PB0147 - FVIII/anti-FVIII immune complexes prevent FVIII-specific activation of B cells in a hemophilia A mouse model

Background: Haemophilia A is an X-linked bleeding disorder characterized by a deficiency of factor VIII (FVIII). Patients may develop anti-FVIII antibodies, rendering FVIII therapy ineffective. Antibody production can be suppressed by targeting CD32 receptors in B cells using immune complexes. Binding FVIII/anti-FVIII immune complexes (FVIII-ICs) to FcγRIIB leads to phosphorylation of the immune receptor tyrosine-based inhibition motif (ITIM) tail of FcγRIIB and send inhibitory signals to B cells, thereby inhibiting antibody production.

Aims: To evaluate the effect of FVIII-ICs administration on the suppression of antibody production by B cells in a mouse model.

Methods: FVIII antibodies were produced by immunization of FVIII KO mice with rFVIII and isolation of plasma containing anti-FVIII antibodies. FVIII-ICs were prepared by incubation of the plasma antibody pool with rFVIII at 1:1 and 1:10 ratios. FVIII KO mice were then injected retro-orbitally with FVIII-ICs, PBS, or rFVIII diluted in naïve plasma once weekly for four weeks (tolerization phase). Next, FVIII was administered retro-orbitally, at a dose of 25 IU/kg once weekly for 4 weeks. Finally, blood samples were collected from all mice and the concentration of anti-FVIII antibodies was determined by ELISA. (Figure 1)

Results: Mice that received the combination of FVIII and naive plasma developed anti-FVIII antibodies (mean increase 6.1 µg/mL, p-value < 0.0001). In contrast, 75% of mice that received 1:1 FVIII-ICs did not develop antibodies (mean increase 1.3 µg/mL, p-value < 0.0723), while 50% of the mice that received 1:10 FVIII-ICs did not develop antibodies (mean increase 2.3 µg/mL, p-value < 0.1030). (Figure 2)

Conclusion(s): In this study we demonstrated the suppression of antibody production via a 1:1 balanced ratio of FVIII-ICs injection. Therefore, antibody feedback may play a role in the immune response to FVIII by suppression of FVIII-specific B cells. Future work will investigate the mechanism of this suppression, particularly the potential role of CD32.