Fig. 2

Identification of Fc variants with optimized pH-dependent hFcRn binding profiles. a Schematic representation of the focused screening strategy using saturation mutagenesis. In saturation mutagenesis-1, mutations at position Q311 were introduced based on the trastuzumab-M428L scaffold [17] to identify key substitutions enhancing hFcRn binding. Following the identification of the trastuzumab-ML variant, saturation mutagenesis-2 targeted position L309 on the trastuzumab-ML scaffold to further refine binding kinetics, resulting in the discovery of YML and EML variants. b Binding profiles of the engineered Fc variants assessed via ELISA. Bar graphs display the binding of Fc variants to hFcRn under mildly acidic (pH 6.0, red) and neutral (pH 7.4, blue) conditions. Trastuzumab-YML and trastuzumab-EML exhibited significantly enhanced hFcRn binding at acidic pH compared to PFc29, while maintaining comparable binding at neutral pH. Error bars represent standard deviations from duplicate experiments