T Cells Engineered to Display on their Surface a Novel Form of Fasl Fail to Induce GVHD Following Transplantation Into Irradiated Allogeneic Hosts (2012)

Introduction: Allogeneic bone marrow transplantation (BMT) has the potential to cure a series of inherited and acquired hematological disorders. The routine application of allogeneic BMT as a therapeutic intervention in the clinic, however, is complicated by GVHD with potential life-threatening complications. T cells specific for alloantigens are the primary culprit of GVHD. Although elimination of T cells from the donor BM inoculum is effective in curtailing GVHD, it results in compromised engraftment. Thus, strategies targeting specific and effective elimination of only the pathogenic T cells may have important implications for routine application of BMT to the clinic. The main objective of this study was to use a novel and practical approach, designated as ProtEx™, to engineer T cells and BMCs to display on their surface a modified form of FasL (SA-FasL) protein with potent apoptotic activity and test the capacity of SA-FasL-engineered cells to engraft in allogeneic recipients without complications of GVHD. We hypothesized that mature T cells in the BM inoculum displaying SA-FasL will respond to the host alloantigens, upregulate the death receptor Fas, and undergo apoptosis following the engagement of SA-FasL with Fas on the same or a different cell, resulting in the prevention of GVHD. Methods: ProtEx™ technology involves generation of chimeric molecules with a core streptavidin (SA), modification of cell membrane with biotin, and the display of chimeric molecules on the cell surface taking advantage of strong interaction between biotin and SA. C57BL/6 T cells and BMCs were biotinylated (5 μM) and engineered with SA-FasL protein (˜100 ng/106 cells). SA-FasL-engineered C57BL/6 T cells were first used in in vitro mixed lymphocyte reactions against irradiated BALB/c stimulators to assess their apoptosis. SA-FasL-engineered BMCs and T cells were then cotransplanted into lethally (1000 cGy) irradiated F1 (C57BL/6×Balb/c) mice for the prevention of GVHD. Controls received cells without engineering or engineered with streptavidin as a control protein. Animals were monitored for GVHD. Results: C57BL/6 T cells engineered to display SA-FasL on their surface underwent apoptosis in response to BALB/c stimulators in vitro. Transplantation of unmodified 10×106 BMCs comixed with 20×106 splenocytes of C57BL/6 mice into lethally irradiated F1 recipients (n=12) resulted in lethal GVHD within 34 days. In marked contrast, transplantation of cells engineered to display SA-FasL on the surface of splenocytes only (n=9) or splenocytes and BMCs (n=9) effectively prevented acute GVHD in all recipients over the 80-day observation period. Conclusions: T cells engineered with SA-FasL undergo apoptosis in response to alloantigens in the recipients, resulting in prevention of GVHD. The practical aspect of this protein display technology combined with its efficacy in preventing GVHD in rodents justifies its further development and testing its efficacy in the prevention of GVHD in the clinic. Funded in parts by NIH (R21 DK61333, R01 AI47864, R21 AI057903, R21 HL080108), and ADA (1-05-JF-56).