Disulfide Scrambling in IgG2 Monoclonal Antibodies: Insights from Molecular Dynamics Simulations

To explore potential non-canonical disulfide linkages feasible in human IgG2 mAbs via molecular dynamics simulations of a model system, Hinge(++). Hinge(++) is derived from the crystal structure of a full-length murine IgG2a antibody by replacing its core hinge region with human IgG2 hinge. Fv and C(H)3 domains were discarded to speed up calculations. Eight independent simulations, grouped in four sets, were performed. In the control set, disulfide bonding is identical to canonical human IgG2 mAb. Different numbers of disulfide bonds were broken in the remaining three sets. Two Fabs move towards Fc asymmetrically repeatedly leading to spatial proximity of LC.Cys214 and HC.Cys128 residues in one Fab with Cys residues in the upper hinge region, which could initiate disulfide scrambling. Local dynamics place the eight hinge region Cys residues in a large number of proximal positions which could facilitate non-canonical inter- and intra- heavy chain disulfide linkages in the hinge region. Consistent with experimental studies, our simulations indicate inter-chain disulfide linkages in human IgG2 mAbs are degenerate. Potential rational design strategies to devise hinge stabilized human IgG2 mAbs are gleaned.