Phosphatidylserine containing liposomes reduce immunogenicity of recombinant human factor VIII (rFVIII) in a murine model of hemophilia A

Abstract

Factor VIII (FVIII) is a multidomain protein that is deficient in hemophilia A, a clinically important bleeding disorder. Replacement therapy using recombinant human FVIII (rFVIII) is the main therapy. However, approximately 15-30% of patients develop inhibitory antibodies that neutralize rFVIII activity. Antibodies to epitopes in C2 domain, which is involved in FVIII binding to phospholipids, are highly prevalent. Here, we investigated the effect of phosphatidylserine (PS)-containing liposomes, which bind to C2 domain with high affinity and specificity, upon the immunogenicity of rFVIII. Circular dichroism studies showed that PS-containing liposomes interfered with aggregation of rFVIII. Immunogenicity of free- versus liposomal-rFVIII was evaluated in a murine model of hemophilia A. Animals treated with s.c. injections of liposomal-rFVIII had lower total- and inhibitory titers, compared to animals treated with rFVIII alone. Antigen processing by proteolytic enzymes was reduced in the presence of liposomes. Animals treated with s.c. injections of liposomal-rFVIII showed a significant increase in rFVIII plasma concentration compared to animals that received rFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between PS-containing bilayers and rFVIII may provide a basis for designing lipidic complexes that improve the stability, reduce the immunogenicity of rFVIII formulations, and permit administration by s.c. route.

Figure 1

Relative % binding of monoclonal ESH4 antibody to liposomal-rFVIII as a function of lipid concentration and composition (DMPC/BPS (70/30)—open circles and DMPC—open triangles). ESH4 was used as the stationary antibody. A rat polyclonal antihuman rFVIII antibody was used as the probe antibody. In all preparations, rFVIII concentration was maintained constant (150 ng/mL). The error bars represent the standard deviation (±SD, n = 3).

Figure 2

(A) Far-UV CD spectra of rFVIII (in 300 mM NaCl, 25 mM Tris, 5 mM CaCl₂, pH 7.0) before (dotted line) and after (solid line) heating to 80°C in the absence (gray line) and presence (black line) of liposomes. The protein concentration was ∼20 μg/mL, and the protein to lipid ratio was ∼1:2500. All spectra were corrected by subtracting the spectrum of the buffer alone. (B) Temperature dependent changes in ellipticity (δθ) at 215 nm for rFVIII (solid line) and rFVIII associated with liposomes (DMPC/BPS, 70/30) (dotted line) during the unfolding process. The transition temperature (T_m) was determined by fitting the thermal unfolding profile to a sigmoid function using WinNonlin (Pharsight Corp., Mountainview, CA).

Figure 3

(A) Total and (B) inhibitory anti-rFVIII antibody titers in hemophilic mice following administration of rFVIII in the absence or presence of liposomes of DMPC/BPS (70/30) of varying sizes at the end of 6 weeks. Treatment was administered via s.c. route. Each point represents values from individual animals and the horizontal bar depicts the mean titer. Blood samples were obtained 2 weeks after the fourth injection.

Figure 4

SDS PAGE analysis of proteolytic (cathepsin-B) pattern of rFVIII and rFVIII associated with liposomes. Position of the light chain is indicated by the arrow.

Figure 5

rFVIII activity levels in the absence (dark bars) and presence (gray bars) of 80 nm DMPC/BPS liposomes following s.c. administration (4000 IU/kg) of the preparations. The data points collected at 3 and 5 h are averages of three animals. Error bars represent standard deviation; * denotes p < 0.05.