Effects of N-Glycan Composition on Structure and Dynamics of IgG1 Fc and Their Implications for Antibody Engineering

Hui Sun Lee,Wonpil Im

doi:10.1038/s41598-017-12830-5

Abstract

Immunoglobulin G1 (IgG1), a subclass of human serum antibodies, is the most widely used scaffold for developing monoclonal antibodies to treat human diseases. The composition of asparagine(N)297-linked glycans can modulate the binding affinity of IgG1 Fc to Fc γ receptors, but it is unclear how the structural modifications of N-glycan termini, which are distal from the binding interface, contribute to the affinity. Through atomistic molecular dynamics simulations of a series of sequentially truncated high-mannose IgG1 Fc glycoforms, we found that the C′E loop and the Cγ2-Cγ3 orientation are highly dynamic, and changes in N-glycan composition alter their conformational ensembles. High-mannose glycoform preferentially samples conformations that are more competent to FcγRIIIa binding, compared to the truncated glycoforms, suggesting a role of IgG1 Fc N-glycan in optimizing the interface with the Fc receptor for efficient binding. The trajectory analyses also reveal that the N-glycan has large amplitude motions and the carbohydrate moiety interconverts between Fc-bound and unbound forms, enabling enzymatic modification of the glycan termini.

Highlights

Immunoglobulin G1 (IgG1) is a subclass of human serum antibodies and is the most widely used platform for developing therapeutic monoclonal antibodies[6]
To explore both challenges, we investigated the impacts of different N-glycan compositions on the structure and dynamics of IgG1 Fc using atomistic molecular dynamics (MD) simulations of a series of sequentially truncated high-mannose IgG1 Fc glycoforms (Fig. 1B): (1) Fc with high-mannose N-glycans (Man8GlcNAc2, Fc-Man8), (2) Fc with five mannoses (Man5GlcNAc2, Fc-Man5), (3) Fc with a single core N-acetylglucosamine (Fc-GlcNAc), (4) aglycosylated Fc (Fc-N297Q), and (5) Fc with an asymmetric glycoform (Fc-Man8/N297Q)
High-mannose glycoform preferentially samples the C′E loop conformations and the Cγ2-Cγ3 orientations that are more competent to FcγRIIIa binding, compared to truncated glycoforms

Summary

Introduction

Immunoglobulin G1 (IgG1) is a subclass of human serum antibodies and is the most widely used platform for developing therapeutic monoclonal antibodies[6]. In addition to the biological significance of elucidating the role of N-glycans in Fc binding to FcγRs, proper N-glycan remodeling is required for the development of generation IgG1-based antibodies through Fc N-glycan optimization[22,23,24] To explore both challenges (antibody biology and engineering), we investigated the impacts of different N-glycan compositions on the structure and dynamics of IgG1 Fc using atomistic MD simulations of a series of sequentially truncated high-mannose IgG1 Fc glycoforms (Fig. 1B): (1) Fc with high-mannose N-glycans (Man8GlcNAc2, Fc-Man8), (2) Fc with five mannoses (Man5GlcNAc2, Fc-Man5), (3) Fc with a single core N-acetylglucosamine (Fc-GlcNAc), (4) aglycosylated Fc (Fc-N297Q), and (5) Fc with an asymmetric glycoform (Fc-Man8/N297Q). We report large amplitude motions of N-glycans, causing most carbohydrate moieties to be detached from the Fc polypeptide surface and making them accessible for enzymatic modifications of the glycan termini

Methods

Results

Conclusion