Abstract During the past years promising clinical outcomes have been reported using immunotherapy for treatment of solid tumors and malignancies of the hematopoietic system. Since T cells act as key players in immune surveillance, an encouraging approach is to genetically engineer T cells with lenti- or retroviral vectors, in order to express artificial chimeric antigen receptors (CARs), directing them against cancer cells and leading to a distinct cytotoxic response. CARs are synthetic fusion proteins consisting of an antigen-binding moiety, commonly a tumor-associated antigen (TAA)-specific single-chain fragment variable (scFv), combined with activating signaling domains like the CD3-zeta chain. Signaling can be enhanced by adding co-stimulatory domains, such as the intracellular regions of CD28 or CD137. Although, CAR T cells showed promising results in patients with B cell malignancies, a major drawback of conventional CARs, bearing a tumor-antigen-binding moiety, is the uncontrollable continuous T cell activity. Hence, CAR T cells can achieve a sustained anti-tumoral response. However, reactivity against any healthy tissues expressing the target antigen might cause severe adverse effects. A chance to gain safety on CAR T cell approaches by retaining efficacy is offered through a novel universal binary CAR system (UniCAR). The system is based on a second generation CAR with an extracellular scFv binding domain, which is directed against a short non-immunogenic peptide motif instead of a cell surface antigen. Thus, engineered T cells are inert per se, remaining in a “sleeping mode” after re-infusion. Small protein links (targeting modules, TMs), consisting e.g. of TAA-specific scFvs harboring the small peptide epitope recognized by the UniCAR are used to redirect UniCAR expressing T cells in an antigen-specific manner. Primary results demonstrate that UniCAR driven T cell activation and cytotoxic effects against tumor cells are strictly dependent on the presence of the antigen-specific TMs. UniCAR T cells show high anti-tumor efficacy even at picomolar TM concentrations and low effector to target cell (e:t) ratios in vitro. After those proof-of-concept studies we disclose positive evidence of high efficacy UniCAR T cells with reactivity against CD123-positive acute myeloid leukemia (AML) and CD19-bearing acute lymphoblastic leukemia (ALL) cells. UniCAR T cell anti-tumor efficacy proofed to be superior when compared to conventional CAR T cells at low e:t ratios adjusted to the situation usually found in patients. Furthermore, UniCAR T cells pre-decorated with CD123-specific TMs in vitro are capable of prohibiting tumor engraftment of CD123-positive AML in vivo and persist in immunodeficient NSG mice. Ex vivo isolated UniCAR T cells retained cytotoxic potential against MOLM-13 cells for at least four month post transplantation fortifying the therapeutic potential in vitro and in vivo. The tremendous benefit of the novel UniCAR platform is the precise control of CAR T cell activity by regulating TM administration and dosage, preventing off side-toxicities arising from conventional CAR approaches. Moreover, the modular UniCAR system also enables combinatorial targeting strategies against several TAAs to increase therapy performance and circumvent the occurrence of tumor resistance mechanisms due to selectable compositions of target structures. Leukemia-related target antigens like CD19, CD20, CD22, CD33 or CD123 are conceivable therapy settings, offering advantages compared to monotherapeutic CAR-based therapies. Taken together, the modular composition of the UniCAR platform maintains the high anti-tumor potential of CAR-engrafted T cells, while introducing tight control mechanisms and unparalleled target flexibility. These features will allow a more sophisticated application of CAR technology and a reduction of adverse events in the clinical setting. Citation Format: Simon Loff, Malte v. Bonin, Josephine Dietrich, Jan-Erik Meyer, Anja Feldmann, Claudia Arndt, Johannes Spehr, Cordula Gründer, Gerhard Ehninger, Michael P. Bachmann, Armin Ehninger, Marc Cartellieri. The UniCAR system: Inducible CAR T cells for precise reactivity and high efficacy against hematopoietic malignancies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B099.