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

Abstract

Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody-antigen (Ab-Ag) interactions are often poorly understood. In this study, we exploit the technical features of a typical surface plasmon resonance (SPR) biosensor to dissect the kinetic and thermodynamic components that govern the binding of single-domain Ab or nanobodies to their target antigen, epidermal growth factor (EGF), a key oncogenic protein that is involved in tumour progression. By carefully tuning the experimental conditions and transforming the kinetic data into equilibrium constants, we reveal the complete picture of binding thermodynamics, including the energetics of the complex-formation transition state. This approach, performed using an experimentally simple and high-throughput setup, is expected to facilitate mechanistic studies of Ab-based therapies and, importantly, promote the rational development of new biological drugs with suitable properties.

Keywords: EGF; SPR; antibody–antigen (Ag–Ab) interactions; biophysics; molecular recognition; nanobodies; thermodynamics.

Figure 1

Binding kinetics of Nb1 (A) and Nb6 (B) to EGF at 25 °C. Nanobodies were injected at increasing concentrations over immobilised EGF, and the association and dissociation phases of all injections were fitted to a 1:1 Langmuir binding model. Two different surface densities were used for k_on, k_off and K_D determination, with the corresponding background responses subtracted. Double-referenced sensorgrams for the highest density channel are shown here. Experiments were performed in duplicates. Residual errors from the fits are shown in the bottom panels.

Figure 2

Temperature dependence of binding kinetics and thermodynamic characterisation. Nb1 (A) and Nb6 (B) were injected over immobilised EGF at the indicated temperatures. For clarity, only sensorgrams of a single analyte concentration (70 nM) are shown. (C) Van’t Hoff plots representing the variation of the equilibrium association constants (K_A) with the inverse of temperature. Data points were fitted to a second-order polynomial equation from which ΔG, ΔH, ΔS and ΔC_p values were determined, following Equation (3). (D) Enthalpy (ΔH) and entropy (−TΔS) contributions to the free energy of binding (ΔG) for each Nb at 25 °C.

Figure 3

Transition state thermodynamics analysis. (A,B) Eyring plots representing the variation of the association constant (k_on) with the inverse of temperature for Nb1 and Nb6. Linear fitting of the experimental data points to Equation (4) (ΔC^≠_p = 0) was performed in (A), while non-linear fitting was performed in (B). (C) Eyring plots for the dissociationn step of Nb1 and Nb6. Data were fitted to a second-order polynomial from which the transition state thermodynamic parameters were derived according to Equation (4). (D) Binding profile of Nb1 and Nb6 illustrating the transitions in ΔG, ΔH, ΔS at 25 °C. [EGF–Nb]^≠ represents the transition state.