Abstract 1944: In vitro analysis of suicide gene expression and function in human T lymphocytes transduced to express a tumor targeted chimeric antigen receptor

Abstract

Abstract Adoptive infusion of T cells genetically modified to express chimeric antigen receptors (CARs) targeted to tumor associated antigens (TAAs) is a promising novel approach to cancer therapy. However, in light of the fact that most TAAs targeted by such modified T cells are expressed, at least to some degree, by normal tissues as well as the fact that clinical experience with this technology is limited, safeguards are needed in the form of additional expression of transduced suicide genes by these T cells to allow for the efficient in vivo abrogation of infused T cells in the setting of unanticipated adverse events which may develop in the context of planned clinical trials. Herein we report the in vitro function of 3 different suicide genes each initially inserted distal to a CAR gene and an IRES (internal ribosome entry site) element cloned into the SFG retroviral vector. Specifically, in this study we evaluated the herpes simplex virus thymidine kinase (HSV-TK SR39), E. coli derived nitroreductase (NTR), and inducible caspase-9 (iCasp-9). We initially found that all 3 suicide genes functioned sub-optimally in the second position of these IRES based bicistronic vectors. In order to enhance suicide gene expression we deleted the IRES elements replacing them with 2A linker peptides, which significantly improved gene expression and the cell growth inhibition upon the addition of respective substrate. In PG13 cells transduced with SFG 1928z. IRES. NTR we found 40% inhibition at 5mM of metronidazole (MTZ), but 90% in PG13 cells transduced with SFG 1928.2A.NTR. Similarly, PG13 (1928z.IRES.HSV-TK) fibroblasts exhibited 60% inhibition at 1µM of ganciclovir compared to 85% inhibition of PG13 (1928z.2A.HSV-TK) fibroblasts. Consistent with these findings, we found that human T cells transduced with either 1928z.2A.NTR or 1928z.2A.HSV-TK demonstrated 90% and 88% inhibition respectively at similar substrate concentrations. In light of this encouraging in vitro data, we are currently in the process of assessing respective suicide gene function in vivo using a syngeneic, immune-competent mouse model recently developed in our laboratory. Ultimately we anticipate that these studies will allow us to rationally select the optimal suicide gene-prodrug combination to be incorporated into future clinical trials utilizing gene modified tumor targeted T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1944.