Towards a New Therapy for Hemophilia A: Evaluation of Multiple Approaches To Prolong the In Vivo Efficacy of Recombinant Factor VIII.

Abstract

Prophylactic therapy with rFVIII has been shown to have a significant positive impact on the treatment of Hemophilia A. One of the impediments to effective prophylaxis is the requirement for frequent injections necessitated by the 12–14 hour circulating half-life of FVIII. We have evaluated a number of approaches to modify FVIII to reduce the need for frequent injections. In one approach, the active form of FVIII was stabilized by addition of a disulfide linkage between the A2 and A3 domains. As previously reported1, these molecules exhibited prolonged FVIII activity in a number of in vitro assays following activation. In vivo characterization of these molecules is in progress. Other approaches focused on increasing the circulating half-life of FVIII. In an attempt to reduce FVIII clearance by the liver receptor LRP, site-directed mutations in the reported LRP binding region of the FVIII A2 domain were generated. Twenty mutants, with single amino acid changes, were analyzed in mouse and rabbit recovery studies and no significant increase in recovery was observed. Since the point mutants may not have covered a large enough surface of the LRP binding domains, polyethlylene glygol (PEG) modification was used to disrupt a larger surface area. Previously, attachment of PEG moieties to FVIII had been shown to lead to an increase in plasma half-life in animal models; however, standard pegylation chemistries result in significant product heterogeneity and thus may not be suitable for commercial production. We have developed a novel method, based on protein engineering, to introduce PEG to specific cysteine residues on the surface of FVIII. Using this method, different molecular weight PEGs have been conjugated to sites in the A1, A2 or A3 domains of FVIII. Analysis of the pegylated proteins confirms that the attachment of PEG is highly specific to the engineered site. The pegylated products retain activity based on the two stage chromogenic assay, but exhibit reduced activity when analyzed by the one stage coagulation assay. Pharmacokinetic studies, performed in mouse and rabbit, show that pegylation increases half-life in a manner that is proportional to PEG molecular weight. Using a number of injury models in hemophilic mice, the pegylated molecules have been shown to be efficacious in stopping bleeds. The prospects for using site-specific pegylation of FVIII to produce a therapeutic for treatment of hemophilia A will be discussed.

Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII

“…(22),alow-affinity siteislocated within the C2 domain (4).In circulating FVIII/VWF complex,the A3 and C2 sites areblocked by VWF bound to LCh (4,23) that is consistent with ashorter half-life of FVIII in patients with vonWillebrand disease (24) and in VWF-deficient mice (5).Exposure of the sites in LCh is drivenb yd issociation of VWFu pon proteolytic activation of FVIII (4). This event also leads to exposure of the high-affinity LRP-binding site within A2 whichi sn ot interactive withL RP withinintactFVIII (25,26). In turn,exposure of the high-affinity sitesfor LRP in FVIIIasuggests that clearancevia LRP may be aspecific mechanism for removalofthe fragments of unstable FVIIIa, i.e.…”

Clearance mechanisms of von Willebrand factor and factor VIII

“…However, binding of the intact FVIII heavy chain was not observed in a study by one of us [10]. It was recently confirmed that intact heavy chain binds poorly, if at all, to LRP1 [27], explaining why site-directed mutagenesis of the LRP1 binding site in the A2 domain leaves in vivo pharmacokinetic parameters of mutated FVIII molecules unaffected [28]. In fact, the LRP1 interactive site within the A2 domain becomes exposed exclusively after proteolysis of the heavy chain [27].…”

The disappearing act of factor VIII

“…Thus, binding of LRP1 and vLDL receptor to the A2 domain is restricted to FVIIIa. This may explain why site‐directed mutagenesis of this interactive site does not result in improved pharmacokinetic parameters when the mutated FVIII procofactor proteins were tested in mice and rats [78]. Noteworthy, this proteolysis‐dependent exposure of the binding site has also been reported for the interaction between FVIII A2 domain and FIXa [79].…”