Abstract
Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.
Highlights
Radical reactions result in breakage of the heavy-light chain linkage and hinge cleavage of an IgG1
The IgG1 used in this study has a mild antibody-dependent cell-mediated cytotoxicity (ADCC) function with an EC50 at ϳ11 ng/ml, and binds to two separate important receptors that are expressed by A431 cells, our results demonstrated that the His/Tyr single substitution in the upper hinge is capable of improving ADCC activity in IgG1
The hinge cleavage and breakage of the heavy chain (HC)-light chain (LC) linkage may follow different reaction pathways, one is driven by the transient radical center His229, and another may result from an electron localization onto the HC-LC bond that was transferred directly from Cys231
Summary
Radical reactions result in breakage of the heavy-light chain linkage and hinge cleavage of an IgG1. Information obtained from these new assessments would be very important for antibody development To this end, a combination of studies is necessary to address the potential impacts of these residues to product quality, pharmacokinetics (PK) and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), because significant effects to the ADCC from hinge substitutions have been observed previously [15]. Our results show that Asp226 maybe not critical for the radical reactions, whereas substitution of His with Tyr reveals a potential strategy for improving stability and function of the IgG1: it maintains a similar pharmacokinetic profile with respect to the native molecule and increases the antibody binding to Fc␥RIII receptors by 2–3-fold, and improves ADCC activity by 2-fold. In combination with our previous observations [10], these results demonstrate the feasibility of engineering the upper hinge to improve the stability and effector function of the IgG1
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