Abstract A037: A novel pharmacologic “ON/OFF” switch to modulate CAR-T-cell function in vitro and in vivo
Authors: Mestermann Katrin , Julian Rydzek , Silke Frenz , Hermann Einsele , Hudecek Michael 
Published: February 2019

Abstract
Background: Immunotherapy with CAR-T-cells (CAR-T) is a powerful novel treatment for hematologic malignancies, but also bound with significant acute and chronic side effects, including potentially life-threatening cytokine release syndrome (CRS) and on-target recognition of normal cells expressing the targeted antigen. This toxicity limits clinical utility and is at least in part caused by the inability to effectively control CAR-T function following infusion. Here, we present a novel strategy of pharmacologic “ON/OFF” switch to precisely control CAR-T function in real-time, which we demonstrate to modulate T-cell activity in vitro and in vivo. Methods: We considered that an effective way for controlling CAR-T function was to interfere with signal transduction though the CAR. We assembled a library of clinically approved drug compounds and screened for their ability to reversible block CAR-T function without affecting CAR-T viability. We performed functional testing with CD8+ and CD4+ CAR-T (n=3 donors) in the presence of titrated doses of the lead compound, and employed CD19- and ROR1-specific CARs comprising 4-1BB or CD28 costimulatory moieties. Results: We identified a lead compound, TCI-1, that stood out through its ability to confer a dose-dependent (partial at lower, complete at higher doses) blockade of all CAR-T effector functions, i.e., cytolytic activity, cytokine secretion and proliferation. We confirmed TCI-1 was effective in both CD8+ and CD4+ T-cells, and in each of the 3 CAR constructs. The onset of CAR-T blockade was immediate after exposure to TCI-1 and was caused by interference with early phosphorylation events in the CAR signaling cascade as demonstrated by Western blot, and interference with the induction of transcription factors, as demonstrated with an NFAT-inducible reporter gene. Intriguingly, blockade of CAR-T function was effective for several days if exposure to TCI-1 was sustained and instantaneously and fully reversible after removal of the compound. Short- and long-term exposure to TCI-1 did not induce a reduction of CAR-T viability, and did not hinder the subsequent ability of CAR-T to exert their functions. We considered that in patients with CRS, CAR-T are in an activated state, and performed comprehensive testing to show that TCI-1 was able to arrest CAR-T that are in the process of executing their effector functions. In addition, we employed a xenograft model in immunodeficient mice (NSG/Raji) to determine whether TCI-1 was capable of controlling the function of CD19 CAR-T-cells in vivo. Indeed, we demonstrate that administration of TCI-1 conferred a functional arrest of CAR-T, and that CAR-T resumed their antitumor function once administration of TCI-1 was discontinued. Conclusions: Our data show that TCI-1 is capable to exert real-time, on/off control over CAR-T function, suggesting the potential to prevent or mitigate side effects of CAR-T therapy in a clinical setting. The reversible complete inhibition of CAR-T function through TCI-1 without compromise to CAR-T viability surpasses the qualities of steroids that are toxic to T-cells and provide only incomplete functional control, and complements suicide-gene strategies that effectively control chronic side effects but also abrogate the antitumor effect of CAR-T.
Citation Format: Katrin Mestermann, Rydzek Julian, Frenz Silke, Einsele Hermann, Michael Hudecek. A novel pharmacologic “ON/OFF” switch to modulate CAR-T-cell function in vitro and in vivo [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A037.