Blocking EGFR Activation with Anti‐EGF Nanobodies via Two Distinct Molecular Recognition Mechanisms

Abstract

One of the hallmarks of cancer is the overproduction of growth factors such as EGF. Despite the clinical success achieved by EGFR-targeted therapies, their long-term efficacy is compromised by the onset of drug-resistant mutations. To address this issue, a family of camelid-derived single-domain antibodies (Nbs) were generated, obtaining the first direct EGF inhibitors that prevent EGFR phosphorylation and pathway activation through this new mechanism of action. The two best Nbs were subjected to a detailed investigation of their interaction mechanism that revealed important differences in their binding kinetics and equilibrium thermodynamics. These distinct behaviors at the biophysical level translate into an equally efficient inhibition of the cellular EGFR phosphorylation, thus proving the efficacy of these Nbs to turn off the initiation of this key oncogenic pathway in cancer cells.

Probing the Kinetic and Thermodynamic Fingerprints of Anti-EGF Nanobodies by Surface Plasmon Resonance

“…We calculated the equilibrium dissociation constants (KD) at 25 °C, by either (I) plotting the level of steady-state binding for each concentration (i.e., equilibrium fitting, Figure S1); or (II) fitting the association and dissociation signals separately, as a function of time, to a given interaction model (i.e., kinetic fitting, Figure 1). For both nanobodies binding to EGF, a 1:1 Langmuir interaction model had been previously confirmed by isothermal calorimetry [19]. In agreement, a 1:1 kinetic model provided a close fit to the experimental data, as shown by the low chi-square values (χ 2 = 0.8-2.9).…”

“…Since experimental injection times in SPR are typically shorter (in our case, 120 s), more reliable KD values are measured by kinetic fitting, which is independent of steady-state levels. It should be noted that these KD values are, to some extent, lower For both nanobodies binding to EGF, a 1:1 Langmuir interaction model had been previously confirmed by isothermal calorimetry [19]. In agreement, a 1:1 kinetic model provided a close fit to the experimental data, as shown by the low chi-square values (χ 2 = 0.8-2.9).…”

“…Our group recently reported the discovery of nanobodies against epidermal growth factor (EGF) [19], a key oncogenic protein involved in the progression of epidermal-based tumours, such as those of the lung, skin and colon, among others [20]. In the present study, we have leveraged the potential of SPR for accurately measuring non-covalent interactions at different temperatures and analyte concentrations to study the kinetics and thermodynamics of two of our leading anti-EGF targeted nanobodies (Nb1 and Nb6).…”

Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics

“…Notably, Rossotti et al ( 57 ) reported DNA immunization-raised EGFR nanobodies with improved functionality compared to protein immunization-raised nanobodies. Nanobodies targeting EGF ( 58 ), HER2 ( 59 , 60 ), CAIX ( 61 ), death receptor 5 (DR5) ( 62 , 63 ), c-Met ( 64 , 65 ), HGF ( 66 ), AgSK1 ( 67 ), mesothelin ( 68 ), proteasome activator complex PA28 ( 69 ), ephrin receptor A4 (EphA4) ( 70 ), CEA-cell adhesion molecule-6 (CEACAM6) ( 71 ), mitochondrial translation elongation factor (TUFM) ( 72 ), protein C receptor ( 73 ), Wnt receptors (LRP5/6) ( 74 ), and CD33 ( 75 ) have also demonstrated delayed tumor growth.…”

Protein Chemical Synthesis Combined with Mirror‐Image Phage Display Yields d‐Peptide EGF Ligands that Block the EGF–EGFR Interaction

“…Hence, EGF has emerged as an alternative target for inhibiting the EGF–EGFR pathway, as demonstrated by the positive results obtained for CIMAvax‐EGF, a vaccine that induces antibodies against self EGF . In the same vein, we have recently described a family of nanobodies that bind EGF with high affinity and are able to block the EGF–EGFR interaction …”