In Vitro analysis of Suicide Gene Expression and Function of 3 Suicide Gene-Prodrug Combinations In Human T Lymphocytes Further Modified to Express the CD19 Targeted 19–28z Chimeric Antigen Receptor.
Authors: Park Jae H. , Yeh Raymond , Rivière Isabelle , Sadelain Michel , Brentjens Renier J. 
Published: October 2019

Abstract
Abstract 3771
Adoptive infusion of T cells genetically modified to express chimeric antigen receptors (CARs) targeted to tumor associated antigens (TAAs) is a promising approach to cancer therapy. However, since TAAs are often expressed by normal tissues, safeguards are needed in the form of additional transduced suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events which may develop in the clinical setting. To this end, we have investigated the in vitro function of 3 different suicide genes each inserted distal to a CAR gene targeted against CD19 (19-28z) and a 2A linker peptide cloned into the SFG gammaretroviral vector. Specifically, we have tested the herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and the E.coli derived nitroreductase (NTR) with the prodrug metronidazole. Cell growth of PG13 murine fibroblasts transduced to express 19–28z CAR with NTR, HSV-TK, and iCasp9 was inhibited by 80% at 1mM of metronidazole, 85% at 1μM of ganciclovir, and 90% at 10nM of CID, respectively, when compared to control PG-13 fibroblasts. The drug concentrations tested in these assays were at physiologically achievable concentrations in humans, and did not affect the growth rate of control PG13 fibroblasts. Consistent with these findings in PG13 fibroblasts, 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. Furthermore, we demonstrate that expression of these suicide genes does not affect the phenotype or function of the 19–28z CAR+ T cells, as assessed in vitro by T cell proliferation and cytotoxicity against CD19-expressing tumor cells. Our studies demonstrate highly effective suicide genes for human T lymphocytes transduced with a tumor targeted CAR, and a novel suicide gene/prodrug (NTR/metronidazole) combination with a comparable efficacy that can potentially serve as a reliable safety mechanism for adoptive T cell immunotherapy. While HSV-TK/ganciclovir has been utilized in various clinical settings, the NTR suicide gene has yet to be used in combination with gene modified tumor-targeted T cells. Furthermore, the NTR suicide gene holds several advantages over the HSV-TK and iCasp9 vectors. First, unlike HSV-TK, the NTR suicide gene is effective in both proliferating and non-proliferating cells. Second, unlike CID that is not commercially available, metronidazole is a widely available antibiotic that is relatively non-toxic. Lastly, metronidazole can be used in patients who may already be taking ganciclovir for cytomegalovirus (CMV) prophylaxis or treatment therefore limiting the application of T cells modified to express the HSV-TK suicide gene. Based on this in vitro data, we are currently testing the function of this suicide gene in vivo in two different animal models. Ultimately we anticipate that further studies with this novel suicide gene/prodrug combination will allow us to enhance safety in future clinical trials utilizing gene modified tumor targeted T cells.

Disclosures:
No relevant conflicts of interest to declare.