Adoptive transfer of donor-derived peripheral blood mononuclear cells (PBMC) can induce durable regressions of monoclonal EBV- induced lymphomas and relapses of CML or AML in marrow allograft recipients. However, treatment with donor PBMC, particularly in matched unrelated or HLC-nonidentical-related hosts, may induce severe or lethal GvHD. Genetic modification of donor T cells to express a gene inducing sensitivity to a drug to which human cells are normally resistant could permit the safe use of donor- derived T cells early after activation or sensitization in vitro when they may still contain alloresponsive T cells. We propose to examine human T cells genetically modified early after activation with lL2 or sensitization to EBV or allogeneic cells. T cells will be transvected with a series of dicistronic vectors encoding a cell surface expressed, mutant nerve growth factor receptor (NTP) and either HSV thymidine kinase (HSV-TK) or cytosine deaminase (CDA). Expression of HSV-TK or CDA renders cells sensitive to granciclovir (GCV) or 5-fluorocytosine (5FC), respectively. Inclusion of NTP in the vector permits immunoaffinity-based detection, isolation and selection of transvected cells expressing this marker both in vitro and in vivo. We will evaluate expression of vector-encoded genes in transvected effector cells and correlate this with their sensitivity to GCV or 5FC over extended culture in vitro using immunocytofluorometric analyses, assays of enzyme protein and RNA and limiting dilution methodologies to qunatitate EBV-specific and allospecific cytotoxic cells before and after drug treatment. The patterns of EBV antigen specificity and HLA restriction exhibited by EBV-reactive T cells transvected early or late after sensitization to autologous EBVLCL will also be analyzed and compared with T cells generated in vivo in patients treated with donor leukocytes or EBV-specific T cell lines as described in Project VI. Transvected human EBV T cells and alloreactive T cells will also be evaluated in vivo in a SCID mouse model, which permits analysis of the capacity of EBV-specific or allospecific T cells to home to and induce regressions of human EBV lymphomas or fresh human ALL, AML or blastic CML xenografts. By treating animals with GCV or 5FC at intervals following adoptive transfer of transvected cells, assessments can be made of the duration of interactions between effector T cells and tangeted tumors required to induce or sustain regressions of disease. These studies are designed to provide pre-clinical in vitro and in vivo data to support future clinical trials evaluating such transvected donor T cells for the treatment of viral infections post-transplant and the use of allospecific T-lymphocytes for controlled GvH responses to eradicate recurrent leukemia or to foster engraftment of T cell depleted marrow transplants.
Showing the most recent 10 out of 452 publications
