Factor IX administration in the skin primes inhibitor formation and sensitizes hemophilia B mice to systemic factor IX administration

Background: Factor IX inhibitor formation is the most serious complication of replacement therapy for the bleeding disorder hemophilia B, exacerbated by severe allergic reactions occurring in up to 60% of patients with inhibitors. Low success rates of immune tolerance induction therapy in hemophilia B necessitate the search for novel immune tolerance therapies. Skin-associated lymphoid tissues have been successfully targeted in allergen-specific immunotherapy. Objectives: We aimed to develop a prophylactic immune tolerance protocol based on intradermal administration of FIX that would prevent inhibitor formation and/or anaphylaxis in response to replacement therapy. Methods: We measured FIX inhibitor, anti-FIX immunoglobulin G1, and immunoglobulin E titers using the Bethesda assay and enzyme-linked immunosorbent assay after 4 weeks of twice-weekly intradermal FIX or FIX-Fc administration followed by 5 to 6 weeks of weekly systemic FIX injections in C3H/HeJ hemophilia B mice. We also measured skin antigen-presenting, follicular helper T, and germinal center B cell frequencies in skin-draining lymph nodes after a single or repeat intradermal FIX administration. Results: Intradermal administration enhanced FIX inhibitor formation in response to systemic administration. We further found that intradermal administration alone triggers inhibitor formation, even at a low dose of 0.4 IU/kg, which is 100-fold lower than the intravenous dose of 40 IU/kg typically required to induce inhibitor development in hemophilia B mice. Also, intradermal administration triggered germinal center formation in skin-draining lymph nodes and sensitized mice to systemic administration. Factor IX-Fc fusion protein did not modulate inhibitor formation. Conclusion: Intradermal FIX administration is highly immunogenic, suggesting that the skin compartment is not amenable to immune tolerance induction or therapeutic delivery of clotting factors.