A Novel Approach to Prevent GvHD: Donor Cells Engineered to Display on Their Surface a Recombinant Form of FasL Protein Effectively Prevent Lethal GvHD in a Mouse Model

Abstract

Allogeneic bone marrow transplantation (BMT) has the potential to cure a series of inherited and acquired hematological disorders and malignancies. BMT can also be used as a cell-based immunomodulatory approach to induce tolerance to foreign and auto-antigens for the prevention and/or treatment of foreign graft rejection and autoimmune disorders. The routine application of allogeneic BMT as a therapeutic intervention in the clinic, however, is complicated by graft-versus-host (GVH) reaction, which is the major cause of graft-versus-host disease (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 bone marrow inoculum can curtail GVH reaction, 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 for the treatment of a variety of diseases. The main objective of this study was to use a novel and practical approach, designated as ProtEx™, to engineer bone marrow cells to display on their surface a modified form of FasL protein with potent apoptotic activity as an immunomodulatory agent and test the capacity of the engineered cells to engraft in allogeneic recipients without complications of GVHD. 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 noncovalent interaction (10−15 M) between biotin and SA. This technology allows for rapid (~ 2 hr) and efficient (100% of the targeted cells) display of exogenous proteins of interest on any cell without compromising the function of the cell or the proteins. In this study, ProtEx™ was used to display SA-FasL protein on C57BL/6 T cells and BMCs and these cells were tested for elimination of alloreactive T cells as well as prevention of acute GVHD following transplantation into lethally (1000 cGy) irradiated F1 (C57BL/6xBALB/c) mice. We hypothesized that mature T cells in the BM inoculum displaying FasL will respond to the host alloantigens, upregulate the death receptor Fas, and undergo apoptosis following the engagement of FasL with Fas on the same or a different cell, resulting in the prevention of GVHD. In support of this hypothesis, we demonstrated specific elimination of C57BL/6 T cells engineered to display SA-FasL on their surface in response to BALB/c antigen presenting cells in mixed lymphocyte cultures. 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 in all recipients within 34 days. In marked contrast, transplantation of cells engineered to display SA-FasL on splenocytes only (n=9) or splenocytes and BMCs (n=9) effectively prevented acute GVHD in all recipients that survived over 80 days of an observation period. The importance of FasL-mediated apoptosis in immune homeostasis and tolerance combined with our ability to use a practical approach, ProtEx™, to display SA-FasL with potent apoptotic activity on the surface of BMC or mature T cells at the protein level to prevent acute GVHD is significant and may have immediate clinical application.