Profiling Hinge Plasticity in Intact Monoclonal Antibodies for Antigen Recognition

Monoclonal antibodies (mAbs) are multidomain glycosylated proteins that mediate antigen binding, among other protein-protein interactions, which makes them successful therapeutics. However, in many cases, adverse physicochemical properties can affect their antigen-binding abilities, compromising therapeutic potential. Hence, structural characterization of these biotherapeutics is strongly desired to predict and possibly redesign better variants. The presence of glycosylation deters the use of isotope labeling, which is required for the structure determination of these intact proteins (MW ∼150 kDa) by Nuclear Magnetic Resonance Spectroscopy (NMR). In this work, NMR-based structural fingerprinting of three therapeutic mAbs at natural abundance was performed. The robustness of the mAb fingerprints was demonstrated by comparing them with two Fc-fusion proteins. A peptide-based assignment methodology was adopted for these intact proteins, which identified the presence of the flexible hinge segment in mAbs in solution. The plasticity of the hinge was demonstrated by the changes in fingerprints in the presence of the cognate antigen and nonantigen. The methodology underlines the importance of the hinge in antigen recognition beyond the canonical role of Complementarity-Determining Regions (CDRs). This methodology lays the dynamic basis of antibody function in solution.