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.
Highlights
Antibody–antigen (Ab–Ag) interactions are one of the most relevant classes of protein-mediated molecular recognition processes
The key strengths of Abs are widely recognised in terms of affinity, specificity and biochemical stability, full-length monoclonal antibodies suffer from some limitations when it comes to their clinical development, such as undesired Fc-mediated cytotoxicity, poor penetration in target tissues, aggregation and stability problems, low batch-to-batch reproducibility, and elevated manufacturing costs, among others [7]
We have interrogated the kinetics and thermodynamics of two anti-epidermal growth factor (EGF) nanobodies by means of surface plasmon resonance (SPR), a technique that is widely implemented in research laboratories to determine binding affinities. This approach combines high sensitivity and throughput with low sample consumption and no need of labels for the ligand or the analyte, allowing the kinetic and thermodynamic profiling of a large number of preclinical Ab candidates. For both nanobodies, the formation of the EGF-Ab complex is characterised by negative changes in enthalpy ∆C and heat capacity ∆Cp (Figure 2D and Table 1), which are caused by the formation of specific contacts between the two molecules, such as hydrogen bonds and electrostatic interactions, and might be accompanied by the burial of hydrophobic surface patches
Summary
Antibody–antigen (Ab–Ag) interactions are one of the most relevant classes of protein-mediated molecular recognition processes. The key strengths of Abs are widely recognised in terms of affinity, specificity and biochemical stability, full-length monoclonal antibodies (mAbs) suffer from some limitations when it comes to their clinical development, such as undesired Fc-mediated cytotoxicity, poor penetration in target tissues, aggregation and stability problems, low batch-to-batch reproducibility, and elevated manufacturing costs, among others [7]. To overcome these issues, a variety of Ab formats have been developed and optimised in recent years, thanks to the remarkable progress achieved in protein engineering and in vitro display methods [8,9]. Compared to mAb, they have a longer variable CDR3 loop, which forms part of a bulging interface that is ideally suited for targeting cavities, grooves and flexible epitopes [12]
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