Factor VIII inhibitors in mild haemophilia A

Source: OAI


The X-linked bleeding disorder haemophilia A is due to a deficiency or functional defect of coagulation factor VIII. The bleeding tendency can be corrected by administration of factor VIII concentrates. A serious complication of factor VIII replacement therapy is the development of anti-factor VIII antibodies (inhibitors) that neutralize factor VIII activity. In recent years, the epitope-specifities and the inhibitory mechanisms of factor VIII inhibitors have gained increasing interest. The generation of factor VIII knock-out mice has opened the possibility of studying the immunobiology of inhibitor formation in murine models of haemophilia A. In spite of these recent developments however, the immunological mechanisms underlying the anti-factor VIII immune response have remained poorly understood so far. Most of our current knowledge is based on studies on inhibitor formation in the severe form of haemophilia. However, inhibitors also occur in patients with mild haemophilia A, in particular after a period of extensive factor VIII replacement therapy. These patients differ from severe haemophiliacs in that they have low levels of circulating factor VIII activity (5-25% of normal). The presence of endogenous factor VIII may have major impact on the immune response to exogenous factor VIII during replacement therapy. The studies presented in this thesis were performed to obtain a better understanding of the immunobiology of inhibitor development in mild haemophilia A. In the introduction (chapter 1), recent studies on the immunobiology of factor VIII inhibitors in haemophilia A patients are summarized and discussed. We have characterized the anti-factor VIII antibodies in patients with mild haemophilia A employing phage display technology. In chapter 2, anti-C2 antibodies were isolated and characterized from the repertoire of a mild haemophilia A patient. Our results provide evidence for the presence of two classes of anti-C2 antibodies that recognize distinct antigenic sites in factor VIII. The characteristics of the anti-C2 antibodies were further analysed in chapter 3, and compared to the epitopes of previously described murine monoclonal antibodies. The first class of anti-C2 antibodies bind to the epitope defined by monoclonal antibody ESH4. The second class of antibodies bind to the epitope defined by monoclonal antibody CLB-CAg 117. Antibodies belonging to this second class of antibodies were also isolated from a different patient with mild haemophilia A (chapter 4). The VH gene segment usage of the antibodies directed at the epitope defined by CLB-CAg 117 is less restricted compared to the first class of anti-C2 antibodies. Based on the long CDR3 region, we argue that this second class of antibodies originates from a pool of polyreactive human antibodies. In chapter 5, we describe the inhibitor development of a patient with mild haemophilia A caused by an Arg593 to Cys mutation. We have isolated and characterized anti-A2 antibodies using phage display and we have performed epitope-mapping studies of anti-factor VIII antibodies in plasma using immunoprecipitation analysis. The data presented in chapter 5 provide a possible explanation for anamnestic responses observed in patients with a history of inhibitor development. We propose that activation of a quiescent pool of memory B cells underlies the rise in inhibitor titer observed in haemophilia A patients with a history of inhibitor development. Chapter 6 describes the epitope specificities of anti-factor VIII antibodies in another patient from our cohort of mild haemophilia A patients with the Arg593 to Cys mutation. Results from this chapter and previous studies show that high responder patients with the Arg593 to Cys substitution develop inhibitory antibodies predominantly directed at the A2 domain of factor VIII. This suggests that inhibitor formation proceeds via a common mechanism in these patients. The role of HLA class II alleles in inhibitor formation was investigated by HLA genotyping of 42 patients with the Arg593 to Cys mutation. Our data suggest a weak association between inhibitor development and HLA class II alleles in mild haemophilia A patients with the Arg593 to Cys mutation. In Chapter 7, we present the characteristics of a mouse transgenic for human factor VIII with the Arg593 to Cys mutation (hufVIII-R593C mouse). The anti-factor VIII immune response was analysed in transgenic hufVIII-R593C mice crossed with factor VIII-deficient mice (exon 16 knock out, or E-16 KO mice). Serial intravenous injections of human factor VIII do not evoke an immune response in hufVIII-R593C/E-16 KO mice. The introduction of the human factor VIII–R593C transgene renders the mice tolerant to human factor VIII. However, when hufVIII-R593C/E-16 KO mice were subcutaneously injected with factor VIII in the presence of an adjuvant, loss of tolerance to factor VIII was observed. The results of chapter 7 demonstrate that hufVIII-R593C/E-16 KO mice provide a valuable model for studies directed at the mechanisms underlying inhibitor development in haemophilia A. Finally, chapter 8 provides a general overview that discusses the implications of our findings.

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