The role of FVIII/Anti-FVIII antibody Immune Complexes in Preventing FVIII-Specific Antibody Production in a Hemophilia A Mouse Model

Abstract

Abstract Background: Hemophilia A is an X-linked bleeding disorder characterized by a deficiency or absence of FVIII. Patients can develop anti-FVIII IgG, rendering FVIII therapy ineffective. Antibody production can be suppressed by targeting CD32b receptors in B cells using ICs. Binding rhFVIII/anti-FVIII immune complexes (rhFVIII-ICs) to FcγRIIB (CD32b) leads to phosphorylation of the immune receptor tyrosine-based inhibition motif (ITIM) tail of CD32b, resulting in the transduction of inhibitory signals to B cells, thereby inhibiting antibody production. Aim: To evaluate the effect of rhFVIII-ICs administration on the suppression of antibody production by B cells in a mouse model. Methods: A high-affinity anti-FVIII IgG pool was obtained by immunizing of FVIII KO mice with rhFVIII (Kovaltry). After immunization, blood was collected from immunized mice, and plasma was obtained. rhFVIII-ICs were prepared by incubating the mice plasma or anti-FVIII monoclonal antibody pool with Kovaltry at 1:1 and 1:10 ratios. FVIII KO mice were injected retro-orbitally with rhFVIII-ICs, PBS, or Kovaltry diluted in naïve plasma once weekly for four weeks (tolerization phase). Next, the mice were administered with FVIII retro-orbitally at a dose of 25 IU/kg once weekly for four weeks. Finally, blood samples were collected from all mice, and ELISA determined the concentration of anti-FVIII IgG. Results: Mice that received the combination of FVIII and naive plasma developed anti-FVIII IgG (mean increase 6.1 μg/mL, p-value = 0.0001). In contrast, 69% of mice that received 1:1 rhFVIII-ICs did not develop antibodies (mean increase 1.3 μg/mL, p-value = 0.0723), while 50% of the mice that received 1:10 rhFVIII-ICs did not develop antibodies (mean increase 2.3 μg/mL, p-value = 0.1030). Conclusion: In this study, we demonstrated that a 1:1 balanced ratio of rhFVIII-ICs injection suppresses antibody production. Thus, antibody feedback may play a role in the immune response to FVIII by suppressing FVIII-specific B cells. Future work will investigate the mechanism of this suppression, particularly the potential involvement of CD32b.