Factor VIII-Fc activates natural killer cells via Fc-mediated interactions with CD16

Abstract

Abstract FactorVIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor usedin replacement therapy of hemophilia A. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasmahalf-life via neonatal Fc receptor (FcRn) binding, other Fc-mediatedinteractions, including engagement with Fc gamma receptors (FcγR), may haveimmunological consequences. Previouslywe and others demonstrated rFVIIIFc interactions with activatingFcγRIIIA/CD16. Here, we investigated ifrFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potentlyactivated NK cells in a CD16-dependent fashion resulting in IFNγ secretion andcytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cellIFNγ secretion levels and the high affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activatedCD16+ NK cells were able to lyse a B-cell clone bearing ananti-FVIII BCR (BO2C11) in an antibody-dependent cellular cytotoxicity (ADCC)assay. These findings provide anunderlying molecular mechanism that may help explain clinical case reports andretrospective studies that suggest rFVIIIFc may be more effective in tolerizinghemophilia A patients with anti-FVIII inhibitory antibodies compared to FVIIInot linked to Fc. Our findings suggest apotential use of Fc-fusion proteins acting via NK cells to target B-cells, inthe management of unwanted immune responses directed against immunogenicself-antigens or therapeutic protein products.