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

ABSTRACT

Purpose

To explore potential non-canonical disulfide linkages feasible in human IgG2 mAbs via molecular dynamics simulations of a model system, Hinge++.

Methods

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.

Results

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.

Conclusion

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.

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Abbreviations

CDR:

complementarity-determining region

Fab:

fragment antigen binding

Fc:

fragment crystallizable

HC:

heavy chain

LC:

light chain

mAb:

monoclonal antibody

MD:

molecular dynamics

PDB:

protein data bank

RMSD:

root mean squared deviation

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ACKNOWLEDGMENTS

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.

Author information

Correspondence to Sandeep Kumar.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

Figure S1

Time series of total energy, temperature, volume and time average of pressure of the simulation system for the eight simulations in the production runs. (DOC 286 kb)

Figure S2

The evolution of radius of gyration (Rg) of Hinge++ with simulation time in all trajectories is shown. (DOC 54 kb)

Figure S3

Time courses for (a) sulphur atom distance for the Cys pair LC2.C214-HC2.C236 from the second set of simulations in the four disulfide-bonded conditions described in the manuscript. The solid black circle at time 0 indicates the initial distance; (b). sulphur atom distance for Cys pairs HC2.C128-HC2.C236 from the first set of simulations in the four disulfide-bonded conditions. (DOC 59 kb)

Figure S4

Contact maps for the eight Cys residues in hinge region. (a) Control simulation; (b) Four-reduced2 simulation; (c) Six-reduced2 simulation; (d) All-reduced2 simulation. Grayscale indicates the frequency of observing a given Cys-Cys contact. A contact is defined by sulphur atom distance for two Cys residues being < 5 Å. Note that the order of Cys residues along the X-axes is opposite to that along the Y axes. Hence, these are not traditional contact maps. Each quadrant is labeled according to the nature of contacts. Inter-heavy chain contacts (inter-HC) are located in upper right or lower left quadrants. The two quadrants are symmetric to each other. Intra-heavy chain contacts (Intra-HC1 and Intra-HC2) reside in the rest two quadrants. Each of the two quadrants is symmetric. (DOC 114 kb)

Table S1

Average (μ), standard deviation (σ) and coefficient of variation (ρ=μ/σ) for the overall RMSD profiles in 5 ns simulation time intervals. (DOC 94 kb)

Supplemental Video

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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Wang, X., Kumar, S. & Singh, S.K. Disulfide Scrambling in IgG2 Monoclonal Antibodies: Insights from Molecular Dynamics Simulations. Pharm Res 28, 3128–3144 (2011). https://doi.org/10.1007/s11095-011-0503-9

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KEY WORDS

  • biotherapeutics
  • hinge
  • immunoglobulin
  • molecular modeling
  • structure