Factor VIII is a circulating plasma protein that plays a critical role in blood coagulation. The hereditary absence of this protein or its activity results in a serious chronic disorder, hemophilia A. Current treatment for the bleeding episodes occurring in this disorder involves replacement of the missing protein by infusion of cryoprecipitate or factor VIII concentrates prepared from normal plasma. A significant complication of this therapy is the appearance of a circulating inhibitor of factor VIII in plasma of some 15-20% of severely effected hemophilia A patients. Once an inhibitor has appeared, treatment of the underlying disorder becomes difficult. Current knowledge about the structure of anti-factor VIII alloantibodies and the immunoregulatory mechanisms which lead to their appearance is limited. The current proposal is to produce human monoclonal anti-factor VIII alloantibodies by human x human fusion of peripheral blood lymphocytes from patients with inhibitors, with a human lymphocytoid line. The type and subtype of the monoclonal antibodies produced by the hybridomas will be established, anti-idiotypic reagents will be produced and used to explore the relatedness of both monoclonal inhibitors and those in the circulation of hemophilia A patients with inhibitors. Primary sequence analysis of a group of these inhibitors will be instituted in order to compare the primary structure. The polypeptide chain of a human factor VIII preparation against which the monoclonal inhibitor is directed will be established. The known sequence of factor VIII will then be employed to choose runs of sequence to be synthesized as potential antigenic determinants. Synthetic peptides will be tested for their ability to inhibit binding of monoclonal inhibitor to factor VIII. Increased understanding of the inhibitor response and the availability of the reagents generated may prove useful in developing strategies to prevent inhibitors from arising or methods of circumventing their action.
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