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Hydroxyl radicals induce hinge cleavage in a human IgG1 molecule via initial radical formation at the first hinge Cys 231 followed by electron transfer to the upper hinge residues. To enable engineering of a stable monoclonal antibody hinge, we investigated the role of the hinge His 229 residue using structure modeling and site-directed mutagenesis. Direct involvement of His 229 in the reaction mechanism is suggested by a 75-85% reduction of the hinge cleavage for variants in which His 229 was substituted with either Gln, Ser, or Ala. In contrast, mutation of Lys 227 to Gln, Ser, or Ala increased hinge cleavage. However, the H229S/K227S double mutant shows hinge cleavage levels similar to that of the single H229S variant, further revealing the importance of His 229 . Examination of the hinge structure shows that His 229 is capable of forming hydrogen bonds with surrounding residues. These observations led us to hypothesize that the imidazole ring of His 229 may function to facilitate the cleavage by forming a transient radical center that is capable of extracting a proton from neighboring residues. The work presented here suggests the feasibility of engineering a new generation of monoclonal antibodies capable of resisting hinge cleavage to improve product stability and efficacy.Availability of the full-length human IgG1 crystal structure has enriched the structural information around the region that connects the Fab and Fc domains (1, 2). The hinge consists of three parts: an upper hinge (Asp-Lys-Thr-His-Thr), a core hinge (Cys-Pro-Pro-Cys), and a lower hinge (Pro-Ala-Glu-LeuLeu-Gly-Gly). The hinge region contains interchain disulfide bonds and provides structural flexibility that facilitates Fab movement. The hinge is required for the function of an IgG molecule, in particular, binding to immune effector molecules (e.g. ADCC, CDC) (3-5). The importance of the core hinge residues was demonstrated by studies that showed a negative impact to C1q binding and complement activation if Cys or Pro was mutated (3,5,6). In contrast, the upper hinge has no significant impact on the effector functions of an IgG1 molecule as demonstrated recently in a systematic study (7). The exposed and flexible upper hinge has been found vulnerable to various degradation mechanisms such as papain cleavage and ␤-elimination reactions (8). Our previous study revealed the hydroxyl radical-mediated hinge cleavage of a human IgG1 molecule (9). In this radical reaction, the first hinge disulfide bond between the two Cys 231 residues was broken, followed by the formation of a thiyl radical at one of the two Cys 231 residues that initiated an electron transfer (ET) 2 to an upper hinge residue where cleavage was observed.Although our previous study sheds light on the hydroxyl radical attack, some critical questions remained pertaining to the mechanism by which the hinge is cleaved. Radical formation at amino acid residues involves the loss of both a proton and an electron that can be transferred among the residues in the protein via either a sin...

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