SignificanceAntibody-based therapies rely not only on antigen binding but also on effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular phagocytosis (CDCP), both of which are strongly shaped by the structure of Fc glycans. While terminal galactosylation is known to improve Fc receptor and complement interactions, strategies to further optimize these effects remain limited. Here, we show that selective fluorination of Fc glycans provides a powerful means to precisely tune antibody effector functions. Using a chemoenzymatic glycoengineering approach, we generated selectively fluorinated antibody glycoforms with enhanced Fc RIIIA and C1q binding, leading to re... More
SignificanceAntibody-based therapies rely not only on antigen binding but also on effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular phagocytosis (CDCP), both of which are strongly shaped by the structure of Fc glycans. While terminal galactosylation is known to improve Fc receptor and complement interactions, strategies to further optimize these effects remain limited. Here, we show that selective fluorination of Fc glycans provides a powerful means to precisely tune antibody effector functions. Using a chemoenzymatic glycoengineering approach, we generated selectively fluorinated antibody glycoforms with enhanced Fc RIIIA and C1q binding, leading to remarkably improved ADCC and CDCP activities. This work establishes fluorination as a unique and powerful tool for designing next-generation therapeutic antibodies.Antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and various complement-dependent activities are critically influenced by the structure and composition of Fc N-glycans. Terminal galactosylation is generally associated with enhanced Fc RIIIA binding and C1q recruitment, thereby improving antibody activities. Recent structural studies suggest that terminal galactose can restrict glycan flexibility and stabilize Fc conformation by interacting with CH2 domain residues, thereby reducing the entropic penalty for Fc RIIIA binding. Motivated by this structural insight, we hypothesized that fine-tuning galactose-mediated Fc glycan-Fc domain interactions via site-selective fluorination could further modulate Fc-receptor and Fc complement interactions. To test this, we developed a chemoenzymatic glycoengineering approach to generate homogeneous antibodies bearing precisely fluorinated Fc N-glycans. Key to this strategy was the chemical synthesis of position-specific fluorinated full-length Fc glycans, which were subsequently installed onto the antibody via enzymatic Fc glycan remodeling catalyzed by a glycosynthase mutant. Using this platform, we constructed a panel of homogeneous fluorinated antibodies and evaluated their functional consequences. ELISA-based binding assays revealed that fluorination at the C2 or C6 position of terminal galactose significantly increased Fc RIIIA affinity. Corresponding enhancements in ADCC were confirmed using a cell-based reporter bioassay. Furthermore, fluorination at these positions also promoted C1q binding and elevated the antibody-dependent cellular phagocytosis potency in whole blood assays. These results collectively demonstrate that selective Fc glycan fluorination represents a unique strategy to enhance antibody effector functions, providing a paradigm for precision glycoengineering in antibody therapeutics.
