We have reported earlier that the non-viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long-term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft-versus-leukemia effector cells, an SB transposon was constructed to coexpress a single-chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti-CD3/CD28 beads. Stable dual-gene expression was confirmed in both T-cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4+ T cells and CD8+ T cells both exhibited specific cytotoxicity against CD19+ leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high-levels of antigen-dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non-viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma.
Bibliographical noteFunding Information:
We thank Luigi Naldini and Mario Amendola (San Raffaele Telethon Institute for Gene Therapy, Milan, Italy) for providing us with the bidirectional PGK/mCMV promoter, Daniel Vallera (University of Minnesota, Minneapolis, MN) for the anti‐CD19 antibodies, Pablo Rubinstein (New York Blood Center, New York, NY) for cord blood units, Arlys Clements (University of Minnesota, Minneapolis, MN) for secretarial assistance, and Cindy Eide (University of Minnesota, Minneapolis, MN) or editing the paper. This work was supported by grants from the Alliance for Cancer Gene Therapy Young Investigator Award, the G & P Foundation for Cancer Research, the National Blood Foundation, the Sidney Kimmel Foundation for Cancer Research Kimmel Scholar Program and, in part, by the Children's Cancer Research Fund in Minneapolis and the University of Minnesota Medical School Dean's Commitment (X.Z.). X.Z. is a recipient of an American Society of Hematology Junior Faculty Scholar Award. This investigation was conducted in a facility constructed with support from a Research Facilities Improvement Program Grant (CO6 CA062526‐01) from the National Center for Research Resources, National Institutes of Health (NIH) (X.Z.) and NIH R01 CA72669 (B.R.B.). C.H.J. is an inventor of a reagent (CD3/CD28 beads) used in the manufacturing of SB‐engineered T cells, and therefore may potentially receive royalties from their commercial use. R.S.M. has a financial interest in Discovery Genomics. The other authors declare no competitive financial interests. This work was presented in abstract no. 722 at the 48th annual meeting of the American Society of Hematology, Orlando, Florida, 9 December 2006, and abstract no. 380 at the Keystone Symposium on the Potent New Anti‐Tumor Immunotherapies, Banff, Canada, 28 March 2007.