Abstract
The classical view of immunoglobulin molecules posits two functional domains defined by the variable (V) and constant (C) regions, which are responsible for antigen binding and antibody effector functions, respectively. These two domains are thought to function independently. However, several lines of evidence strongly suggest that C region domains can affect the specificity and affinity of an antibody for its antigen (Ag), independent of avidity-type effects. In this study, we used isothermal titration calorimetry to investigate the thermodynamic properties of the interactions of four V region-identical monoclonal antibodies with a univalent peptide antigen. Comparison of the binding of IgG1, IgG2a, IgG2b, and IgG3 with a 12-mer peptide mimetic of Cryptococcus neoformans polysaccharide revealed a stoichiometry of 1.9-2.0 with significant differences in thermodynamic binding parameters. Binding of this peptide to the antibodies was dominated by favorable entropy. The interaction of these antibodies with biotinylated peptides manifested greater enthalpy than for native peptides indicating that biotin labeling affected the types of Ag-Ab complexes formed. Our results provide unambiguous thermodynamic evidence for the notion that the C region can affect the interaction of the V region with an Ag.
Highlights
Antibody (Ab)4 binding to its antigen (Ag) is a fundamental step for the development of protective adaptive immune responses
The classical view of Ab function was that of a bifunctional molecule with the V domains being solely responsible for Ab affinity and specificity, whereas the C region was responsible for the biological properties such as complement activation, Fc receptor binding, avidity, and serum half-life [4]
In recent years this dogma has unraveled with the accumulation of new data, which suggest that the CH region can affect V region structure, thereby affecting Ab affinity and specificity [5,6,7,8,9,10,11,12,13]
Summary
These Abs have identical V regions but differ in their CH domains [10]. ITC simultaneously and directly determines the enthalpic and entropic contributions, as well as the binding constant and stoichiometry in solution. ITC revealed differences in the binding energetics of V region-identical mAbs differing in isotype for a peptide mimetic establishing the influence of the CH region in Ag-Ab binding interactions. The results have important implications for Ab engineering and for the use of therapeutic Abs of different isotype
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