Abstract: Our goal is to develop improved approaches for safe adoptive transfer of genetically modified T cells. While transfer of anti-human CD19SFv chimeric antigen receptor (hCD19-CAR) expressing T cells have provided dramatic effects in CD19+ B lymphoid malignancies, complications include prolonged B cell aplasia and fatal severe systemic inflammatory response syndrome. We propose to solve these complications by using a novel approach to regulate CAR function in vivo using an FDA approved drug at sub-therapeutic concentrations. Tstem memory (Tsm) cells have the highest potency for anti-tumor responses due to their in vivo longevity. Expanded CAR+ T cells lose Tnaive and Tsm phenotypes. We hypothesize that strategies to retain or drive the Tsm state of CAR-T cells will improve outcomes. Human T cells have been reprogrammed into inducible pluripotent stem cells (IPSCs) that can be readily genetically modified and re-differentiated into mature T cells. We hypothesize that Tsm generated IPSCs can provide a virtually limitless source of long- lasting Tsm CAR T cells and that starting IPSC generation from Tsm cell state will retain the epigenetic landscape and developmental plasticity of Tsm cells in the final CAR-T cell product. Expanded T cells differentiate into Teffectors or exhausted T cells, associated with profound, global changes in gene expression. The transcription factor (TF) Bcl6 is expressed at high levels in CD8 Tsm, decreasing with progressive differentiation, while Prdm1 expression reciprocally increases. We hypothesize that inducing high Bcl6 and loss of Blimp-1 protein in IPSC T cell progeny will optimize Tsm CAR efficacy. Additional control is mediated by the epigenome. We will use a combination of epigenetic and transcription analysis of Tsm, IPSC and T-IPSC progeny to identify novel TFs that regulate the differentiation of Tsm from T-IPSC progeny. Specific aims will test the hypotheses that: 1. Drug regulation of anti-hCD19SFv CAR in T cells can clear malignant CD19 B cells by dimerizing receptors for an SFv with one containing an intracellular signaling domain (1A) and CAR persistence is not fully dependent upon tumor- or host- derived B cell antigenic signals (1B). Competitor drug infusion can rapidly halt CAR signaling by precluding dimerization, minimizing side-effects (1C); 2. Human Tsm cells can be reprogrammed into IPSCs to provide a self-renewable, regulated anti-CD19 SFv CAR-Tsm cells for adoptive tumor immunotherapy (2A). IPSCs can be induced to express a TF profile (bcl6hi; prdm1lo) facilitating re-differentiated, ex vivo expanded Tsm CAR T cells to retain a non-senescent state (2B); 3. Integrated analysis of gene expression, epigenetic analysis and TF ?foot-printing? during in vitro Tsm differentiation will identify (3A) and validate (3B) essential regulators of the Tsm state to optimize Tsm based CAR therapy. These 3 aims will create an ?off-the-shelf? product for CAR Tsm T cell therapy that can overcome critical side-effects of CAR therapy and identify and validate key, novel TFs that will guide the optimization of CAR therapy for future clinical trials in this PPG and establish a new paradigm of cancer treatment.