Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the Factor VIII coagulation protein (FVIII). Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma derived FVIII. These treatments have substantially improved the outcome of hemophilia. More recently long-acting recombinant FVIII concentrates have become available in the clinic, but hemophilia A patients still require the repeated intravenous injections frequently 2-3 times per week. Furthermore, the development of inhibitors which are associated with significant mortality and morbidity remains a serious complication for approximately 30% of patients with severe hemophilia A, highlighting the need for new FVIII products with reduced immunogenicity. The development of recombinant FVIII molecules with a lower inhibitor risk profile would present an important advance in the care of patients with hemophilia A. rVIII-SingleChain (AFSTYLATM), is the first and only single-chain molecule purposely designed to provide key features such as increased dosing intervals and high clinical efficacy. The present study has focused on evaluating the immunogenic profile of rVIII-SingleChain in comparison to other rFVIII products (both full length and B-domain-truncated/deleted). Several key characteristics have been identified: The highly pure and homogeneous rVIII-SingleChain with a covalently linked heavy chain and light chain is less likely to contain dissociated FVIII chains (or fragments thereof) that cannot bind to von Willebrand Factor (VWF) and are thus available for uptake by antigen presenting cells (APC). The high degree of sulphation of the predicted six tyrosine sulphation sites in rVIII-SingleChain (as compared to other rFVIII molecules) not only provides a basis for optimal functionality but also (with respect to Tyr1680) ensures maximal VWF binding of the majority of rVIII-SingleChain molecules. The improved binding of rVIII-SingleChain to VWF as compared to full-length rFVIII may more effectively reduce antigen uptake and processing by antigen-presenting cells. In this regard, rVIII-SingleChain pre-complexed with plasma-derived VWF, is less efficiently internalized by human monocyte-derived dendritic cells (MoDCs) as compared to several rFVIII products tested. Further, in a ProImmune ProPresent® antigen presentation assay to identify potentially immunogenic regions in rVIII-SingleChain as compared to full length rFVIII protein revealed a lower number of HLA-DR/DP/DQ-restricted epitopes derived from rVIII-SingleChain as compared to the full length rFVIII protein (33 c.f. 47 for DR; 24 c.f. 32 for DP and 26 c.f. 32 for DQ). In addition, when compared to BDD-rFVIII comparators in the ProImmune ProReveal® T cell stimulation assay, rVIII-SingleChain was found to contain fewer HLA-DR/DP/DQ-restricted epitopes derived from the linker region that triggered T-cell proliferation (15% as compared to 25-35% for the comparators). Collectively, these features should contribute to the reduced immunogenic potential of rVIII-SingleChain. Importantly, these pre-clinical studies are supported by findings from the completed rVIII-SingleChain clinical studies in previously treated patients (PTPs) in which no inhibitor development has been observed. In summary, the immunogenicity of rVIII-SingleChain has been extensively characterized in PTPs over 28,418 exposure days equivalent to more than 233 treatment years. As of 01 August 2016, no PTP subject has developed inhibitors after being exposed to rVIII-SingleChain. All available data support the hypothesis that the unique structural attributes of rVIII-SingleChain may translate into reduced immunogenicity when used for replacement therapy in patients with hemophilia A. The previously untreated patient (PUP) arm in the ongoing extension study is designed to generate data that are necessary to prove this significant clinical benefit. rVIII-SingleChain could potentially offer hemophilia A patients a recombinant product with an inhibitor risk profile that is more favorable than that of currently available products.


Maraskovsky:CSL Limited: Employment. Verhagen:CSL Limited: Employment. Huynh:CSL Limited: Employment. Baz Morelli:CSL Limited: Employment. Schmidbauer:CSL Behring: Employment. Zollner:CSL Behring: Employment. Powell:CSL Behring: Employment. St Ledger:CSL Behring: Employment. Veldman:CSL Behring: Employment. Hofmann:CSL Behring: Employment.

Author notes


Asterisk with author names denotes non-ASH members.