Reduction of the antigenicity of factor VIII toward complex inhibitory antibody plasmas using multiply-substituted hybrid human/porcine factor VIII molecules

Factor VIII (fVIII) circulates as a heavy chain/light chain (A1-A2-B/apA3-C 1-C2) heterodimer. The 41-residue light chain activation peptide, ap, is cl eaved from fVIII during proteolytic activation by thrombin or factor Xa, We constructed 7 active recombinant hybrid B-domainless human/porcine fVIII m olecules that contained combinations of porcine sequence replacements withi n the A2, ap-A3, and C2 domains. The cross-reactivity of 23 high-titer inhi bitory antibodies between human fVIII and the hybrids was inversely related to the degree of porcine substitution, In all plasmas, the substitution of all 3 regions yielded cross-reactivities that were not significantly diffe rent from those of porcine fVIII. To differentiate between inhibitor bindin g to the ap region and the A3 domain, we constructed 2 additional hybrids t hat contained porcine A2 and C2 domain substitutions and either porcine A3 or porcine ap substitutions. The porcine ap segment was less antigenic than the human ap segment in several plasmas that had activity against the ap-A 3 region. This indicates that some inhibitor plasmas contain antibodies dir ected against the fVIII ap segment in addition to A2, A3, and C2 domain epi topes identified In previous studies. Substitution of porcine sequences wit hin the A2, A3, C2, and ap regions of human fVIII is necessary and sufficie nt to achieve a maximal reduction in antigenicity relative to porcine fVIII with respect to most inhibitory antibody plasmas.