The development of neutralizing alloantibodies (inhibitors) to infused factor VIII (FVIII) remains the most significant complication of therapy in hemophilia A (HA). Despite some insights into genetic and environmental risk factors associated with inhibitor development, the immunological mechanisms behind this complication remain incompletely understood. Given that infused FVIII is likely encountered in the periphery, transitional B cell response to FVIII may regulate inhibitor development. Transitional B cell survival, maturation and proliferation are tightly regulated by the cytokine BLyS (B-lymphocyte stimulator). Elevated levels of BLyS have been described in both auto- and allo-immune disease processes and may be genetically determined in a subset of patients with lupus and multiple sclerosis. In prior experiments, we noted that BLyS levels are higher in HA patients with inhibitors as opposed to 10 other pro- and anti-inflammatory cytokines. However, BLyS levels normalized amongst those who underwent successful immune tolerance induction. Thus, we hypothesized that BLyS may regulate FVIII inhibitor development or persistence. Here, we present results from animal studies aimed at 1) preventing inhibitor development in naïve HA mice and 2) maintaining long-term tolerance.
For prevention experiments, HA C57Bl/6 mice (n=8-10/group) were given a single dose of anti-BLyS neutralizing antibody (Sandy-2, Adipogen) or PBS control one week prior to immunization with recombinant human FVIII (rhFVIII) at 80 IU/kg via tail vein injection every 14 days for four injections (Fig 1). Plasma samples were obtained at baseline and 7 days after last immunization and analyzed for FVIII antibody by Bethesda assay, anti-FVIII IgG by ELISA, and BLyS levels by ELISA. To test long-term tolerance, 22 weeks after initial anti-BLyS antibody injection, mice without inhibitors were challenged with 4 additional rhFVIII intravenous infusions and plasma obtained 7 days following each injection (Fig 1). Data was analyzed using Prism and is reported as median (IQR) with Mann-Whitney test for two-group comparisons and Mantel-Cox log-rank test for Kaplan-Meier survival analysis.
Anti-BLyS treated mice achieved nadir BLyS levels at 14 days post-injection compared to control (0.2 [0.09-0.3] vs 8.2 [7.3-8.5] ng/ml, respectively, p < 0.0001), and levels equalized by day 28 between groups. Only 2 of 9 mice in the anti-BLyS treated group developed inhibitors to FVIII (0 [0-12.5] BU) compared to 9 of 10 in the treated group (21.1 [2.5-157.3] BU) with a significant log-rank survival of 0.0007 and favorable hazard ratio of 0.16 (95% CI 0.05-0.56) after initial immunization. Total anti-FVIII IgG was lower in the treated group (4.7 [1.1-11.5] mcg/ml) compared to control mice (27.6 [24-42] mcg/ml) with p of 0.003. Of the anti-BLyS treated inhibitor-free mice that have been challenged with rhFVIII, no inhibitors were detected until after the fourth rhFVIII injection. In these mice, there was a trend towards lower inhibitor titers compared to controls with a range of 1-1.8 BU vs. 85.6-1016 BU and lower FVIII IgG (23 [20.9-25.2] vs 69.9 [65.1-129.9] mcg/ml), respectively.
Here, we demonstrate that BLyS depletion prior to FVIII exposure in naïve HA mice can prevent antibody development and may result in long-term sustained FVIII tolerance. As BLyS is central to the survival and maturation of transitional B cells, including marginal zone B cells which have recently been demonstrated to be important in inhibitor formation, this may provide a strategy for both antibody prevention and eradication in HA patients. Notably, the anti-BLyS monoclonal antibody belimumab is already FDA approved for the treatment of patients with lupus. Further studies to confirm the duration of immune tolerance obtained with anti-BLyS therapy in animal models may allow for translation of this strategy to provide long-lasting FVIII tolerance to patients with high risk of inhibitor development.
Doshi:Bayer Hemophilia Awards Program: Research Funding.
Asterisk with author names denotes non-ASH members.