Recombinant fusion of a single molecule of either FVIII or FIX to the Fc domain of IgG1 (rFVIIIFc or rFIXFc, respectively) has been shown to decrease clearance compared to rFIX or rFVIII in an FcRn-dependent manner (Blood (2010) 115: 2057; Blood (2012) 119:3024). The long-lasting procoagulant activity of these two molecules relies on a natural pathway in somatic (endothelial) and hematopoietic (monocyte) cells that recirculate antibodies into the blood stream. In hemophilia A subjects, rFVIIIFc has demonstrated ∼1.6-fold longer half-life compared to recombinant full length FVIII (Advate®) (Blood (2012) 119:3031), while rFIXFc shows a ∼3-fold longer half-life than that reported for current rFIX products (Blood (2012) 119:666). In the current study we determined the biodistribution of rFVIIIFc in hemophilia A (HemA) mice and sought to identify the cell types and tissues that contribute to FcRn-mediated protection and recycling of rFVIIIFc or rFIXFc.
The pharmacokinetics of rFVIIIFc or rFIXFc were compared to IgG1 in bone marrow chimeric mice of wild-type and FcRn-KO mice. The plasma FVIII activity was determined using a chromogenic assay, and the decay of antigen levels of rFIXFc or IgG1 in plasma post dosing was determined by ELISA. The biodistribution of iodinated rFVIIIFc in HemA mice was evaluated by Quantitative Whole Body Autoradiography (QWBA).
We have shown that the rFVIIIFc half-life is ∼2-fold longer in wild type mice than in FcRn-KO mice. However, bone marrow transplantation of FcRn-expressing hematopoietic cells into FcRn-deficient mice did not substantially increase the half-life of rFVIIIFc. Also, bone marrow transplantation of FcRn-deficient cells into wild-type mice did not decrease the half-life of rFVIIIFc, suggesting a substantional role of somatic FcRn. In contrast, while rFIXFc has a ∼3 fold longer half-life in FcRn wild-type mice than in knockout mice, both FcRn-deficient mice receiving wild-type bone marrow, as well as wild-type mice receiving FcRn-deficient bone marrow, displayed clearance levels of rFIXFc intermediate to that of wild-type and FcRn-KO mice. These studies suggest that rFVIIIFc is recycled via FcRn predominantly in somatic cells, while rFIXFc, similar to IgG1, is recycled approximately equally in both FcRn-expressing hematopoietic and somatic cells.
Furthermore, QWBA using 125I-labelled rFVIIIFc in HemA mice show extensive localization of rFVIIIFc in the liver, suggesting that somatic cells in the liver may mediate rFVIIIFc recycling through Fc – FcRn interaction. In summary, these data suggest that similar to IgG1, rFVIIIFc and rFIXFc are recycled in both somatic and hematopoietic FcRn-expressing cells. However, the relative contributions of the various cell types are dependent on the particular Fc-fusion protein, suggesting additional regulation at the level of protein-specific interactions for cellular uptake. Characterization of the cell-type specific recycling pathways for rFVIIIFc and rFIXFc is in progress to support further delineation of the mechanism of action for these two novel therapeutics currently in registrational trials for treatment of severe Hemophilia A and B, respectively.
van der Flier:BiogenIdec: Employment. Liu:BiogenIdec: Employment. Drager:BiogenIdec: Employment. Vallee:former BiogenIdec: Employment. Chen:BiogenIdec: Employment. Tan:BiogenIdec: Employment. Wharton:former BiogenIdec: Employment. Light:biogenidec: Employment. Jiang:biogenidec: Employment.
Asterisk with author names denotes non-ASH members.