Optimizing the graft versus leukemia effect for pediatric ALL

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Under the first aim of this project we have utilized a precursor B cell leukemia derived from mice that express an E2aPBX1 transgene. E2aPBX1 leukemia cells from a single mouse were initially passaged in vivo and subsequently conditioned to grow in vitro. Injection of these cells intravenously reproducibly results in leukemia development from as few as 10,000 cells with distribution to bone marrow, blood, lymph nodes, spleen, liver and central nervous system. A subline has been transfected with luciferase and leukemia development can be tracked in situ using imaging. The cell line was confirmed to be immunogeneic as vaccination with irradiated leukemia cells protects against subsequent challenge with E2aPBX1 but not other tumors. Using monoclonal antibodies to deplete cell subsets, we demonstrated that protection in immunized mice requires both CD4 and CD8 T cells and is impaired following NK cell depletion. Under aim 2 we next performed syngeneic bone marrow transplantation experiments in which mice were injected with E2aPBX1 one week following transplant. This was followed by adoptive transfer of T cells from donors primed with an irradiated E2aPBX1 vaccine. While leukemia development was delayed compared to BMT recipients receiving purified T cells from unprimed mice, all recipients eventually succumbed to leukemia. Together, this data indicates that whole-cell vaccination induces a tumor-specific, T cell mediated immune response that is unable to prevent the develop of leukemia following syngeneic BMT. Importantly, these experiments also demonstrate that ALL can be targeted by T cells but requires prior priming to leukemia antigens The use of allogeneic bone marrow rather than syngeneic bone marrow introduces minor histocomaptibility antigens potential targets on leukemia cells. Indeed, allogeneic transplantation followed by E2aPBX1 leukemia challenge and subsequent transfer of primed allogeneic T cells resulted in cure of leukemia in all mice but weight loss and histologic changes consistent with moderate GVHD. Interestingly, priming T cell donors with recipient (and leukemia) strain non-malignant B cells did not cure the mice indicating that both minor antigens and leukemia-associated antigens are responsible for cure in this model. We next sought to separate the anti-leukemic GVL effect from GVHD by selecting for T cells subsets. Neither CD4 or CD8 T cells from primed donors alone were sufficient to cure all of the mice. However, selection for CD62L (l-selectin) positive T cells (consistent with a central memory subtype in primed donors) was capable of mediating a selective, curative GVL effect without GVHD. Aim 3 is ongoing and involves identification of leukemia-associated targets. we have established that E2aPBX1 overexpresses the Wilm's Tumor 1 gene, also overexpressed on approximately 70-80% of human leukemias and validated as potential target in patients. In order to obtain large numbers of WT-1 specific T cells, we are in the process of generating mice that express a T cell receptor specific for the dominant epitope derived from WT-1. The T cell receptor has been cloned and multiple founders have been generated that express large numbers of WT-1 specific T cells. Once the murine lines have been expanded, T cells from these mice will be tested in the E2aPBX1 model. If, successful, subsequent experiments following allogeneic transplantion will help validate and characterize the use of leukemia directed T cells as a modality to treat or prevent relapse.