Fucosylation limits ADCC in clinically used anti-RhD monoclonal antibodies

Background: Haemolytic disease of the fetus and newborn (HDFN) is caused by maternal alloantibodies, often targeting the D antigen on fetal red blood cells. Maternal immunization is preventable with timely administration of anti-D polyclonal antibodies (pAbs). Although the mechanisms of action for pAbs are not completely known, clinical efficacy has been suggested to be associated with afucosylated anti-D IgG and strong Fc-receptor-mediated antibody-dependent cellular cytotoxicity (ADCC). Anti-D pAbs are derived from hyperimmunized individuals, which makes the supply expensive and donor dependent. Monoclonal antibodies (mAbs) offer an alternative, but none have successfully prevented HDFN; some of them even enhanced alloimmunization. Despite lacking food and drug administration / European medicines agency (FDA/EMA) approval, two mAbs—Rhoclone™ and Trinbelimab (TBL) (Anti D®)—are widely used in low/middle-income countries. Study Design and Methods: Here we investigated functional and structural properties of these mAbs, including epitope mapping, glycan composition, and de novo sequencing by liquid chromatography tandem mass spectrometry (LC–MS/MS). Then, antibody engineering was employed to enhance ADCC potential. Results: Both Rhoclone (Rho) and TBL recognized RhD-epitope 5.5. Amino acid sequencing revealed these mAbs to be identical at the protein level, and that TBL had lower fucosylation (86%) than Rho (96%). Both mAbs had lower ADCC activity than anti-D pAb Rhophylac®. ADCC performance was correlated with fucosylation levels: afucosylated engineered anti-D > pAbs > TBL > Rho ≈ fucosylated anti-D control. Glycoengineered versions with low fucose showed markedly enhanced natural killer cell (NK)-cell-mediated ADCC. Discussion: Afucosylation of anti-D monoclonals mimics polyclonal anti-D and enhances their ADCC. Future efforts should focus on determining if these functional differences translate to clinical efficacy.