Adoptive cell therapy with donor leukocytes can induce regressions of EBV-associated B-cell lymphomas in marrow transplant recipients. Similarly, donor leukocytes containing T cell reactive against alloantigens on host leukemic cells can induce remissions in patients with CML and certain forms of acute leukemia who relapse following marrow transplant. However, such infusions carry a significant risk of severe acute and chronic GvHD, particularly with leukocytes from an """"""""HLA-matched"""""""" unrelated or HLA-Disparate related marrow donors are used. Genetic modifications of donor T-cells to express a gene inducing a sensitivity to drugs to which human cells are normally resistant could permit the safe use of in vitro expanded virus-specific T-cells early in their generation, even if populations still contain alloreactive T-cells. Similarly, alloreactive """"""""suicide vector"""""""" modified T-cells could be used to eradicate leukemia through a transient reversible GvH reaction. Over the last grant period, we developed a series of dicistronic vectors encoded a mutated NGFR and HSV-TK, defined optimal orientation for expression of the genes and developed efficient techniques for transvection and selection of vector-modified human T-cells. We have shown that the NIT retroviral vector can be used to rapidly select and enrich for desired EBV-virus specific T-cells during their initial proliferation in response to sensitization and to select against contaminating alloreactive T-cells. These T-cells are HLA restricted, antigen-specific and highly cytotoxic against virus-transformed targets in vitro. Upon adoptive i.v. transfer, they specifically migrate to and induce regressions of EBV lymphomas bearing appropriate HLA restrict elements in xenografted SCID mice. Expression of NGFR and HSV-TK is sustained for over 16 weeks in vitro and in vivo. Treatment of mice with ganciclovir post adoptive transfer of human T-cells bearing HSV-TK selectively eliminates these T-cells from blood and targeted tissues. We propose to explore novel strategies, incorporating genetically modified antigen presenting cells to develop novel, broadly accessible approaches for rapid generation of virus- antigen-specific T-cells of desired specificity and HLA restriction (Specific Aim 1). Using similar strategies we will generate suicide- vector-modified T cells specific for minor alloantigens expressed on human ALL cells (Specific Aim 2). We will then comparatively evaluate virus-specific and minor alloantigen-specific, vector modified T-cells generated by these novel strategies, for their capacities to migrate to and induce regression of targeted human EBV lymphomas and leukemias xenografted in SCID mice (Specific Aim 3). Lastly, we propose to initiate clinical trials of vector-modified and selected virus-antigen-specific and leukemia-associated minor alloantigen specific T cells in the treatment of EBV lymphomas and leukemic relapses complicating marrow allografts (Specific Aim 4).
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