Monoclonal anti-D induces low efficiency trogocytosis: Implications for the prevention of Hemolytic disease of the fetus and newborn (HDFN)

Introduction/Objective

Hemolytic disease of the fetus and newborn (HDFN) is an alloimmune condition provoked by maternal IgG that crosses the placenta and causes fetal red blood cell (RBC) destruction. Donor-derived Rhesus Immune Globulin (RhIG) is the only licensed option available to prevent HDFN through a phenomenon called antibody-mediated immune suppression (AMIS). The mechanism as to how RhIG induces AMIS is poorly understood and this has hampered the successful development of monoclonal antibodies to replace RhIG. Our recent murine data suggests that trogocytosis-induced antigen loss could play a central role in AMIS induction. The present work aims to compare the ability of anti-D monoclonal antibody, clinically assessed, to promote in vitro trogocytosis under AMIS conditions compared with RhIg.

Design and Methods

RhD⁺human RBCs (RBCs) were fluorescently labeled with PKH67 and sensitized with different concentrations of RhIG (WinRho, SDF) or the IgG1 BRAD5 monoclonal anti-D antibody. Sensitized vs non-sensitized RBCs were incubated with or without THP-1-CD16A macrophages for 30 min and 3 hours. Fluorescent RBCs were recovered, macrophages washed, and remaining RBCs lysed. The ability of BRAD5 vs RhIG to induce RBC membrane fluorescence loss and RBC membrane transfer to the macrophages (ie., trogocytosis) as well as phagocytosis were evaluated. Median fluorescence intensity (MFI) of PKH67 on the RBC recovered, the percentage of PKH67⁺ macrophages, as well as their PKH67 MFI, were determined by flow cytometry. Confocal cell microscopy to visualize the interaction between macrophages and anti-D sensitized RBCs was performed.

Results

We observed that RBCs sensitized with ≥110 ng/mL of RhIG showed significant phagocytosis while lower concentrations primarily demonstrated significant trogocytosis-driven antigen loss. As the theoretical plasma concentration of anti-D in patients administered RhIG is below 100 ng/mL, our findings indicate that trogocytosis is the probable in vivo mechanism under AMIS conditions. In the case of BRAD5, this antibody, like RhIg, was capable of inducing both phagocytosis and trogocytosis. However, much higher concentrations of BRAD5 were necessary to achieve a comparable degree of trogocytosis as compared to RhIg.

Conclusions

This work demonstrates that RhIG has the capacity to induce trogocytosis at clinically relevant concentrations, with minimal to no phagocytosis. Conversely, the BRAD5 monoclonal antibody although capable of trogocytosis, was over ten times less efficient than RhIG, mirroring its poorer efficacy in prior clinical studies for preventing RhD alloimmunization.