Allosteric effects of CDR-H3 mutations modulate binding and neutralization of a conserved SARS-CoV-2 RBD-targeting antibody.

SARS-CoV-2 antigenic evolution continues to erode the activity of first-generation monoclonal antibodies, underscoring the value of antibodies that recognize conserved features within the spike receptor-binding domain (RBD). As a model for breadth-oriented engineering, we assessed the RBD-directed antibody XG83, which has a CDRH3-dominated paratope. Utilizing the XG83-Wuhan RBD crystal structure as a reference, we integrated MOE alanine scanning and residue scanning with CDRH3 (A116-Y132) to identify chemically reasonable replacements and test interaction to an Omicron BA.1 RBD. The parental CDRH3-centric pose in comparative docking had a better score (-280.18) than the mutant MuXG83 (-254.2), along with 100-ns molecular dynamics showed that MuXG83 was less stable (with higher RMSD/RMSF with less favorable interaction energy). The ELISA results against Omicron BA.1 RBD demonstrated that XG83 had 40% stronger binding than MuXG83; convergence happened only at the highest concentration, concordant with SPR studies and the fact that mutation increased dissociation. According to this binding gap, BA.1 pseudovirus neutralization demonstrated that XG83 was much more powerful than MuXG83, showing that the E118L/F130H CDR-H3 alterations decreased functional activity against Omicron BA.1. These results imply that allosteric influences on interface stability and conformational dynamics by non-epitope CDR-H3 residues can affect antibody performance. Functional testing was confined to Omicron BA.1; therefore, larger variant-panel studies are needed to ascertain if such mutations affect antibody breadth. Our findings highlight a structure-guided approach for optimizing paratopes and indicate that non-epitope (potentially allosteric) changes to CDRH3 are also important while investigating potential development and neutralization before advancement. IMPORTANCE: This study shows how modest allosteric characteristics in CDR-H3 control the delicate balance between neutralizing potency and breadth, making a timely and significant addition to SARS-CoV-2 antibody engineering. Using crystallography, alanine scanning, residue scanning, docking, molecular dynamics, and experimental ELISA and neutralization assays, this study offers a structure-guided framework for rational paratope optimization. The discovery of CDR-H3 residues that regulate long-range stability rather than just direct epitope contacts reveals an unappreciated aspect of antibody design and explains why some alterations improve anticipated interactions but degrade functional performance. Importantly, the comparison of wild-type XG83 and modified MuXG83 shows how allosteric tuning affects antibody-antigen compatibility in developing variations like Omicron. These findings illuminate conserved RBD recognition and offer strategies for building next-generation therapeutic antibodies that are more resistant to viral evolution.