Tolerance Induction to B Domain-Deleted and Full-Length F.VIII by Co-Administration with Rapamycin
Authors: Moghimi Babak , Sack Brandon , Cao Ou , Herzog Roland W 
Published: October 2019

Abstract
Abstract 21
Hemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII. Standard treatment is periodical IV administration of recombinant F.VIII to maintain therapeutic levels in moderate and severe patients. Treatment is complicated by the formation of inhibitory antibodies (inhibitors), which occurs in ∼25% of patients and diminishes responsiveness to factor concentrates. Long-term management of patients with inhibitors requires immune tolerance induction (ITI) via the administration of repetitive high doses of F.VIII with or without immunosuppressive therapy. Appropriate immunosuppressive regimen have been challenging step in designing tolerance protocols, and no prophylactic protocols are available. We propose the use of rapamycin, which inhibits the mTOR pathway of T cell activation, resulting in suppression of T cell proliferation and programmed cell death. In addition, antigen presentation in the presence of rapamycin expands CD4+CD25+ regulatory T cells (Treg). We tested this approach in hemophilia A mice. Initially, we carried out a regimen of 3 IP injections per week for 4 weeks of antigen/rapamycin/IL-10 cocktail using recombinant B-domain deleted, BDD, F.VIII protein (1 IU). Subsequently, weekly IV injections of 1U BDD F.VIII were performed, which mimics human treatment and results in ∼70% correction of coagulation. However, after 4 weeks, the mice had formed high-titer inhibitors (39-58 BU) despite immune suppression. Therefore, the experiment was repeated in a modified form. Hemophilia A mice (n=5) were fed rapamycin at 2 mg/kg for 4 weeks (5 feedings per week). During the last 2 weeks, 0.3 IU of BDD F.VIII was IV twice a week. No antibody against F.VIII had formed at that time. Finally, the animals received weekly IV injections of 1U BDD F.VIII for 4 weeks. The mice were left untreated for 6 weeks, whereupon they were challenged once more with 1U of FVIII. At that time, flow cytometric analysis of peripheral blood cells showed normal B and T cell numbers. Mice failed to form anti-F.VIII IgG or inhibitors during any of these time points, including after the last challenge. Control mice that had been treated with F.VIII but had not received immune modulation formed on average 5mg anti-F.VIII IgG after 4 weeks of treatment with an average inhibitor titer of 86 BU (ranging from 37–133 BU). Mice that received rapamycin without F.VIII still formed high-titer inhibitors after subsequent challenge, thus excluding the possibility of non-specific immune suppression. We were similarly successful in tolerance induction for full-length F.VIII (Kogenate). To show reconstitution of immune function treated mice with our tolerance protocol received subcutaneous injection of Adenovirus expressing LacZ. We observed no significant difference in neutralizing antibody titers against adenovirus in control and treated mice (titer range 1/32-1/128), confirming immune function was restored during challenge process. Splenic cells isolated from tolerized mice were stimulated in vitro with FVIII and analyzed for cytokine expression by quantitative RT-PCR. Control mice with inhibitors showed IL-2, IL-4 and IL-6 responses. These were completely (IL-2, IL-4) or partially (IL-6) prevented in tolerized animals, which instead had increased CD25, Foxp3 and CTLA4 expression, demonstrating a prevention of Th2 in favor of Treg responses by our protocol. We propose that this protocol could be useful for prophylactic tolerance induction in patients at high risk for inhibitor formation, in patients requiring intensive factor treatment for surgery, and potentially for treatment of acquired/autoimmune hemophilia A.

Disclosures:
No relevant conflicts of interest to declare.