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Chimeric antigen receptor T cells (CAR-T) targeting CD19 or B cell maturation antigen (BCMA) are highly effective against B cell malignancies. However, application of CAR-T to less differentially expressed targets remains a challenge due to lack of tumor-specific antigens and CAR-T controllability. CD123, a highly promising leukemia target, is expressed not only by leukemic and leukemia-initiating cells, but also by myeloid, hematopoietic progenitor, and certain endothelial cells. Thus, CAR-T lacking fine-tuned control mechanisms pose a high toxicity risk. To extend the CAR-T target landscape and widen the therapeutic window, we adapted our rapidly switchable universal CAR-T platform (UniCAR) to target CD123. UniCAR-T efficiently eradicated CD123+ leukemia in vitro and in vivo. Activation, cytolytic response, and cytokine release were strictly dependent on the presence of the CD123-specific targeting module (TM123) with comparable efficacy to CD123-specific CAR-T in vitro. We further demonstrated a pre-clinical proof of concept for the safety-switch mechanism using a hematotoxicity mouse model wherein TM123-redirected UniCAR-T showed reversible toxicity toward hematopoietic cells compared to CD123 CAR-T. In conclusion, UniCAR-T maintain full anti-leukemic efficacy, while ensuring rapid controllability to improve safety and versatility of CD123-directed immunotherapy. The safety and efficacy of UniCAR-T in combination with TM123 will now be assessed in a phase I clinical trial (ClinicalTrials.gov: NCT04230265)., Graphical Abstract, CAR-T cell treatment of acute leukemia beyond CD19 remains challenging. Loff et al. demonstrate that rapidly switchable universal CAR-T cells in combination with soluble CD123-specific adaptors achieve robust anti-leukemic responses, while hematotoxicity is rapidly reversible, enabling safe targeting of such less differentially expressed target antigens and broadening of the therapeutic window.

Despite remarkable therapeutic success using chimeric antigen receptor modified T-cells (CAR-T) in hematologic malignancies, targeting solid tumors still remains challenging, given their heterogeneity and immunosuppressive milieu, including the expression of inhibitory immune checkpoints such as PD-1/PD-L1. Preclinical models demonstrate that combining CAR-T therapy with checkpoint inhibitors targeting the PD-1/PD-L1 axis could be a promising strategy to enhance anti-cancer activity of CAR-T in solid tumors and on-going phase I trials are clinically evaluating this strategy. However, primary and acquired resistance to checkpoint blockade is frequently observed limiting its broad applicability and efficacy. In addition, lack of specificity results in frequent occurrence of immune-related adverse events affecting several organs. Alternatively, expression of PD-L1 by solid tumor cells could be an attractive target for CAR-T therapy, especially in order to prevent or reverse tumor re-growth and relapse after successful initial tumor cell lysis. The development of a rapidly switchable universal CAR-T platform (UniCAR) with enhanced safety features allowed us to explore the possibility to directly attack PD-L1 expressing solid tumor cells. An inherent key feature of the UniCAR-T platform is a rapid and reversible turn off mechanism (less than 4 hours) determined by the short pharmacokinetic half-life of soluble targeting modules (TMs) and fast internalization of cell-bound TMs. TMs provide the antigen-specificity for UniCAR gene-modified T-cells (UniCAR-T) and consist of an antigen-binding moiety, e.g. an scFv, linked to a small peptide motif recognized by the UniCAR. For redirecting UniCAR-T against the PD-1/PD-L1 axis, a PD-L1 specific TM (TM-PD-L1) was developed and its functionality confirmed in binding assays to human and murine PD-L1 using surface plasmon resonance as well as in cell-based cytotoxicity assays. Half maximal killing efficacy (EC50) mediated by TM-PD-L1 redirected UniCAR-T was in the low picomolar range. As PD-L1 is also expressed on healthy tissue, toxicity studies were performed in a humanized mouse model with human UniCAR-T. Humanized mice treated with TM-PD-L1 twice a day for ten consecutive days did not reveal any signs of toxicity. In a prostate cancer (PCa) xenograft model efficient suppression of tumor growth could be demonstrated by administration of TM-PD-L1. Our data are the first to demonstrate safety and feasibility of active targeting of PD-L1 expressed by solid tumors with CAR-T and that this approach is capable of inducing a significant anti-tumor response. Citation Format: Josephine Dietrich, Simon Loff, Johannes Spehr, Julia Riewaldt, Cordula Gründer, Maria Schreiber, Michael Bachmann, Gerhard Ehninger, Michael Pehl, Marc Cartellieri, Armin Ehninger. Rapidly switchable universal CAR-T cells with improved safety profile allow for active targeting of PD-L1 expressing solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2209.

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.

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.