Fusion partners matter: Factor VIII light chain binding enables CD16-mediated recombinant Factor VIII-Fc fusion protein natural killer cell activation.

Abstract Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with multiple Fc gamma receptors (FcγR) resulting in activation or inhibition a wide variety FcγR expressing immune cells. We activates natural killer (NK) cells via mediated interactions FcγRIIIA CD16. Here, we human NK cell lines and primary enriched from peripheral blood leukocytes to study rFVIIIFc-mediated activation. Following overnight incubation rFVIIIFc, assessed cellular by measuring inflammatory cytokine secretion (IFNγ ELISA) degranulation (flow cytometry-based surface LAMP1/CD107a). In some cases, introduced specific blocking molecules (anti-FVIII, anti-Fc, anti-CD16) into our assays act as inhibitors identified FVIII light chain playing key role CD16 +NK rFVIIIFc. Using domain-specific inhibitors, demonstrate C1 C2 domain-mediated membrane binding potently inhibits activation, while targeting heavy domains does not. Our results suggest chain-mediated tethering allows Fc-CD16 proceed, This working model may help explain previous where observed recombinant IX-Fc exhibited different CD16-signaling properties despite having identical IgG1 domains.