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.

© 2025 The Author(s). Transfusion published by Wiley Periodicals LLC on behalf of AABB.