Engineering affinity-matured variants of an anti-polysialic acid monoclonal antibody with superior cytotoxicity-mediating potency

ABSTRACT Monoclonal antibodies (mAbs) that specifically recognize cell surface glycans associated with cancer and infectious disease hold tremendous value for both basic research and clinical applications. However, high-quality anti-glycan mAbs, especially those with sufficiently high affinity and specificity, remain scarce, highlighting the need for protein engineering approaches based on rational design or directed evolution that enable optimization of antigen-binding properties. To this end, we sought to enhance the affinity of a polysialic acid (polySia)-specific antibody called mAb735, which was raised by animal immunization and possesses only modest affinity, using a combination of rational design and directed evolution. The application of these approaches led to the discovery of affinity-matured IgG variants with up to ∼7-fold stronger affinity for polySia relative to the parental antibody. The higher affinity IgG variants were observed to opsonize polySia- positive cancer cells more avidly, which in turn resulted in significantly greater cytotoxicity as determined by both antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. Collectively, these results demonstrate the effective application of both rational and random molecular evolution techniques to an important anti-glycan antibody, providing insights into its carbohydrate recognition while at the same time uncovering variants with greater therapeutic promise due to their enhanced affinity and potency.