Razvan D. Miclea

Role of glycosylation in conformational stability, activity, macromolecular interaction and immunogenicity of recombinant human factor VIII.

Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein–lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.

Effect of route of administration of human recombinant factor VIII on its immunogenicity in Hemophilia A mice.

Factor VIII is a multi-domain glycoprotein and is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes Hemophilia A, a bleeding disorder. Replacement using exogenous recombinant Factor VIII (FVIII) is the first line of therapy for Hemophilia A. Immunogenicity, the development of binding (total) and neutralizing (inhibitory) antibody against administered protein is a clinical complication of the therapy. There are several product related factors such as presence of aggregates, route and frequency of administration and glycosylation have been shown to contribute to immunogenicity. The effect of route of administration of FVIII on antibody development in Hemophilia A is not completely understood. Here we investigated the effect of route of administration (s.c. or i.v.) on immunogenicity in Hemophilia A mice. The total and inhibitory titers were determined using ELISA and modified Bethesda Assay respectively. The results indicated that s.c. is more immunogenic compared to i.v. route in terms of total antibody titer development (binding antibodies) but no significant differences in inhibitory titer levels could be established.

Passive Transfer of Polyethylene glycol to Liposomal-Recombinant Human FVIII Enhances its Efficacy in a Murine Model for Hemophilia A

The replacement therapy using recombinant human FVIII (rFVIII) is the first line of therapy for hemophilia A. Approximately 15-30% of the patients develop inhibitory antibodies. Recently, we reported that liposomes composed of phosphatidylserine (PS) could reduce the immunogenicity of rFVIII. However, PS containing liposomal-rFVIII is likely to reduce the systemic exposure and efficacy of FVIII due to rapid uptake of the PS containing liposomes by the reticuloendothelial system (RES). Here, we investigated whether phosphatidylserine (PS) liposomes containing Polyethylene glycol (PEG) (PEGylated), could reduce the immunogenicity of rFVIII and reverse the reduction in systemic exposure of rFVIII. Animals given PEGylated liposomal-rFVIII had lower total and inhibitory anti-rFVIII antibody titers, compared to animals treated with rFVIII alone. The mean stimulation index of CD4+ T-cells from animals given PEGylated liposomal-rFVIII also was lower than for animals that were given rFVIII alone. Pharmacokinetic studies following intravenous dosing indicated that the systemic exposure (area under the activity curve, AUAC(0-24h)) of PEGylated liposomal-rFVIII was approximately 59 IU/mL x h and significantly higher than that of non-PEGylated liposomal-rFVIII (AUAC(0-24h) approximately 36 IU/mL x h). Based on these studies, we speculate that PEGylated PS-containing liposomal rFVIII may improve efficacy of rFVIII.

Immunogenicity and pharmacokinetic studies of recombinant Factor VIII containing lipid cochleates

Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15–30% of patients develop inhibitory antibodies against administered rFVIII, which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previous studies developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. It was hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. This study investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as a delayed release delivery vehicle with an input peak of cochleates showed limited RES uptake and associated rFVIII displayed a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.