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. 2013 Jul 2;21(7):1214-24.
doi: 10.1016/j.str.2013.05.008. Epub 2013 Jun 20.

Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains

Karl R Schmitz  1 , Atrish Bagchi, Rob C Roovers, Paul M P van Bergen en Henegouwen, Kathryn M Ferguson
Affiliations

Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains

Karl R Schmitz et al. Structure. .

Abstract

The epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs. Here, we describe X-ray crystal structures of the extracellular region of EGFR in complex with three inhibitory nanobodies, the variable domains of heavy chain only antibodies (VHH). VHH domains, the smallest natural antigen-binding modules, are readily engineered for diagnostic and therapeutic applications. All three VHH domains prevent ligand-induced EGFR activation, but use two distinct mechanisms. 7D12 sterically blocks ligand binding to EGFR in a manner similar to that of cetuximab. EgA1 and 9G8 bind an epitope near the EGFR domain II/III junction, preventing receptor conformational changes required for high-affinity ligand binding and dimerization. This epitope is accessible to the convex VHH paratope but inaccessible to the flatter paratope of monoclonal antibodies. Appreciating the modes of binding and inhibition of these VHH domains will aid in developing them for tumor imaging and/or cancer therapy.

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Figures

Figure 1
Figure 1. Cross competition of VHH, Fab and EGF binding to sEGFR
A. BIAcore analysis of the effect of added competitor upon binding of sEGFR to immobilized EGF or Fab. For CM5 sensor chips to which EGF, FabC225 or Fab425 had been amine coupled, the SPR responses for 100 nM sEGFR plus 5 μM VHH (7D12, EgA1 or 9G8) or 10 μM mAb (cetuximab or mAb 425) are shown, normalized to the SPR response for 100 nM sEGFR alone. Error bars indicate the standard deviation on at least three independent measurements. B. Sample of 5 μM sEGFR alone and mixtures of 5 μM sEGFR with (i) 5 μM EgA1, (ii) 5 μM FabC225, or (iii) 5 μM EgA1 plus 5 μM FabC225 were subject to velocity ultracentrifugation. Sedimentation velocity c(S) species analysis shows that sEGFR forms 1:1 complexes with EgA1 (5.4 S) and FabC225 (6.2S), and a ternary EgA1:FabC225:sEGFR (6.6S) when VHH and Fab are added. C. Similar velocity centrifugation shows a ternary 2xVHH:sEGFR complex (5.9S) for mixtures of sEGFR with EgA1 and 7D12, whereas only 1:1 VHH:sEGFR complex (5.4S) is seen for samples containing sEGFR, EgA1 and 9G8. See also Figure S1.
Figure 2
Figure 2. The 7D12 binding site on domain III of sEGFR
A. Cartoon is shown with 7D12 colored green and sEGFRd3 colored gray. CDRs are highlighted in light green and labeled. B. In this view the structure has been rotated approximately 180° about a vertical axis relative to A. The expected locations of domains I, II and IV of sEGFR are in light blue, based on the structure of tethered sEGFR in PDB ID 1NQL (Ferguson et al., 2003). C. View of the interface region between 7D12 and sEGFRd3 in a similar orientation to A. Side chains that participate in key interactions are shown as sticks, as is the sugar group on sEGFRd3. Predicted salt bridge (≤ 4.5 Å) or hydrogen bond (≤ 3.5 Å) interactions are indicated with dashed lines. Kabat numbering is used. See also Figure S2.
Figure 3
Figure 3. Comparison of the interactions of EGF, FabC225 and 7D12 with EGFR
A. Cartoon of the 7D12:sEGFRd3 complex. Orientation is similar to Figure 2B. On the right hand panel the footprint (green) of 7D12 on the surface of domain III of EGFR highlights all atoms within 4Å of the bound 7D12. This view has been rotated by 90° about a horizontal axis so as to look down on to the flat domain III binding surface. B. The interaction of EGF with domain III. Domain III from PDB ID 1IVO was overlaid with domain III in the 7D12 complex. Orientations are as in A. The two groups of interactions between EGF and domain III (sites 2 and 3) as defined by Ogiso et al. (Ogiso et al., 2002) are ringed. The footprint for EGF is in grey. C. The interaction of FabC225 with domain III. Domain III from PDB ID 1YY9 was overlaid with domain III in the 7D12 complex. The footprint for FabC225 is in red. See also Table S1.
Figure 4
Figure 4. EgA1 and 9G8 have highly divergent CDR3s but bind to almost identical epitopes on sEGFR
A. Overview of the EgA1:FabC225:sEGFR complex showing a cartoon plus transparent molecular surface. The sEGFR is in grey, FabC225 is colored orange (heavy chain) and yellow (light chain) and EgA1 is in blue. The right hand panel shows a close up view rotated approximately 90° about a vertical axis in which the location of EgA1 in a cleft between domains II and III can be appreciated. In this panel a solid surface has been rendered on sEGFR and domains I and II are colored light blue for contrast. B. Close up view of the interface between EgA1 and sEGFR in approximately the same orientation as in the right hand panel of part A. Only CDR1 (dark blue) and CDR3 (light blue) participate in the interaction. Key side chains are shown as sticks and predicted salt bridge (≤ 4.5 Å) or hydrogen bond (≤ 3.5 Å) interactions are indicated with dashed lines. C. The same view of the 9G8 complex is shown. CDR1 is identical and colored as in B. CDR3 is different (Figure S2) and colored light purple. See also Figure S3.
Figure 5
Figure 5. Conformational changes in EgA1 upon binding to sEGFR
A. Superposition of free EgA1 (dark grey) and EgA1 bound to sEGFR (white), with CDRs in dark and light blue respectively. The N-terminus of free EgA1 packs against CDR1 and the hydrophobic core of the Ig fold, whereas in the bound structure this segment is oriented away from the VHH and makes (presumed) polar contacts with domain III. Detailed views of the differences in side chain orientations near the N-terminus (B) and in CDR2 and CDR3 (C). See also Figure S4
Figure 6
Figure 6. Comparison of the interactions of EgA1, 9G8, matuzumab and EGF with sEGFR
For each case the right hand panel shows a cartoon of just domain III of sEGFR (grey) with bound VHH, Fab or ligand. The left hand panel shows a surface representation of domain III in the same orientation with the footprint of VHH, Fab or ligand highlighted. A. EgA1 (blue); B. 9G8 (violet); C. matuzumab Fab (teal, PDB ID 3C09); D. EGF (dark red, PDB ID 1IVO). See also Figure S5.
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Comment in

  • Magic bullets from llamas.
    Leahy DJ. Leahy DJ. Structure. 2013 Jul 2;21(7):1072-3. doi: 10.1016/j.str.2013.06.008. Structure. 2013. PMID: 23823325 Free PMC article.

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