434. Adoptive Treg Cell Therapy Using Factor VIII-Specific CAR Regulatory T Cells Regulates Anti-Factor VIII Immune Responses in Hemophilia A Mice
Authors: Richard Y. Fu, Meghan J. Lyle, Xuefeng Wang, Carol H. Miao
Journal: Molecular Therapy (2016)

The immune response to factor VIII (FVIII; F8 in constructs) protein limits the effectiveness of treatments for hemophilia A (HemA) patients. Our previous studies demonstrated that regulatory T cells (Tregs) play a pivotal role in modulation of anti-FVIII immune responses. In particular, adoptive transfer of Tregs isolated from FVIII-primed HemA/Foxp3 mice attenuated anti-FVIII immune responses induced by gene transfer of FVIII plasmid in HemA mice. For developing adoptive Treg therapy, we successfully expanded FVIII-sensitized polyclonal Tregs using an FVIII-specific expansion protocol in vitro and showed that these cells had increased FVIII-specific suppressive activity compared with nonspecific Tregs. However, FVIII-specific Tregs in the polyclonal population are still in very small numbers. In this study, we explored the strategy to generate FVIII-specific Tregs using the chimeric antigen receptor (CAR) approach. Lentiviral vector (LV) incorporating a high-binding anti-FVIII antibody-derived variable region (scFv) linked to signaling and costimulatory moieties of immune receptors (third generation CAR) and fused with a murine Foxp3 cDNA (F8CAR-Foxp3-LV) was prepared and used to transduce murine CD4+T cells. Flow cytometry analysis confirmed extracellular scFv and intracellular Foxp3 expression in transduced cells (F8CAR-Tregs). In vitro suppressive assay showed that transduced CD4+T cells had significantly higher FVIII-specific suppressive activity than untransduced cells towards FVIII-specific CD4+ effector T cells (Teffs). In addition, 1×106 transduced cells and untransduced cells were adoptively transferred into HemA mice. One day after cell transfer, the treated mice were challenged with FVIII plasmid injected hydrodynamically. The anti-FVIII antibody titers are evaluated overtime. It is expected that F8CAR-Foxp3-LV transduced cells will prevent or decrease the production of anti-FVIII antibodies. We have also prepared a LV incorporating only the F8CAR region (F8CAR-LV). CD4+CD25− and CD4+CD25+ cells isolated from HemA mice were transduced with F8CAR-LV to generate FVIII-specific Teffs and Tregs, respectively. In vitro FVIII-specific suppressive assays using CD4+ Teffs from FVIII-primed HemA mice or F8CAR-LV transduced Teffs as responder cells are performed to compare the suppressive function of F8CAR-LV transduced CD4+CD25+ cells and F8CAR-Foxp3-LV transduced CD4+ cells. CD4+CD25+ cells isolated from FVIII-primed HemA mice and untransduced CD4+CD25+ and CD4+ cells from HemA mice are used as control cells. These experiments compare the extent of suppression towards specific F8CAR Teffs and polyclonal FVIII-specific Teffs as well as the potency of suppressive function between two differently engineered F8CAR Tregs. Finally, adoptive transfer experiments into HemA mice using the transduced and control cell populations are performed to evaluate their in vivo function to protect the HemA mice from anti-FVIII antibody production. We anticipate that compared with nonspecific and polyclonally expanded Tregs, FVIII-specific CAR Tregs will exert superior suppressive activity towards anti-FVIII immune responses without triggering systemic immune suppression.