Survival gene transfer into leukemia specific donor lymphocytes for chemoprotection post allogeneic marrow transplantation

ALL is the least responsive of all leukemias to immunotherapy with DLI after allogeneic stem cell transplantation. The absence of the GVL effect is probably due to 1) poor immunogenicity of ALL cells and 2) a high proliferative capacity of the leukemia, resulting in the need for frequent chemotherapy to control the leukemic clone which has the deleterious effect of eradicating any expanding ALL-specific cytotoxic T-lymphocytes (CTLs). We have shown that the immunogenicity of ALL cells can be increased by upregulating essential (co)stimulatory molecules (B7.1&2, CD40, ICAM-1, LFA-3) using CD40 ligand. CD40 ligated ALL cells are highly efficient in inducing allogeneic T-cells to proliferate compared to untreated ALL cells. CD40ligated ALL cells can also be used to generate ALL-specific CTLs from the donor. The CTLs are protected from the toxic effects of methotrexate (MTX) or trimetrexate (TMTX) chemotherapy by introducing a drug resistant variant of dihydrofolate reductase (L22YDHFR). Gene modified ALL-specific CTLs will allow for the administration of antifolate chemotherapy which will control the leukemia and reduce the leukemia burden while the ALL-specific CTLs will be protected from the chemotherapy, expand, and eradicate the leukemia. Transduction efficiency of T-cells was measured by flow cytometry after gene modification with a replication incompetent lentivirus-based vector encoding Green Fluorescent Protein & L22YDHFR. After TMTX selection the transduction efficiency was 50% compared to 13% of T-cells not exposed to TMTX. We are currently investigating if L22YDHFR transduction significantly alters the function of antigen specific CTLs.