Factor VIII BAR T-Cell Recognition By Antibodies to Different Domains

Up to 30% of patients with hemophilia A develop inhibitory antibodies to therapeutic FVIII and these antibodies block the efficacy of this critical protein. To target the B cells specific for FVIII, we previously generated cytotoxic cells expressing the FVIII C2 or A2 immunodominant domains as chimeric receptors. We termed these antigen-expressing engineered T cells, "BARs", for B-cell Antibody Receptor. These could directly interact and kill with A2 or C2 FVIII-specific B cells as well as FVIII-specific hybridomas in vitro and in vivo. To increase the number of B-cell targets that could be killed, we recently engineered human and murine BAR CD8 T cells to express the FVIII light chain (LC), which includes A3 and C1, as well as C2 domains. In addition, since C2-expressing BAR T cells showed tonic signaling, we hypothesized that LC expressing BARs might not. Our data suggest that LC-expressing BAR T cells do not display tonic signaling but they can be stimulated to proliferate by anti-LC monoclonal antibody (mAb 5G12), for example. Flow cytometry data showed that LC-BAR T cells are stained by anti-C2 mAb (3G6) but this mAb did not stimulate them to proliferate. These data reflect the accessibility of different FVIII domains in BAR T cells, and are important in terms of the utility of FVIII domain expressing BARs to target specific B cells in vivo in the presence of inhibitors. (Supported by NIH grant R01 HL126727)