225. Activation Signals from CD19-CAR Permit NFAT-Controlled Inducible Expression of Transgenes in PBMCs

Abstract

Adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) is a promising cell-based anticancer therapy. Although clinical studies of this approach show therapeutic efficacy, additional genetic modification is necessary to enhance the efficacy and safety of CAR-T cells. For example, producing an antitumor cytokine from CAR-T cells can enhance their tumor-killing activity, but there are concerns that constitutive expression of anticancer molecules will cause systemic side effects. Therefore, it is important that exogenous gene expression is confined to the tumor locality. Such an approach may lead to therapeutic strategies that are safe and effective. In this study, we aimed to develop a switch promoter driven by activation signals from a CAR. We prepared a switch cassette that was arranged in the order of two modified SV40 early polyA sequences as a background reduction signal, four NFAT-responsive elements, a minimal interleukin-2 (IL-2) promoter, a reporter gene, and a BGH polyA sequence. Transgene expression in peripheral blood mononuclear cells (PBMCs) transduced with the CD19-targeted CAR and switch cassette (PBMCs/CAR/iReporter) was only induced strongly by coculture with CD19-positive target cells. Furthermore, after antigen stimulation, PBMCs/CAR/iReporter produced approximately the same amounts of IL-2 and interferon-γ as PBMCs expressing the CAR only. The cells also showed redirected cytolysis toward CD19-positive, but not CD19-negative, tumor cells. These results indicated that the switch promoter was selectively driven by activation signals from the CAR. Furthermore, transduction with the switch cassette did not affect the original effector activity including IL-2 and IFN-γ production and antitumor activity of CAR-redirected cytotoxic T lymphocytes. In summary, we developed a retroviral vector that incorporates a CAR-derived, activation signal-dependent promoter to drive exogenous gene expression. This switch cassette permits visualization and quantification of the activation status in CAR-expressing PBMCs.