Histidine Residue Mediates Radical-induced Hinge Cleavage of Human IgG1

Hydroxyl radicals induce hinge cleavage in a human IgG1 molecule via initial radical formation at the first hinge Cys231 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 His229 residue using structure modeling and site-directed mutagenesis. Direct involvement of His229 in the reaction mechanism is suggested by a 75–85% reduction of the hinge cleavage for variants in which His229 was substituted with either Gln, Ser, or Ala. In contrast, mutation of Lys227 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 His229. Examination of the hinge structure shows that His229 is capable of forming hydrogen bonds with surrounding residues. These observations led us to hypothesize that the imidazole ring of His229 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.