Biophysical Characterization of the Contribution of the Fab Region to the IgG-Fc gamma RIIIa Interaction

The cell-surface receptor Fc gamma RIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with Fc gamma RIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant Fc gamma RIIIa ligands. A van't Hoff analysis revealed that compared to the interaction of the papain-digested Fc fragment with Fc gamma RIIIa, the interaction of commercially available, full-length rituximab with Fc gamma RIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower off rate. Similar results were obtained from analyses of IgG1 molecules and an IgG1-Fc fragment produced by Expi293 cells. For further validation, we also prepared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc was nearly equal to that of the IgG1-Fc fragment for the interaction with Fc gamma RIIIa, indicating that such alternatives to the Fab domains as MBP do not positively contribute to the IgG-Fc gamma RIIIa interactions. Our investigation strongly suggests that the Fab region directly interacts with Fc gamma RIIIa, resulting in an increase in the binding enthalpy and a decrease in the dissociation rate, at the expense of favorable binding entropy.