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Application of autologous T cells genetically engineered to express CD19-specific chimeric antigen receptors (CAR-T) is highly effective in the treatment of B cell malignancies. To this date, application of CAR-T therapy beyond CD19 remains challenging due to the inability to control CAR-T reactivity in patients and the lack of tumor-associated antigens exclusively expressed by malignant cells. The interleukin-3 receptor alpha chain (CD123) is a promising immunotherapeutic target and associated with leukemia-initiating compartments in myeloid- or lymphoid derived diseases. However, in contrast to CD19, CD123 is a precarious target due to its prevalent expression on healthy hematopoietic stem and progenitor cells (HSPC) as well as endothelial cells. Thus, CAR-T lacking any fine-tuned control mechanisms are at risk to cause life threatening toxicities or can only act as bridging therapy to an allogeneic stem cell transplantation. To extend application of CAR-T therapy and safely redirect CAR-engineered T cells to challenging targets such as CD123, a switch-controllable universal CAR-T platform (UniCAR) was recently introduced. The UniCAR system consists of two components: (1) a non-reactive inducible second generation CAR with CD28/CD3ζ stimulation for an inert manipulation of T cells (UniCAR-T) and (2) soluble targeting modules (TM) enabling UniCAR-T reactivity in an antigen-specific manner. Here we provide late stage pre-clinical data for UniCAR-T in combination with a CD123-specific TM (TM123) for treatment of acute leukemia. Primary patient-derived CD123-positive leukemic blasts were efficiently eradicated by TM123-redirected clinical-grade manufactured UniCAR-T in vitro and in vivo. Activation, cytolytic responses and cytokine release were proven to be strictly switch-controlled. Moreover, anti-leukemic responses of UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro. In contrast to conventional CD123 CAR-T, TM123-redirected UniCAR-T discriminate between CD123high malignant cells and CD123low healthy cells with negligible toxicity towards HSPC in vivo. As 4-1BB mediated co-stimulation is known to enhance CAR-T activity in vivo, a novel CD123-specific targeting module bearing a covalently bound trimeric 4-1BB ligand (4-1BBL) was developed and characterized for co-stimulation at the leukemic site in trans. Specific binding of TM123-4-1BBL was demonstrated against native 4-1BB as well as CD123-positive leukemic blasts. In long-term tumor eradication models, TM123-4-1BBL ameliorated the killing capability of UniCAR-T in vitro. Additionally, the increased hydrodynamic radius of trimeric 4-1BBL-coupled TM123 prolonged plasma half-life and enhanced bioavailability in vivo. In conclusion, UniCAR-T maintain high anti-leukemic efficacy, while adding a sophisticated mechanism for immediate control to improve safety and versatility of CD123-directed CAR-T therapy. Moreover, switching between several TMs from short to moderate plasma half-life allows for an individualized treatment of various leukemic settings while minimizing potential adverse effects. Disclosures Loff: GEMoaB Monoclonals GmbH: Employment. Meyer:Cellex Patient Treatment GmbH: Employment. Dietrich:Cellex Patient Treatment GmbH: Employment. Spehr:Cellex Patient Treatment GmbH: Employment. Julia:Cellex Patient Treatment GmbH: Employment. Gründer:GEMoaB Monoclonals GmbH: Employment. Franke:GEMoaB Monoclonals GmbH: Employment. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ehninger:GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Equity Ownership. Cartellieri:Cellex Patient Treatment GmbH: Employment.