Neutralizing antibody formation against transgene products can represent a major complication following gene therapy with treatment of
genetic diseases, such as
hemophilia A. Although successful approaches have been developed to prevent the formation of anti-
factor VIII (FVIII)
antibodies, innovative strategies to overcome pre-existing anti-FVIII immune responses in FVIII-primed subjects are still lacking. Anti-FVIII
neutralizing antibodies circulate for long periods in part due to persistence of memory B-cells. Anti-CD20 targets a variety of B-cells (pre-B-cells to mature/memory cells); therefore, we investigated the impact of B-cell depletion on anti-FVIII immune responses in
hemophilia A mice using anti-CD20 combined with regulatory T (Treg) cell expansion using IL-2/IL-2mAb complexes plus
rapamycin. We found that anti-CD20 alone can partially modulate anti-FVIII immune responses in both unprimed and FVIII-primed
hemophilia A mice. Moreover, in mice treated with anti-CD20+IL-2/IL-2mAb complexes+
rapamycin+FVIII, anti-FVIII antibody titers were significantly reduced in comparison to mice treated with regimens targeting only B or T cells. In addition, titers remained low after a second challenge with FVIII plasmid. Treg cells and activation markers were transiently and significantly increased in the groups treated with IL-2/IL-2mAb complexes; however, significant B-cell depletion was obtained in anti-CD20-treated groups. Importantly, both FVIII-specific antibody-secreting cells and memory B-cells were significantly reduced in mice treated with combination
therapy. This study demonstrates that a combination regimen is highly promising as a treatment option for modulating anti-FVIII
antibodies and facilitating induction of long-term tolerance to FVIII in
hemophilia A mice.