The Complexity of the Humoral Immune Response to Factor VIII in Hemophilia A Open Access
Markovitz, Rebecca Claire (2014)
Abstract
The humoral immune response is the most serious complication in the treatment of hemophilia A patients. Approximately thirty percent of patients with severe hemophilia A develop anti-factor VIII (fVIII) antibodies (Abs), termed inhibitors, with associated increases in both morbidity and mortality. The Abs most commonly target the A2 and C2 domains of fVIII. Consequently, we used a murine immunogenicity model to study the humoral immune response to the A2 domain of fVIII. This study confirmed the importance of the immunodominant epitope between 484-508. In addition, by identifying novel inhibitory mechanisms, we gained a better understanding of the pathogenicity of fVIII inhibitors. We identified anti-A2 MAbs that inhibited cleavage at Arg372 and Arg1689, thus inhibiting thrombin's activation of fVIII cofactor function and fVIII's dissociation from von Willebrand Factor (VWF) respectively. In vivo studies with anti-A2 MAbs then demonstrated that the epitopes and mechanisms of inhibition of the MAbs in combination with inhibitory titer may better correlate with in vivo pathogenicity than inhibitory titer alone. Our collaborators then used the MAbs to compare the antigenicity of different fVIII molecules. They found that ovine fVIII (ofVIII) could be a good alternative treatment for some patients with inhibitors to both porcine and human fVIII. In addition to reducing antigenicity, we were interested in reducing the immunogenicity of fVIII and in inducing tolerance in murine models of fVIII inhibitor formation. We hypothesized that by targeting fVIII-specific naïve and memory B cells, we could decrease or inhibit the formation of anti-fVIII Abs. We chose the toxin saporin, a type I ribosome inactivating protein (RIP), and conjugated it to fVIII using N-succinmidyl-3-(2-pyridyldithio)propionate (SPDP) a heterobifunctional cross linker. We have used both an adoptive transfer and naïve mouse model to test our hypothesis with variable results. This may be due to experimental error or due to a flaw in our hypothesis. If ultimately successful this strategy could be a novel mechanism of inhibiting anti-fVIII Ab formation as well as inducing tolerance to fVIII after inhibitors have formed. In addition, it could increase knowledge regarding mechanisms of tolerance to fVIII.
Table of Contents
Introduction 1
Diagnosis of Neutralizing Anti-FVIII Antibodies in Hemophlia A Patients 3
Risk of Inhibitor Development 5
How the Immune System Sees fVIII 8
The Diversity of the Immune Response to FVIII 13
Immune Tolerance Therapy 16
Possible Mechanisms of ITI and Alternative Mechanisms of Tolerance 19
References 33
Chapter I: The Diversity of the Immune Response to the A2 Domain of Human Factor VIII 40
Abstract 41
Introduction 42
Materials and Methods 44
Results 48
Discussion 55
References 61
Figures 70
Chapter II:High Titer Type I but not Type II Anti-Factor VIII A2 Domain Antibodies Are Pathogenic in a Murine in vivo Bleeding Model 81
Abstract 82
Introduction 83
Materials and Methods 86
Results 91
Discussion 94
Figures 97
References 101
Chapter III:Expanding the Ortholog Approach for Hemophilia Treatment Complicated by Factor VIII Inhibitors 104
Introduction 107
Materials and Methods 111
Results 115
Discussion 122
Figures 127
References 138
Conclusion 145
Summary 145
Key Results from the Anti-A2 MAb Studies 146
FVIII-Saporin In vivo Experiments 149
Future Directions 153
References 157
Appendix 159
Figures 165
Appendix Results 168
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