Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

Abstract

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ϳ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5-to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation. (Blood. 2012;119(13):3024-3030) IntroductionHemophilia A is an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII) activity. 1 The disease is characterized by spontaneous hemorrhage and excessive bleeding after trauma. Over time, repeated bleeding into muscles and joints, which begins in early childhood, results in hemophilic arthropathy and irreversible joint damage. This damage is progressive and leads to pronounced musculoskeletal morbidity. 1 Prophylaxis significantly reduces joint damage and long-term sequelae, and improves quality of life in comparison to on-demand treatment. 2,3 However, the short half-life (10-12 hours) of FVIII necessitates dosing every other day or 3 times per week by IV injection for full prophylaxis. 2,4,5 Therefore, a longer-acting FVIII would represent a key advancement in the management of hemophilia A.We have developed a recombinant factor VIII-Fc (rFVIIIFc) fusion protein to extend the half-life of FVIII by leveraging a naturally occurring biologic pathway. rFVIIIFc is a heterodimeric protein comprising a single B-domain-deleted (BDD) FVIII linked recombinantly to the Fc domain of human IgG1 (IgG1). The Fc domain enables binding to the neonatal Fc receptor (FcRn), which is responsible for protection of IgG from degradation and facilitates its recycling, 6,7 resulting in a half-life for IgG of ϳ 3 weeks in humans. The Fc domain of IgG1 has been fused to growth factors, cytokines, enzymes, and ligand-binding regions of receptors [8][9][10] ; several of these fusion proteins have been approved as therapeutics (eg, etanercept, abatacept, belatacept, alefacept, rilonacept, aflibercept, and romiplostim). However, traditional dimeric Fc fusions, created through the fusion of the monomeric effector p...

A von Willebrand factor fragment containing the D′D3 domains is sufficient to stabilize coagulation factor VIII in mice

“…The t 1/2 of recombinant FVIII or recombinant human B-domain deleted FVIII infused into mice is significantly shorter in the absence than presence of VWF (18 minutes vs 5.9-7.6 hours, respectively). [24][25][26] We confirm this relationship and demonstrate that a minimal, N-terminal VWF fragment comprising D9D3 domains (S764-P1247) is sufficient to stabilize endogenous FVIII in Vwf 2/2 mice. Extending the circulatory lifetime of D9D3 by Fc fusion markedly prolongs endogenous FVIII survival in Vwf 2/2 mice but is insufficient to extend the FVIII t 1/2 in HA mice.…”

“…These data would also explain the longer t 1/2 of transfused FVIII we observed in HA mice (129S1/SvImJ genetic background) compared with previous reports using HA mice on a C57BL/6 genetic background. [24][25][26] The stoichiometry of VWF:FVIII is ;50:1 in human plasma. 43 Assuming a human plasma FVIII concentration of ;1 nM 43 and that FVIII levels in C57BL/6J mice are ;0.26-fold of human levels, 40 we estimate a similar stoichiometry (;57:1) for the VWF/FVIII complex in C57BL/6J mice.…”

“…21,22 Several approaches have attempted to engineer FVIII with an extended plasma half-life (t 1/2 ). [23][24][25][26] Recombinant human B-domain deleted FVIII infused into a C57BL/6 mouse has a similar t 1/2 (4.3 hours) to that of endogenous murine VWF (;4.5 hours). 25,27,28 Selective conjugation of polyethyleneglycol (PEG) 24,26 or fusion of FVIII to the Fc domain of immunoglobulin G (IgG) 25 increases FVIII plasma t 1/2 by ;1.5-to 2-fold in mice [24][25][26] and humans.…”

“…[23][24][25][26] Recombinant human B-domain deleted FVIII infused into a C57BL/6 mouse has a similar t 1/2 (4.3 hours) to that of endogenous murine VWF (;4.5 hours). 25,27,28 Selective conjugation of polyethyleneglycol (PEG) 24,26 or fusion of FVIII to the Fc domain of immunoglobulin G (IgG) 25 increases FVIII plasma t 1/2 by ;1.5-to 2-fold in mice [24][25][26] and humans. 29 Here, we report the effect of a set of VWF fragments on FVIII binding in vitro and survival in vivo and demonstrate a markedly extended plasma t 1/2 for an Fc fusion to a 484-amino acid N-terminal VWF fragment, with a concomitant increase in plasma FVIII levels in VWF-deficient but not HA mice.…”

Expressão gênica empregando pseudopartículas em células de mamíferos (HEK 293T e Huh 7.0) cultivadas em diferentes meios de cultura livres de soro.

“…A célula HEK 293, é uma linhagem derivada de células embrionárias humanas de rim, transformada com adenovírus tipo 5 humano, foi desenvolvida há 37 anos (GRAHAM et al, 1977), mas apenas nos últimos anos tem sido extensivamente usada, sendo a linhagem humana provavelmente mais utilizada para pesquisas relacionadas a expressão de proteínas recombinantes (ADAM et al, 2008;DUMONT et al, 2012;FISCHER et al, 2012;LOIGNON et al, 2008;SUN et al, 2006;SWIECH et al, 2011;WAJIH;OWEN;WALLIN, 2008). Esta linhagem exibe uma morfologia epitelial e uma das razões para sua larga aplicação é o fato destas células crescerem facilmente em suspensão e em SFM disponíveis comercialmente (DUROCHER; BUTLER, 2009;LIU et al, 2006;MEI et al, 2006;ZHU, 2012 VAN DER VALK et al, 2004).…”