PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor (CAR)-T cell therapies have dramatically shifted the treatment paradigm for many patients with cancer. Broader use of autologous CAR-T cell therapies, however, is hindered by complicated logistics, variable quality, T cell exhaustion, antigen escape and on-target/off-tumor effects and other toxicities, like CRS. The broad objective is to address these issues by the development of a safe and effective off-the- shelf cell therapy. To accomplish this, we propose a different cellular vehicle, specifically Regulatory T Cells (Tregs). Advantages of Tregs include (a) an inherent ability to suppress the release of proinflammatory cytokines and host mediated allogeneic rejection, and (b) proliferative capacity for large-scale expansion and genetic engineering, ideal characteristics of an allogeneic immune effector cell (IEC) therapeutic. Based on the safety and efficacy profile of third party Tregs previously tested in phase I/II trials for prevention of graft-versus- host disease (GVHD), and their tumoricidal potency in preclinical models, this strategy addresses some of the grand challenges facing allogeneic IECs. During the current funding period, in proof of concept studies, we demonstrated the tumoricidal activity of Treg expressing CARs targeting CD19+ and CD83+ hematological malignancies. Our central hypothesis is that allogeneic Tregs can be engineered for enhanced persistence and maximal tumor killing with reduced risks of adverse side effects compared to conventional T cells. Specifically, we will establish the safety profile of CAR19 Tregs at the maximal tolerable dose without and with rhIL-2 in patients with relapsed/refractory CD19+ acute lymphocytic leukemia (Aim 1A, Aim 1B), assessing in vivo pharmacokinetics and persistence as well as tumoricidal activity. To avoid the potential side effects of systemic rhIL-2 and avoid bystander activation, we will engineer CAR19 Tregs to express an orthoIL2Rβ responsive to orthoIL-2 to maximize persistence and tumor killing (Aim 1C). In Aim 2, we will test the potency of nanoCAR Tregs targeting CD83 on AML cells and not myeloid progenitors (Aim 2A). Additionally, to minimize Treg exhaustion from tonic stimulation as well as enhance safety, Tregs will be engineered with a drug inducible CAR permitting control over its activation (Aim 2B). These IND enabling studies will support future clinical testing. IMPACT. The availability of safer and more effective allogeneic ‘off-the-shelf’ tTreg IECs would address a critical unmet need, overcoming some of the principal limitations of individualized products. 1