[Show abstract][Hide abstract] 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 Cys231 directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His229-mediated hinge cleavage. On the other hand, the substitution of His229 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.