The Fab portion of immunoglobulin G has sites in the CL domain that interact with Fc gamma receptor IIIa.

The interaction between IgG and Fc gamma receptor IIIa (FcγRIIIa) is essential for mediating immune responses. Recent studies have shown that the antigen binding fragment (Fab) and Fc are involved in IgG-FcγRIII interactions. Here, we conducted bio-layer interferometry (BLI) and isothermal titration calorimetry to measure the kinetic and thermodynamic parameters that define the role of Fab in forming the IgG-FcγRIII complex using several marketed therapeutic antibodies. Moreover, hydrogen/deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS) were used to clarify the interaction sites and structural changes upon formation of these IgG-FcγRIII complexes. The results showed that Fab in IgG facilitates the interaction via slower dissociation and a larger enthalpy gain. However, a larger entropy loss led to only a marginal change in the equilibrium dissociation constant. Combined HDX-MS and XL-MS analysis revealed that the CL domain of Fab in IgG was in close proximity to FcγRIIIa, indicating that this domain specifically interacts with the extracellular membrane-distal domain (D1) and membrane-proximal domain (D2) of FcγRIIIa. Together with previous studies, these results demonstrate that IgG-FcγRIII interactions are predominantly mediated by the binding of Fc to D2, and the Fab-FcγRIII interaction stabilizes complex formation. These interaction schemes were essentially fucosylation-independent, with Fc-D2 interactions enhanced by afucosylation and the contribution of Fab slightly reduced. Furthermore, the influence of antigen binding on IgG-FcγRIII interactions was also investigated. Combined BLI and HDX-MS results indicate that structural alterations in Fab caused by antigen binding facilitate stabilization of IgG-FcγRIII interactions. This report provides a comprehensive understanding of the interaction between IgG and FcγRIII.

Effect of UVC Irradiation on the Oxidation of Histidine in Monoclonal Antibodies.

We oxidized histidine residues in monoclonal antibody drugs of immunoglobulin gamma 1 (IgG1) using ultraviolet C irradiation (UVC: 200-280 nm), which is known to be potent for sterilization or disinfection. Among the reaction products, we identified asparagine and aspartic acid by mass spectrometry. In the photo-induced oxidation of histidine in angiotensin II, 18O atoms from H218O in the solvent were incorporated only into aspartic acid but not into asparagine. This suggests that UVC irradiation generates singlet oxygen and induces [2 + 2] cycloaddition to form a dioxetane involving the imidazole Cγ - Cδ2 bond of histidine, followed by ring-opening in the manner of further photo-induced retro [2 + 2] cycloaddition. This yields an equilibrium mixture of two keto-imines, which can be the precursors to aspartic acid and asparagine. The photo-oxidation appears to occur preferentially for histidine residues with lower pKa values in IgG1. We thus conclude that the damage due to UVC photo-oxidation of histidine residues can be avoided in acidic conditions where the imidazole ring is protonated.