B-Cell Depletion Using IgG1 Anti-CD20 Spares MZ B Cells and Promotes Tolerance to Human FVIII In a Murine Model of Hemophilia A

Abstract 2206

Preventing and reversing inhibitor formation remains one of the major challenges for hemophilia A therapy. Anti-CD20 mAb (Rituximab) has been reported to be beneficial for hemophilia A patients who failed immune tolerance induction (ITI). However, the evaluation of anti-CD20 therapy often is complicated in the clinical setting by concomitant use of other immune modulating drugs, such as hydrocortisone and IVIG. In this study, we tested the effect of B-cell depletion per se on tolerance induction to FVIII in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential effects were used. We previously showed that IgG1 anti-CD20 selectively depleted follicular (FO) B cells and spared marginal zone (MZ) B cells, while IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice (inhibitor titer = 30.7 ± 4.8 BU/ml), a single dose of IgG1 anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice given daily, high dose FVIII i.v. injection as a model for ITI. Surprisingly, only a marginal effect was achieved when we repeated the same protocol using IgG2a anti-CD20 for B-cell depletion, which efficiently depletes both FO and MZ B cells. To examine tolerance to FVIII, we re-challenged the treated mice with 2 μg FVIII intraperitoneally three months after the initiation of B cell depletion using IgG1 anti-CD20 when the number of peripheral B cells had recovered 60 % or more. The inhibitor titers remained significantly lower in the IgG1 anti-CD20 group after this FVIII boost injection (60.9 ± 33.2 versus 190.3 ± 33.5 BU/ml in control IgG1 group; p = 0.02). Importantly, after the mice were subcutaneously challenged with an unrelated antigen, OVA in CFA, there was no significant difference in anti-OVA IgG titers between the two groups. Taken together, these results suggested that selectively depletion of FO B cells by IgG1 subtype anti-CD20 mAb treatment may facilitate the tolerance induction to FVIII. (Supported by NIH R01 HL061883 and a fellowship from the American Heart Association)