Fc mutagenesis enhances the functionality of anti-RhD monoclonal antibodies.

Hemolytic disease of the fetus and newborn (HDFN) due to Rhesus D (RhD) antigen mismatch between the mother and fetus has been a significant cause of neonatal jaundice, recurrent miscarriage, and stillbirth throughout history. Polyclonal anti-RhD immunoglobulin G (RhD-pIgG), derived from the plasma of RhD-negative donors immunized with RhD-positive red blood cells (RBCs), has reduced the incidence of HDFN, but this approach is currently restricted to developed countries. Monoclonal antibodies (mAbs) offer a promising alternative to address this pressing need, but prior attempts to develop effective anti-RhD mAbs have failed, in some cases, due to differences in fucosylation patterns between mAbs produced in cell lines and RhD-pIgG. Chinese hamster ovary (CHO) cell lines, commonly used for pharmaceutical protein production, induce high levels of fucosylation, reducing the antibody-dependent cellular cytotoxicity (ADCC) activity crucial for clearing RhD-positive RBCs. In contrast, RhD-pIgG has lower fucosylation levels, which enhances ADCC activity. Regulating the glycan levels of mAbs during production requires specialized cell lines and culture conditions. In this study, we took an alternative approach through antibody engineering. The Fragment crystallizable (Fc) regions of 2 existing anti-RhD mAbs (Brad3 and Fog1) were subjected to mutagenesis to introduce ADCC-enhancing mutations and then expressed in CHO cells under standard conditions. We demonstrate that targeted Fc mutagenesis significantly enhanced ADCC compared with the wild-type mAbs, while preserving RhD binding and efficient production in CHO cells. Furthermore, these Fc variants achieved comparable efficacy with RhD-pIgG, suggesting a new strategy for producing anti-RhD mAbs with improved functionality, without the need for glycoengineering.