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Factor VIII Fc Fusion Protein but not FVIII Drives Human Monocyte-Derived Dendritic Cell Activation via Fc?RIIa.

ABSTRACT: This study compares the effect of recombinant Factor VIII Fc fusion protein (rFVIII-Fc) with recombinant FVIII (rFVIII) on monocyte-derived dendritic cells (moDC's). Cells treated with rFVIII-Fc showed morphological changes typical for cell activation, had a significant up-regulation of cell activation markers and produced higher levels of pro-inflammatory cytokines. Even after stimulation with Lipopolysaccharides, the addition of rFVIII-Fc led to increased expression of activation markers, indicating that rFVIII-Fc is capable of amplifying the maturation signal. On the contrary, cultivation of moDC's with rFVIII did not alter cell morphology or increase surface activation marker expression and pro-inflammatory cytokine production. The binding of the Fc domain to the activating Fc? receptor IIa (Fc?RIIa) can cause cell activation. Therefore, the effect of rFVIII-Fc on Fc?RIIa was analyzed in detail. Cultivation of moDC's with rFVIII-Fc led to increased phosphorylation of Fc?RIIa, which was not detected for rFVIII. Blocking Fc?RIIa prior to the cultivation with rFVIII-Fc significantly reduced the activating effect of rFVIII-Fc, indicating that rFVIII-Fc-induced moDC activation was caused by Fc?RIIa. Moreover, rFVIII-Fc bound to FCGR2A-transfected human embryonic kidney 293 cells. Taken together, our data present a new mechanism of moDC activation by rFVIII-Fc via Fc?RIIa.

SUBMITTER: Kannicht C

PROVIDER: S-EPMC7000470 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Factor VIII Fc Fusion Protein but not FVIII Drives Human Monocyte-Derived Dendritic Cell Activation via FcγRIIa.

Kannicht Christoph C Danßmann Ilona I Weilandt Constanze C Derkow Katja K Kohla Guido G Geyer Henriette H

HemaSphere 20200103 1

This study compares the effect of recombinant Factor VIII Fc fusion protein (rFVIII-Fc) with recombinant FVIII (rFVIII) on monocyte-derived dendritic cells (moDC's). Cells treated with rFVIII-Fc showed morphological changes typical for cell activation, had a significant up-regulation of cell activation markers and produced higher levels of pro-inflammatory cytokines. Even after stimulation with Lipopolysaccharides, the addition of rFVIII-Fc led to increased expression of activation markers, indi ...[more]

PMID: 32072146

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Project description:We recently developed a longer lasting recombinant factor VIII-Fc fusion protein, rFVIIIFc, to extend the half-life of replacement FVIII for the treatment of people with hemophilia A. In order to elucidate the biological mechanism for the elongated half-life of rFVIIIFc at a cellular level we delineated the roles of VWF and the tissue-specific expression of the neonatal Fc receptor (FcRn) in the biodistribution, clearance and cycling of rFVIIIFc. We find the tissue biodistribution is similar for rFVIIIFc and rFVIII and that liver is the major clearance organ for both molecules. VWF reduces the clearance and the initial liver uptake of rFVIIIFc. Pharmacokinetic studies in FcRn chimeric mice show that FcRn expressed in somatic cells (hepatocytes or liver sinusoidal endothelial cells) mediates the decreased clearance of rFVIIIFc, but FcRn in hematopoietic cells (Kupffer cells) does not affect clearance. Immunohistochemical studies show that when rFVIII or rFVIIIFc is in dynamic equilibrium binding with VWF, they mostly co localize with VWF in Kupffer cells and macrophages, confirming a major role for liver macrophages in the internalization and clearance of the VWF-FVIII complex. In the absence of VWF a clear difference in cellular localization of VWF-free rFVIII and rFVIIIFc is observed and neither molecule is detected in Kupffer cells. Instead, rFVIII is observed in hepatocytes, indicating that free rFVIII is cleared by hepatocytes, while rFVIIIFc is observed as a diffuse liver sinusoidal staining, suggesting recycling of free-rFVIIIFc out of hepatocytes. These studies reveal two parallel linked clearance pathways, with a dominant pathway in which both rFVIIIFc and rFVIII complexed with VWF are cleared mainly by Kupffer cells without FcRn cycling. In contrast, the free fraction of rFVIII or rFVIIIFc unbound by VWF enters hepatocytes, where FcRn reduces the degradation and clearance of rFVIIIFc relative to rFVIII by cycling rFVIIIFc back to the liver sinusoid and into circulation, enabling the elongated half-life of rFVIIIFc.

Dataset Information

Factor VIII Fc Fusion Protein but not FVIII Drives Human Monocyte-Derived Dendritic Cell Activation via Fc?RIIa.

Publications

Factor VIII Fc Fusion Protein but not FVIII Drives Human Monocyte-Derived Dendritic Cell Activation via FcγRIIa.

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