downregulation is likely to arise from enhanced degradation/recycling ratio. Experimental data suggest that receptor internalization is not increased, as monovalent clone B and downregulating D-B constructs exhibit equivalent intracellular accumulation. Moreover, the kinetics of downregulation (τ1/2
= 0.4–1.4 h) are comparable to constitutive receptor internalization kinetics. Thus, although receptor internalization may be increased slightly, it does not appear to be a significant contributor to downregulation. Enhanced degradation could conceivably result from the presence of receptor clusters that either inhibit recycling or drive degradation. In fact, AlexaFluor488-conjugated 225 antibody exhibits reduced recycling in the presence of downregulating heterobivalent A-D as compared to co-treatment with monomer A or non-downregulating C-B (Supplementary data
This panel of engineered fibronectin domains should provide useful reagents for a variety of applications. The small size of these agents should provide rapid clearance for in vivo
imaging applications and close proximity of binding site and fluorophore for Förster resonance energy transfer studies. The engineered domains are cysteine-free with primary amines located distal to the presumed binding site with two exceptions: clone H contains a cysteine and lysine in the FG loop and clone D contains adjacent cysteines in the FG loop. Thus, the domains are amenable to thiol and amine chemical conjugation to fluorophores, nanoparticles, drug payloads and chemically modified surfaces for drug delivery, diagnostic and biotechnology applications. The single-domain architecture readily enables protein fusion such as the heterodimers discussed herein and immunotoxins (Pirie et al., 2011
). The picomolar to low nanomolar binding of these domains is sufficient for most applications. The breadth of epitopes targeted is useful for biophysical studies and dual binding such as for receptor clustering or sandwich immunoassays.
Downregulation decreases the amount of receptor available for ligand binding, receptor homo- and hetero-dimerization and constitutive activation, thereby decreasing the opportunity for receptor signaling. Downregulation is sufficient to inhibit ligand-induced phosphorylation of ERK, a downstream signaling molecule on a pathway that leads to proliferation and migration. The heterodimers reported here inhibit proliferation and migration of a cell line with autocrine signaling, and this inhibitory activity can be augmented by combination treatment with antibody 225, which can provide additional crosslinking and ligand competition. The ability to significantly downregulate EGFR with minimal agonism, while decreasing cell proliferation and cell migration, highlight the promise of these engineered heterodimers as components of novel potential cancer therapeutics.