Michela Leoni

The treatment of hemophilia has been greatly improved over the past decades. Non-factor replacement therapies such as the bi-specific antibody emicizumab are now available for prophylactic treatment of hemophilia A patients. FVIII administration is however still needed for treatment of breakthrough bleeds and/or surgery putting patients at risk of developing FVIII inhibitors. It has been shown that around 25% of patients with severe hemophilia A develop FVIII inhibitors in response to FVIII replacement therapy. The development and persistence of FVIII inhibitors remains an unresolved challenge. Inhibitory antibodies directed towards FIX develop in around 3% of patients with hemophilia B. Our current knowledge on the mechanism driving immune responses to FVIII and FIX is still very limited.

In Chapter 1 we provide an overview of the etiology of hemophilia A and B. We also describe currently available treatment options for patients with hemophilia A and B. In the last part of Chapter 1 we described the development of anti-FVIII and anti-FIX antibodies in patients with hemophilia A and B in response to FVIII or FIX replacement therapy.

In Chapter 2 we describe the properties of liver sinusoidal endothelial cells (LSEC) which have been shown to synthesize the vast majority of circulating FVIII. LSEC are highly fenestrated cells and lack a basal membrane which allows for a rapid exchange of nutrients present in blood and hepatocytes and stellate cells. Many scavenger receptors are expressed on LSEC which serve to rapidly clear cellular debris and lipoproteins from the circulation. Interestingly, stabilin-2, one of the major scavenger receptors on LSEC has been implicated in clearance of the FVIII-VWF complex. Importantly, LSEC have evolved as an unique population of antigen presenting cells which can promote the differentiation of naïve CD4+ T cells into regulatory T cells thereby promoting tolerance. This unique property positions LSEC as a potential target for innovative therapies to induce tolerance to FVIII.

In Chapter 3 we studied the interaction of stabilin-2 with FVIII. We demonstrate that stabilin-2 is able to bind to FVIII in a von Willebrand factor independent manner. We also show that the Link domain of stabilin-2 contributes to the binding of FVIII.

In Chapter 4, we explored a novel strategy for inducing immune tolerance to FVIII by exploiting the physiological clearance of red blood cells (RBCs). Recombinant proteins in which FVIII domains (C2, A2 or A3-C1-C2) were fused to Annexin V (AnxA5) were designed and shown to bind to phosphatidylserine (PS) exposed on RBCs. FVIII-AnxA5 fusion protein loaded RBCs were rapidly endocytosed by macrophages. Since macrophage-mediated RBCs turnover is an inherently tolerogenic process, this approach can potentially create a favorable environment for induction of FVIII-specific tolerance.

In Chapter 5 we focus on inhibitor development in hemophilia B. We pulsed monocyte derived dendritic cells with FIX and analyzed the repertoire of FIX derived peptides that was presented on HLA-DR. Peptides derived from different domains of FIX were presented on HLA-DR. Surprisingly, we identified a single peptide 295-NVIRIIPHHNYNA-306 that was presented by multiple donors with different HLA-DR alleles. This promiscuously presented FIX peptide may be used for the design of novel therapies focusing on prevention and/or eradication of FIX inhibitors in patients with hemophilia B.

In Chapter 6, we analyze the potential of modulating the interaction of anti-FVIII antibodies with specific Fc receptors. We mutated six amino acids in the Fc region of EL14-IgG1, an anti-FVIII antibody that targets the C2 domain. This modification specifically enhances the binding to the inhibitory FcγRIIB receptor that is highly expressed on LSEC as well as other antigen presenting cells. Based on our findings we argue that anti-FVIII antibody that can bind with high affinity to the inhibitory FcγRIIB receptor as well as next generation FVIII-Fc fusion proteins with increased affinity for FcγRIIB may be used to more efficiently restore tolerance when compared to currently available treatment strategies.

Finally, in Chapter 7 we discuss the findings reported in this PhD thesis with reference to other studies. Knowledge obtained on FVIII clearance mechanisms as well as antigen presentation of FVIII and FIX may help to design novel approaches for reducing inhibitor formation and improving long-term outcomes in patients with hemophilia.