Development of Inducible CD19-CAR T Cells with a Tet-On System for Controlled Activity and Enhanced Clinical Safety

Abstract

The tetracycline regulatory system has been widely used to control the transgene expression. With this powerful tool, it might be possible to effectively control the functional activity of chimeric antigen receptor (CAR) T cells and manage the severe side effects after infusion. In this study, we developed novel inducible CD19CAR (iCAR19) T cells by incorporating a one-vector Tet-on system into the CD19CAR construct. The iCAR19 T cells showed dox-dependent cell proliferation, cytokine production, CAR expression, and strong CD19-specific cytotoxicity. After 48 h of dox induction, the relative CAR expression of induced cells was five times greater than that of uninduced cells. Twenty-four hours after dox removal, CAR expression significantly decreased by more than 60%. In cytotoxicity assays, dox-treated cells induced significantly higher specific lysis against target cells. These results suggested that the activity of iCAR19 T cells was successfully controlled by our Tet-on system, offering an enhanced safety profile while maintaining a robust anti-tumor effect. Besides, all manufacture processes of the lentiviral vectors and the T cells were conducted according to the Good Manufacturing Practice (GMP) standards for subsequent clinical translation.

Bispecific T-Cell Redirection versus Chimeric Antigen Receptor (CAR)-T Cells as Approaches to Kill Cancer Cells

“…The CAR, however, is not fully activated until binding of the targeting scFv to the tumor cell surface antigen triggers CD3ε signaling as well [345]. Similarly, there are multiple versions of a tetracycline (doxycycline)-inducible (tet-inducible) CAR, in which CAR expression is completely dependent on the presence of the tetracycline [346,347]. The tet-inducible system, however, appears to be leaky with a significant background expression level in the absence of the inducer [347].…”

Emerging Approaches for Regulation and Control of CAR T Cells: A Mini Review

“…It has also been suggested to regulate CARs at the transcriptional level. Several groups have shown that a drug-inducible CAR system can be generated by controlling CAR transcription using the TET-on system, allowing reversible control of CAR T cells (76)(77)(78). CAR mRNA is thus only produced in the presence of doxycycline, although some background CAR expression was observed (76).…”

Cancer Vaccines and Oncolytic Viruses Exert Profoundly Lower Side Effects in Cancer Patients than Other Systemic Therapies: A Comparative Analysis

“…CAR T-cell technologies, although having considerable therapeutic potential, are also faced with severe toxicity problems (irAEs of 3-4). To reduce these, new strategies aim at introducing inducible gene switches [108,109].…”