Immunohistochemical Staining of rFVIIIFc and rFVIII in Liver Cells Differentiates Between a VWF-Dependent and Independent Clearance Pathway in Mice

rFVIIIFc is a fully recombinant fusion protein consisting of a single B domain-deleted human FVIII covalently attached to the dimeric Fc domain of human IgG1. rFVIIIFc has a 1.5-fold extended half-life and decreased clearance compared to rFVIII in patients with hemophilia A (Powell, Blood 2012). The Fc region of rFVIIIFc binds to the neonatal Fc receptor (FcRn), which is part of a naturally occurring pathway that cycles IgG back into circulation, delaying lysosomal degradation. Our previous studies with FcRn-chimeric mice showed that the decreased clearance of rFVIIIFc is mediated by FcRn expressed in somatic cells and not in hematopoietic cells. Biodistribution studies with ¹²⁵I-rFVIIIFc identified liver as the major organ for rFVIIIFc disposition. Furthermore, qPCR studies showed that three different cell types in liver all express FcRn: hepatocytes (HC), liver sinusoidal endothelial cells (LSEC) and Kupffer cells (KC). In primary liver cell co-cultures, rFVIIIFc and rFVIII were both internalized by somatic LSEC and HC, but not KC, in the absence of von Willebrand factor (VWF). It has been reported that in vivo, 95% of FVIII circulates as a non-covalent complex with VWF (Lenting, JTH 2007) and we found that VWF delays liver uptake of FVIII and improves the circulating half-life of rFVIIIFc in mice.

Compare the cellular localization of rFVIII and rFVIIIFc in the murine liver, in the presence and absence of VWF, in order to identify the cells responsible for the prolonged half-life of rFVIIIFc.

FVIII KO (HemA) and FVIII/VWF DKO mice were dosed with rFVIII, rFVIIIFc or the mutants rFVIIIFc-IHH and rFVIIIFc-N297A that are deficient in interacting with FcRn and FcgR, respectively. The cellular localization in the liver of rFVIII, rFVIIIFc and mutants was investigated by immunohistochemistry, using a panel of anti-human FVIII monoclonal antibodies or anti-human-IgG (Fc) for detection, along with markers for LSEC, KC, and VWF.

In HemA mice with circulating endogenous VWF, the majority of both rFVIII and rFVIIIFc signal is found in KC, which co-stain for VWF. In contrast, neither rFVIII nor rFVIIIFc is detected in the endothelial cells of the large vessels that stained for VWF in the Weibel-Palade bodies. Closer examination shows faint vesicular staining by rFVIII in HC in contrast to a diffuse staining by rFVIIIFc in the liver sinusoid. However in FVIII/VWF DKO mice lacking VWF, neither rFVIII nor rFVIIIFc is detected in KC consistent with the notion that internalization of FVIII by KC is mediated by VWF. The majority of rFVIII is found in HC, whereas rFVIIIFc again appears as a diffused liver sinusoidal staining pattern that is more intense than that observed in the HemA mice expressing VWF. These findings suggest free rFVIII is internalized and cleared by HC, while rFVIIIFc is cycled out of the HC and localizes in the Space of Disse, hence its sinusoidal localization. Alternately, in the absence of VWF, rFVIIIFc may cycle through LSEC rather than HC. In order to distinguish these two pathways, an rFVIIIFc variant, rFVIIIFc-IHH, which is not competent to bind FcRn, was tested. In a similar manner as rFVIII, the rFVIIIFc-IHH mutant localizes into HC in DKO mice and is found predominantly in KC in HemA with endogenous VWF. In contrast, rFVIIIFc-N297A, which is not competent to bind FcgR, localizes similarly to rFVIIIFc in DKO mice, excluding a role for FcgR.

Our current immunohistochemical studies and previous biodistribution and PK studies in mice indicate that there are two parallel, linked clearance pathways for rFVIII and rFVIIIFc. rFVIII or rFVIIIFc complexed with endogenous VWF is internalized predominantly by macrophages, including KC. However, because the VWF-FVIII complex is in constant equilibrium, a fraction of free rFVIII or rFVIIIFc is available for clearance by HC. We propose that this free fraction of rFVIIIFc entering HC is then cycled by FcRn back into the liver sinusoid and into circulation, in contrast to the free rFVIII entering the HC. Staining of the rFVIIIFc-IHH mutant in HC suggests that these cells play a dominant role in vivo, however LSEC may also contribute to cycling of VWF-free rFVIIIFc. The fate of the VWF-bound rFVIIIFc fraction internalized by KC is less clear, however data using FcRn-chimeric mice suggest that FcRn expressed in hematopoietic cells, including KC contributes only marginally to the delayed clearance of rFVIIIFc.

van der Flier:Biogen Idec: Employment, Equity Ownership. Liu:Biogen Idec: Employment, Equity Ownership. Light:Biogen Idec: Employment, Equity Ownership. Jiang:Biogen Idec: Employment, Equity Ownership.