Adoptive transfer of donor derived peripheral blood mononuclear cells (PBMC) or virus-specific immune T lymphocytes can induce durable regressions of monoclonal EBV-induced lymphomas developing in marrow allograft recipients. Infusions of donor-derived PBMC have also induced durable clinical and molecular remissions in 70-80% of patients relapsing with chronic myelogenous leukemia after an allogeneic marrow graft. However, treatment with donor PBMC, particularly in recipients of matched unrelated or HLA-non-identical related donors, may induce severe or lethal GvHD. Genetic modification of donor T-cells to express a gene inducing sensitivity to drugs to which human cells are normally resistant could permit the safe use of PBMC or in vitro expanded virus- specific T-cells even when such populations may still contain significant numbers of alloresponse T-cells. Alloreactive T-cells modified in this way might also be used to induce a controlled and reversible graft vs. host response to eradicate residual host leukemic cells. In this project, we propose to examine the biology and function of EBV-specific and allospecific T-cells genetically modified with a series of dicistronic vectors encoding a cell surface expressed, mutant nerve growth factor receptor (NTP) and either HSV thymidine kinase or cytosine deaminase. Expression of HSV-TK or cytosine deaminase renders cells sensitive to ganciclovir or 5-fluorocytosine, respectively. Inclusion of NTP in the vector permits rapid and efficient immunoaffinity-based detection and isolation and selection of transfected cells expressing this marker both in vitro and in vivo. We propose to evaluate the extent of selection and function of transfected effector cells and their sensitivity to ganciclovir or 5-fluorocytosine in vitro, using immunocytofluorometric analyses and limited dilution methodologies to quantitative EBV-specific and allospecific cytotoxic and IL-2 secreting T-cells. The growth, level of sustained gene expression and function of transfected human T-cells will be assessed in vivo in a murine model of GvHD and in a preclinical model in SCID mice which permits analysis of the capacity of EBV- specific or allospecific T-cells to home to and induce regression of human EBV +lymphomas and primary human leukemia xenografts. By treating animals with ganciclovir or 5-FC at intervals following adoptive transfer of transvected cells, assessments will also be made of the reversibility of antiviral or anti-tumor responses and GvHD, and of the relative contributions of virus or alloantigen specific T-cells vs. other adaptively transferred cell populations to the initiation and the maintenance of durable anti-tumor reactions and GvHD.
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