Sleeping Beauty (SB) Transposon Mediated Umbilical Cord Blood (UCB) T Cell Therapy for Refractory Acute Lymphoblastic Leukemia (ALL).

UCB is a promising alternate source of hematopoietic stem cell transplantation due to a readily available graft and low risk of GVHD. However, the incidence of ALL relapse in children is relatively high (40% for high risk ALL). To test whether UCB T cells can be genetically modified as GVL effector cells, the SB transposon system was used as a delivery vehicle since we have shown that this system can mediate genomic integration and long-term reporter gene expression in 5–20% of human primary T cells without prior activation, thus reducing duration of in vitro culture and enhancing T cell function (Huang et al., Blood. 2006, 107:483). A SB bidirectional transposon was constructed to co-express a single chain chimeric antigen receptor for CD19, commonly expressed in B-ALL, and human CD20, a marker for in vitro selection of transfected T cells and a “suicide” gene for in vivo elimination by Rituxan when necessary. In preclinical studies, UCB and peripheral blood (PB) mononuclear cells were nucleofected with the bidirectional SB transposon and a SB10 transposase-expressing plasmid, and then activated and expanded by anti-CD3/CD28 beads in culture. Flow cytometric analyses confirmed the stable dual gene expression in both transfected T cell types. After sorting for the dual gene expression, engineered T cells demonstrated specific cytoxicity against CD19⁺ leukemia and lymphoma cell lines but not CD19⁻ myeloid leukemia and multiple myeloma cells. Furthermore, SB engineered T cell killing was found to be CD19-specific as evidenced by killing K562 cells stably expressing CD19 but not K562 and K562 cells stably expressing eGFP. We also demonstrated that the unsorted PB T cells killed CD19⁺ target cells as effectively as the sorted PB T cells, suggesting that transfected T cells can be immediately infused into patients without selection and extended in vitro culture. While the mechanisms responsible for anti-leukemia cytolysis are under investigation, it is clear that both engineered CD4 and CD8 PB T cells and CD8 UCB T cells, but not engineered CD4 UCB T cells, killed CD19⁺ target cells. In vivo experiments are in progress to determine efficacy of CD19⁺ leukemia cell reduction by the engineered human T cells and the efficiency of Rituxan-mediated elimination of adoptively transferred T cells using a bioluminescent imaging technique. We conclude that the SB transposon-engineered UCB and PB T cells can stably express the therapeutic genes and mount potent anti-leukemia and lymphoma responses in vitro. Safety and therapeutic potential of engineered UCB T cells will be tested in the treatment of high risk CD19⁺ ALL as a strategy for reducing risk of relapse without the risk of increased acute GVHD. In addition, our approach is novel (transposon-based), simple (naked DNA), efficient (no prior T cell activation and less immunogenic), stable (integrating), and probably safe (random integration) compared to retroviral vectors and conventional plasmids.