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The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic "drugs" for cancer therapy. CAR T therapies generally rely upon manufacturing approaches that include prior T cell activation through engagement of the TCR and costimulatory receptors followed by ex vivo expansion of patient-derived T cells over days to weeks. We previously reported that CD3/CD28 stimulation prior to transduction promotes progressive T cell effector differentiation over time in culture with loss of CAR T cell potency (Ghassemi et al. 2018 PMID: 30030295). Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that lentiviral transduction of quiescent T cells without prior activation will enhance engraftment and persistence of CART cells that is associated with long-term leukemia control. Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We showed using a non-optimized process that a mean of 6.5% (range 2%-10%) of freshly isolated quiescent T cells can be transduced using an infrared red fluorescent protein (iRFP)-expressing lentiviral vector with slower kinetics of expression compared with activated T cell transduction (peak at 96 hrs vs. 48 hrs for quiescent and activated T cells, respectively). Although substantially less efficient compared to activated T cells, transduction was detected across all T cells subsets with central memory T cells showing the greatest transduction efficiency with a mean of 4-fold greater transduction compared with naïve T cells. Somewhat unexpectedly, CART19 cells generated from quiescent T cells using a CD19-specific CAR vector showed a 3-5 fold greater transgene expression compared with iRFP vectors transduced at similar MOI. However, we show that CAR expression can occur in quiescent T cells even without reverse transcription or integrase function, so called "pseudotransduction". Importantly, we show that this CAR expression produces T cells with cytolytic activity and effector cytokine production in response to antigen that is similar to activated and transduced CAR T cells. Using the well-characterized Nalm6 model of acute lymphoblastic leukemia, we show that CART19 cells generated by transduction of quiescent T cells for 16 hours followed by washing to remove vector exhibit dose-dependent anti-leukemic activity that is durable with injection of as little as 2x105 total T cells. We estimate the latter to contain ~2x104 T cells with integrated lentiviral vector based upon transduction efficiency determined in studies using non-tumor bearing mice. (Fig 1). In summary, our results support the need for further investigation of CAR T cells that are generated using engineering of quiescent T cells. Taking advantage of the ability of lentiviral vectors to transfer genes to quiescent T cells, the highly abbreviated and simplified manufacturing approach described here has the potential to enhance therapeutic potency while also substantially reducing the materials and labor costs associated with current manufacturing approaches that use activated and expanded T cells. In addition, the rapid nature of this manufacturing has the potential to extend the population of patients that may be treated with these therapies by shortening the interval between aphaeresis collection and re-infusion of CAR T cells, which prevents the treatment of some patients with rapidly progressive disease. Disclosures Ghassemi: Novartis: Patents & Royalties. Milone:Novartis: Patents & Royalties, Research Funding.

Adoptive cell therapy employing T cells equipped with a chimeric antigen receptor (CAR) containing a single chain antibody fragment fused to T cell signaling domains 4-1BB and CD3zeta (CTL019) has shown great potency against various hematopoietic malignancies, e.g. B cell acute lymphoblastic leukemia (ALL). However, it has not shown the same response rate in other malignancies such as chronic lymphocytic leukemia (CLL). We recently demonstrated that the in vivo expansion and persistence of CAR T cells is an important predictor of response to CTL019 in CLL (PMID: 26333935) and ALL (Thudium et al., ASH 2016; Fraietta et al., ASH 2016). Furthermore, it is well known that prolonged culture of T cells negatively impacts the in vivo expansion of the adoptively transferred cells. We therefore hypothesized that minimizing the ex vivo manipulation of T cells would improve the efficacy of CAR T cells. We tested this hypothesis by generating CART19 cells using our standard 9-day manufacturing process plus two abbreviated versions. Cells from normal donors (n=9) and from patients with adult ALL (n=6) were stimulated on day 0 followed by transduction with the CAR19-encoding lentiviral vector on day 1. Cells were harvested on days 3, 5, and 9. Cryopreserved aliquots were evaluated for T cell differentiation using polychromatic flow cytometry, cytokine secretion profile using Luminex, cytolytic ability against a leukemia cell line (NALM6), proliferative ability upon restimulation with CD19-expressing target cells, and in vivo control of our well-established xenogeneic ALL model employing NALM6 as the target. Our data show that all cultures contain a substantial proportion (40%-80%) of na•ve-like CD45RO-CCR7+ T cells that progressively differentiate leading to the accumulation of predominantly (60%-90%) central memory T cells by the end of expansion. Comparative assessment of the CART19 cells at all three time points demonstrated that the cells from the shorter cultures displayed a superior in vitrocytolytic activity, and proliferative response compared to the standard process. In addition,the cells from our standard and shortened cultures all secreted comparable levels of type I cytokines (i.e. IFN-g, IL-2, and TNF-α). Importantly, we investigated the therapeutic potential of cells harvested at day 3 versus later time points. We treated NALM6 xenograftmice with a low dose (0.5 x106 CAR+ T cell I.V.) or standard dose (3 x106 CAR+ T cell I.V.).We demonstrate that day 3 CART19 cells show superior anti-leukemic activity compared to day 5 or day 9 cells. Additionally, we show that mice treated at a low dose with day 3 cells exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia (Figure 1). Our preclinical findings provide evidence that extended ex vivo manipulation of T cells negatively affects their in vivo potency.In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction provides the most efficacious T cells for adoptive T cell immunotherapy. Figure 1 Figure 1. Disclosures Ghassemi: Novartis: Research Funding. Scholler:Novartis: Patents & Royalties; University of Pennsylvania: Patents & Royalties: FAP-CAR US Patent 9,365,641 for targeting tumor microenvironment. Nunez-Cruz:Novartis: Research Funding. Barrett:Novartis: Research Funding. Bedoya:Novartis: Patents & Royalties. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. June:Johnson & Johnson: Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership. Milone:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding.