The Complexity of the Humoral Immune Response to Factor VIII in Hemophilia A Open Access

Markovitz, Rebecca Claire (2014)

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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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