Antigen binding triggers long-range conformational changes in monoclonal antibodies

Monoclonal antibodies (mAbs) are glycoproteins whose therapeutic efficacy depends on antigen recognition and the activation of effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). These activities are tightly regulated by post-translational modifications, most notably N-glycosylation at the conserved Asn297 residue1. In addition, the light-chain (LC) isotype (κ or λ) has been identified as a key determinant of global antibody flexibility2. Although antigen binding is traditionally viewed as a localized Fab-mediated event, growing evidence supports an integrated structural model in which Fab and Fc regions are functionally coupled3. Understanding how antigen engagement, glycosylation, and LC isotype jointly reshape global antibody architecture is essential for the rational design of optimized biotherapeutics. Here, we used 1 μs accelerated molecular dynamics (aMD) simulations to investigate the structural and dynamic consequences of antigen binding in two therapeutic IgG1s: adalimumab and avelumab. To capture the impact of post-translational modifications and structural variability, each antibody was analyzed in both fucosylated (G0F) and afucosylated (G0) states, considering their different LC isotypes (κ for adalimumab and λ for avelumab). The sampled conformational space was characterized through free energy landscapes, covariance analysis, and Principal Component Analysis (PCA). We found that antigen engagement stabilizes mAbs in a canonical Y-shaped conformation, distinct from the more compact T-shaped arrangements often observed in the unbound state2. Covariance analysis revealed consistent long-range positive correlations between the variable domains (VH/VL) and distal regions of the hinge and Fc, supporting an allosteric communication network triggered by antigen binding. Antigen binding also increased the exposure of key Fc residues involved in FcγRIIIa recognition, as indicated by solvent exposure metrics and 3D-RISM hydration analyses. This effect was markedly modulated by glycosylation and LC isotype: in avelumab, the λ-LC isotype conferred higher rigidity via increased CH1-CL contacts, partially limiting motion propagation to the Fc compared with the κ-LC isotype in adalimumab. Overall, these findings indicate that antigen binding acts as a structural modulator, reshaping global mAb architecture through coordinated long-range motions. By showing that the Fab region can allosterically regulate Fc accessibility, this work provides computational guidance for the design of therapeutic antibodies with tuned effector potential. Bibliography Ferrara C, et al. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose. Proc Natl Acad Sci U S A. 2011;108(31):12669-12674. doi:10.1073/pnas.1108455108 Saporiti S, et al. Effect of Fc core fucosylation and light chain isotype on IgG1 flexibility. Commun Biol. 2023;6(1):237. Published 2023 Mar 3. doi:10.1038/s42003-023-04622-7 Corrada D, Morra G, Colombo G. Investigating allostery in molecular recognition: insights from a computational study of multiple antibody-antigen complexes. J Phys Chem B. 2013;117(2):535-552. doi:10.1021/jp310753z