Modulating mAbs Function through Glycosylation and Isotype: A Molecular Dynamics Perspective

The activity of monoclonal antibodies (mAbs) emerges from a finely tuned interplay between structural dynamics, post-translational modifications, and immune receptor recognition. In collaboration with Merck Serono S.p.A., we have employed advanced molecular dynamics (MD) simulations to unravel how glycosylation patterns, light- chain (LC) isotype, and antigen binding collectively shape the conformational landscape and effector function of therapeutic IgG1 antibodies. In our initial study (Biophysical Journal, 2021 https://doi.org/10.1016/j.bpj.2021.10.026), we explored the structural basis by which Fc N-glycans modulate antibody flexibility. Classical MD simulations revealed that core fucosylation not only influences local Fc dynamics but also governs large-scale antibody motions, thereby modulating Fc accessibility to effector receptors. This work offered one of the first atomistic descriptions of glycan-mediated conformational regulation. Building on these findings, we integrated classical and accelerated molecular dynamics (cMD + aMD) to examine the interplay between glycosylation and light-chain isotype (κ vs λ) in shaping IgG1 flexibility. Our goal was to identify the molecular determinants in FcγR engagement across antibody subclasses (Communications Biology, 2023, 10.1038/s42003-023-04622-7). The simulations uncovered distinct hinge dynamics and Fab-Fc orientations depending on both glycan status and isotypes, suggesting a structural rationale for differential receptor binding. This study marked one of the first applications of enhanced-sampling MD to probe the conformational diversity of full-length antibodies. Expanding this approach, we investigated the IgG1::FcγRIIIa complex to directly assess how fucosylation and light chain isotype influence receptor recognition (Computational and Structural Biotechnology Journal, 2025, DOI: 10.1016/j.csbr.2025.100034). Our analysis identified novel interfacial residues contributing to complex stability and provided a detailed molecular explanation for the reduced FcγRIIIa affinity observed in afucosylated mAbs. Most recently, we turned our attention to antigen engagement as a potential allosteric modulator of mAb conformation. Using commercial antibodies in complex with their respective antigens, and comparing G0 and G0F glycoforms via accelerated MD, we uncovered a consistent antigen-induced allosteric network linking Fab and Fc domains. Antigen binding shifted the conformational equilibrium toward more open, Y-shaped Fc states, enhancing receptor accessibility, particularly in afucosylated κ-LC antibodies. This effect was attenuated in λ-LC systems, where stronger CH1-CL contacts constrained Fab motion and limited long-range allosteric propagation. Collectively, these studies establish a detailed mechanistic framework for IgG1 function: from intrinsic Fc dynamics to receptor recognition and antigen-triggered allosteric regulation. This evolving paradigm suggests that Fab engineering, alongside Fc and glycan modifications, may represent a promising frontier for optimizing antibody effector functions.