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

Abstract

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