Thermodynamic profiling of antibody-antigen binding and affinity measurements

Abstract

While high-affinity antibody-antigen interactions are essential for therapeutic and diagnostic applications, weak-affinity interactions dominate regulatory networks highlighting the importance of comprehensive binding characterization. This research employed Isothermal Titration Calorimetry (ITC) to determine complete thermodynamic profiles for 40 antibody-antigen pairs spanning four orders of magnitude in affinity (Kd = 0.1 nM to 1 μM). Binding was characterized by dissection into enthalpic (ΔH) and entropic (TΔS) components, revealing distinct thermodynamic signatures across affinity ranges. High-affinity interactions (Kd < 10 nM) were predominantly enthalpy-driven with ΔH contributions averaging -12.4 kcal/mol, while moderate-affinity interactions showed greater entropic contributions. Enthalpy-entropy compensation was observed across the dataset (r = 0.89), limiting the achievable free energy range despite diverse molecular interactions. Surface area burial correlated with enthalpic contribution (r = 0.72), providing structural rationalization for thermodynamic observations. These findings establish quantitative relationships between structural features and binding thermodynamics applicable to antibody engineering efforts.