Functional Analysis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor

Ryosuke Uchibori,Takeshi Teruya,Hiroyuki Ido,Ken Ohmine,Yoshihide Sehara,Masashi Urabe,Hiroaki Mizukami,Junichi Mineno,Keiya Ozawa

doi:10.1016/j.omto.2018.11.003

Ryosuke Uchibori, Takeshi Teruya + Show 7 more

Open Access

https://doi.org/10.1016/j.omto.2018.11.003

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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, production of an antitumor cytokine from CAR-T cells can potentially 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. Here, we aimed to develop an inducible promoter driven by activation signals from a CAR. Transgene expression in T cells transduced with the CD19-targeted CAR and an inducible promoter, including inducible reporter genes (CAR-T/iReporter), was only induced strongly by co-culture with CD19-positive target cells. CAR-T/iReporter cells also showed redirected cytolysis toward CD19-positive, but not CD19-negative, tumor cells. Overall, our study indicated that the inducible promoter was selectively driven by activation signals from the CAR, and transduction with the inducible promoter did not affect original effector activities including interleukin-2 and interferon-γ production and the antitumor activity of CAR-redirected cytotoxic T lymphocytes. Moreover, this inducible promoter permits visualization and quantification of the activation status in CAR-T cells.

Highlights

Adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) is a promising cell-based anticancer therapy.[1,2,3,4,5] This approach involves both cellular and humoral immune responses by assembly of an antigen-binding moiety, most commonly a single chain variable fragment derived from a monoclonal antibody, together with an activating immune receptor, such as the intracellular domain from CD3z and/or CD28
Once the CAR is expressed at the surface of modified T cells and upon binding of the scFv to its antigen, an activation signal is transmitted into the T cell, which in turn triggers its effector functions against the target cell.[6,7,8]
CD19-CAR expression was observed in Jurkat-1928z cells, but not in Jurkat cells (Figure 1B)

Summary

Introduction

Adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) is a promising cell-based anticancer therapy.[1,2,3,4,5] This approach involves both cellular and humoral immune responses by assembly of an antigen-binding moiety, most commonly a single chain variable fragment (scFv) derived from a monoclonal antibody, together with an activating immune receptor, such as the intracellular domain from CD3z and/or CD28. Once the CAR is expressed at the surface of modified T cells and upon binding of the scFv to its antigen, an activation signal is transmitted into the T cell, which in turn triggers its effector functions against the target cell.[6,7,8] As a result, T cells are acti-. Oncolytics Vol 12 March 2019 a 2018 The Author(s). After 24 hr, ZsGreen[1] expression was monitored by flow cytometry. (E) Percentage of ZsGreen1-positive cells and (F) mean fluorescence intensity were calculated by flow cytometric analysis. *p < 0.05, yp < 0.01 Data are presented as means ± SEM

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