title: Lower inhibitor development in hemophilia A mice following administration of recombinant factor VIII-O-phospho-L-serine complex.
abstract: Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.
authors.affiliation: Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, New York 14260-1200, USA.
authors.forename: Vivek S
authors.initials: VS
authors.lastname: Purohit
authors.forename: Karthik
authors.initials: K
authors.lastname: Ramani
authors.forename: Rita
authors.initials: R
authors.lastname: Sarkar
authors.forename: Haig H
authors.initials: HH
authors.lastname: Kazazian
authors.forename: Sathyamangalam V
authors.initials: SV
authors.lastname: Balasubramanian
mesh_terms: D000818:Animals; D005169:Factor VIII / Q000008:administration & dosage* / Q000276:immunology* / Q000627:therapeutic use; D005260:Female; D006467:Hemophilia A / Q000188:drug therapy* / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000096:biosynthesis*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010768:Phosphoserine / Q000008:administration & dosage* / Q000031:analogs & derivatives*
chemical_list: D007518:Isoantibodies; D010768:Phosphoserine; C078147:F8 protein, human; D005169:Factor VIII
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