Structural insights into the non-inhibitory mechanism of the anti-EGFR EgB4 nanobody

Abstract

Background The epidermal growth factor receptor (EGFR) is involved in various developmental processes, and alterations of its extracellular segment are associated with several types of cancers, in particular glioblastoma multiforme (GBM). The EGFR extracellular region is therefore a primary target for therapeutic agents, such as monoclonal antibodies and variable domains of heavy chain antibodies (VHH), also called nanobodies. Nanobodies have been previously shown to bind to EGFR, and to inhibit ligand-mediated EGFR activation. Results Here we present the X-ray crystal structures of the EgB4 nanobody, alone (to 1.48 Å resolution) and bound to the full extracellular EGFR-EGF complex in its active conformation (to 6.0 Å resolution). We show that EgB4 binds to a new epitope located on EGFR domains I and II, and we describe the molecular mechanism by which EgB4 plays a non-inhibitory role in EGFR signaling. Conclusion This work provides the structural basis for the application of EgB4 as a tool for research, for targeted therapy, or as a biomarker to locate EGFR-associated tumors, all without affecting EGFR activation.

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells

“…The most likely explanation for this significant increase in Q86c-HL488 BRET induced by EGFR ligands is that it represents a conformational change related to the agonist-induced extended conformation of EGFR and exposure of the dimerization interface in domain II leading to receptor homodimerization ( 5 – 10 ). This is in keeping with the recent receptor X-ray crystal structures of Q86 (EgB4) alone and bound to the full extracellular EGFR-EGF complex in its extended active conformation ( 30 )). It is also interesting that Q86c can induce a small enhancement of Q44c-HL488 binding (but not EGF-AF488 or EGF-AF647 binding) which suggests that there are subtle differences in the binding of EGF and Q44c to domain III of EGFR.…”

A Nanobody‐on‐Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR)

“…The two different NBs do not compete for binding to EGFR (Supporting Figure S1) and can thus be used to detect the soluble EGFR ectodomain in immunological sandwich assays. NB1 binds sEGFR and sEGFRvIII in the cleft formed between domains II and III , [36, 37] whereas NB2 only binds to domains I and II of sEGFR [36, 38] . Conjugation to the different tags did not affect their affinity for EGFR (Supporting Figure S2 and Table S1).…”

A Nanobody‐on‐Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR)

“…NB1 binds sEGFR and sEGFRvIII in the cleft formed between domains II and III, [36,37] whereas NB2 only binds to domains I and II of sEGFR. [36,38] Conjugation to the different tags did not affect their affinity for EGFR (Supporting Figure S2 and Table S1). The spectral properties of the Lumi4-Tb (Tb) FRET donor and QD acceptors used for the FRET immunoassays are shown in Supporting Figure S3.…”