Influence of aggregation on immunogenicity of recombinant human Factor VIII in hemophilia A mice.
Recombinant human factor VIII (rFVIII), a multidomain glycoprotein is used in replacement therapy for treatment of hemophilia A. Unfortunately, 15%-30% of the treated patients develop inhibitory antibodies. The pathogenesis of antibody development is not completely understood. The presence of aggregated protein in formulations is generally believed to enhance the immune response. rFVIII has a tendency to aggregate but the effect of such aggregation on the immunogenicity of rFVIII is not known. We have, therefore, characterized aggregated rFVIII produced by thermal stress and evaluated its effect on the immunogenicity of rFVIII in hemophilia A mice. Aggregated rFVIII alone and mixtures of rFVIII with aggregated rFVIII were less immunogenic than native rFVIII. In vitro Th-cell proliferation studies and cytokine analyses conducted on splenocytes obtained from immunized animals suggest that aggregated rFVIII behaves as a unique antigen compared to native monomeric rFVIII. The antigenic properties of the aggregated and native rFVIII were compared using ELISAs (epitope availability) and cathepsin-B (an antigen processing enzyme) digestion. The data suggest significant differences in the antigenic properties of rFVIII and aggregated rFVIII. Overall it appears that aggregated rFVIII does not enhance the immunogenicity (inhibitor development) of rFVIII in hemophilia A mice but rather acts as a distinct antigen.
Purohit
Vivek S
VS
The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, 14260-1200, USA.
Middaugh
C Russell
CR
Balasubramanian
Sathyamangalam V
SV
D000818:Animals; D002401:Cathepsin B / Q000276:immunology; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000235:genetics / Q000276:immunology*; D005260:Female; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011994:Recombinant Proteins / Q000276:immunology; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; C078147:F8 protein, human; D005169:Factor VIII; D002401:Cathepsin B