Disulfide Scrambling in IgG2 Monoclonal Antibodies: Insights from Molecular Dynamics Simulations
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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 CH3 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.
KEY WORDSbiotherapeutics hinge immunoglobulin molecular modeling structure
fragment antigen binding
protein data bank
root mean squared deviation
We appreciate the anonymous referees for their constructive criticism of the research work and for suggestions to improve the manuscript. We thank Drs. Sandeep Nema, Sa V Ho, James Carroll, B. Muralidhara, Patrick Buck and Kevin King for several helpful discussions and for critical reading of this manuscript. A postdoctoral fellowship for Xiaoling Wang in Biotherapeutics Pharmaceutical Research and Development, Pfizer Inc. is gratefully acknowledged. High Performance Computing Support received from Pfizer Research Informatics played an essential role in this project.
The video is from the production run of All-reduced1 simulation. In the video, the sulphur atoms from the six pairs of Cys residues involved in canonical inter-chain disulfide bonds are highlighted as CPK spheres. The sulphur atoms are colored the same as their respective heavy (green and blue) and light (purple and red) chains. Hinge++ molecular model represents only the middle portions of human IgG2 mAbs (see the text for details). During the course of the video, a sulphur atom from a light chain Cys 214 (purple) momentarily moves away from its canonical heavy chain (green) partner and becomes close to other sulphur atoms (green) in upper hinge region. (MPG 18949 kb)
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