Frequency and epitope specificity of anti–factor VIII C1 domain antibodies in acquired and congenital hemophilia A

Joerg Kahle, Aleksander Orlowski, Diana Stichel, John F. Healey, Ernest T. Parker, Marc Jacquemin, Manuela Krause, Andreas Tiede, Dirk Schwabe, Pete Lollar and Christoph Königs

Key Points

  • The fVIII C1 domain contributes significantly to the immune response against fVIII in acquired and congenital hemophilia inhibitor patients.

  • B-cell epitopes identified for monoclonal murine and human C1 inhibitors are recognized by antibodies present in patients with hemophilia.


Several studies showed that neutralizing anti–factor VIII (anti-fVIII) antibodies (inhibitors) in patients with acquired hemophilia A (AHA) and congenital hemophilia A (HA) are primarily directed to the A2 and C2 domains. In this study, the frequency and epitope specificity of anti-C1 antibodies were analyzed in acquired and congenital hemophilia inhibitor patients (n = 178). The domain specificity of antibodies was studied by homolog-scanning mutagenesis (HSM) with single human domain human/porcine fVIII proteins and antibody binding to human A2, C1, and C2 domains presented as human serum albumin (HSA) fusion proteins. The analysis with HSA-fVIII domain proteins confirmed the results of the HSM approach but resulted in higher detection levels. The higher detection levels with HSA-fVIII domain proteins are a result of antibody cross-reactivity with human and porcine fVIII leading to false-negative HSM results. Overall, A2-, C1-, and C2-specific antibodies were detected in 23%, 78%, and 68% of patients with AHA (n = 115) and in 52%, 57%, and 81% of HA inhibitor patients (n = 63). Competitive binding of the human monoclonal antibody (mAb) LE2E9 revealed overlapping epitopes with murine C1-specific group A mAbs including 2A9. Mutational analyses identified distinct crucial binding residues for LE2E9 (E2066) and 2A9 (F2068) that are also recognized by anti-C1 antibodies present in patients with hemophilia. A strong contribution of LE2E9- and 2A9-like antibodies was particularly observed in patients with AHA. Overall, our study demonstrates that the C1 domain, in addition to the A2 and C2 domains, contributes significantly to the humoral anti-fVIII immune response in acquired and congenital hemophilia inhibitor patients.

  • Submitted November 15, 2016.
  • Accepted April 16, 2017.
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