Differential Immunodominance of Human and Porcine Factor VIII in Hemophilia A Mice.

Hemophilia A inhibitor patients and patients with acquired hemophilia A recognize immunodominant epitopes in the A2 and C2 domains of human factor VIII (fVIII). Hemophilia A mice also recognize A2 and C2 domain epitopes when immunized with human fVIII using a dosing schedule that mimics clinical use. We compared the immune responses of hemophilia A mice to human and porcine fVIII using a domain specific ELISA. In this assay, monoclonal antibodies are tested against a panel of six single human fVIII domain hybrid human/porcine fVIII molecules that contain the human A1, A2, ap, A3, C1 or C2 domains. With anti-human antibodies, a positive signal with one of the single human domain proteins identifies domain specificity, whereas loss of signal indicates domain specificity of anti-porcine fVIII antibodies. Exon16 (E16) - disrupted hemophilia A mice (n = 3) received six weekly μ10 g/kg intravenous injections of recombinant B-domain deleted human fVIII and a final 25 μg/kg boost. To obtain comparable inhibitor titers, E16 mice (n = 3) received six weekly injections of μ40 g/kg of recombinant B-domain deleted porcine fVIII. Spleens from high titer mice were fused with NS1 mouse myeloma cells and 485 of the resulting hybridomas were analyzed for fVIII domain specificity (Table). Only two hybridomas secreted antibodies specific for the ap domain. Human fVIII elicited a significantly greater number of antibodies to the A2 domain, whereas porcine fVIII elicited a significantly greater number of antibodies to the A1 and A3 domains (p < 0.01, chi square test). The greater number of anti-C2 antibodies to human fVIII was not significant at the 95% confidence level (p = 0.08). The differential immunodominance of human and porcine fVIII epitopes suggests that it may be possible to design a recombinant hybrid human/porcine fVIII molecule that is less immunogenic than human fVIII in the treatment of patients with hemophilia A.

Domain Specificity of Anti-FVIII MAbs