BAR-CD8 T-Cell Mediated Targeted Killing of Inhibitor Producing FVIII-Specific B Cells

Anti-FVIII antibody (inhibitor) formation is a major complication in the treatment of Hemophilia A. Our lab has focused on developing novel approaches to modulate inhibitor formation by regulation of FVIII-specific T effectors that recognize FVIII and “help” B cells to produce antibodies to FVIII.  As an alternative approach, in this presentation, we directly target anti-FVIII B cells.  Therefore, we have developed a novel chimeric antigen-like receptor called B-cell antibody recognizing receptor (BAR) to target and directly kill the FVIII-specific B cells and antiobody-producing cells . Our BAR comprises of FVIII-domain C2 attached to T cell signaling CD28 and CD3ζ elements. We reasoned that CD8 T cells transduced to express the FVIII C2 should trigger cytotoxicity upon recognition by and interaction with specific B cells. We expressed the C2-BAR or Ova-BAR (control) in human T cells and demonstrated expression by flow cytometry.  These BAR CD8 T cells also proliferated upon addition of anti-C2 or anti-Ova, respectively post 72 hrs. For proof of principle that these BAR-expressing CD8 T cells can kill specific B cells, the following basic experiment was designed. Mouse CD8 T cells transduced to express C2-BAR were co-cultured with FVIII-specific hybridomas (expressing surface IgG anti-C2) at a various ratios. After 5 hours, we measured antibody secretion by ELIspot assay. Our results demonstrated suppression of antibody secretion by C2 BAR expressing CD8 T cells, but not by control Ova-expressing BAR CD8s.  These results provide evidence that BAR CD8 T cells expressing FVIII domains can be used to target specific B cells to suppress inhibitor formation, and may also provide a platform to treat other undesirable antibody responses. (Supported in part by a Pfizer ASPIRE award and NIH grant HL061883)