Systematic Optimization and Modification on Human Primary T Cells

Abstract

Adoptive T cell transfer (ACT) has become a promising and integral therapeutic option for patients with leukemia and lymphoma. However, challenges remain in the application of this mode of therapy to other cancer types. One inherent obstacle is the limited replicative lifespan of T-cells with little progress in the development of durable engraftment of T-cells. Decades of work have been devoted to improvements in the genetic modification of prolonging T-cells lifespan yielding multiple technologies. Universal viral vectors (e.g. lentivirus) system are at a disadvantage for low capacity, lengthy manufacturing and low cost-performance for both research and clinical use. Attractive new transposons system (e.g. piggybac) provide better options but integration efficiency was limited and the therapeutic engineering for human primary T cells remains clouded. In this study, we are aiming to identify genetic modification that enhance function and survival of primary human T cells. In order to do this, we have established a systematic Leap-In/Neon engineering system. We were able to show that this system was able to efficiently and stably modify human T cells. We next generated a list of all recurrent mutations that have been associated with T-cell lymphoproliferative states. Finally, we introduced these genes into primary human T cells using the transposons and tested the function and characteristics of the integrated cells. Our system may thus be used to express multiple Integrated genes and/or combinations that will enhance the function and survival of primary human T cells. We showed the stable transduction of Jurkat and primary T-cells using the Leap-In/Neon engineering system with efficiencies of ~60% and ~20% respectively. We have identified 6 gene candidates that are able to confer proliferative and/or survival advantages to transfected cells in vitro. Moreover, we demonstrated that the single candidate gene STAT3 Y640F and the gene candidate combo of Fas-TM-41BB and Casp7-DN may exert a great pro-proliferation advatage on primary CD8+ T cell modification with maintained original cell characteristics and functions.