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BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

Meeting abstracts T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of

Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL). In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to address a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells expressing membrane bound IL-21, by employing a novel methodology single-cell nanowell screening that determines their cytotoxic ability and cytokine secretion capability at single-cell resolution. We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells and 9 ± 3% killed three target cells within the 6 hour window of observation. In parallel, microengraving was used to determine the cytokine secretion profile of these same cells. Hierarchical clustering of the two functions indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity or the ability of T cells to kill multiple target cells. Simultaneously, monitoring apoptosis on CAR+ T cells allowed us to quantify their activation-induced cell death (AICD). CAR+ T cells that secreted IFNγ upon target ligation did not undergo AICD whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). Dynamic time-lapse imaging of the interactions between CAR+ T cells and tumor cells indicated that the majority of CAR+ T cells have high basal motility, form long-lived interactions with tumor cells (50 - 100 min) that lead to motility arrest and subsequent tumor-cell apoptosis. However, contact lifetimes or overall contact duration were not reliable predictors of subsequent tumor-cell apoptosis. Finally, kinetics of serial killing suggest that motile CAR+ T cells that form short-lived contacts exhibit rapid killing with very little motility arrest in vitro. In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to: (1) determine in vitro functions of CAR+ T cells that correlate with clinical efficacy and (2) inform CAR design to maximize effector functionality while minimizing AICD.

Meeting abstracts Humanized monoclonal antibodies (mAb) targeting tumor antigens have paved the way to complementary strategies in the treatment of cancer by harnessing the immune system towards a more specific response against tumors. Such mAb can mobilize natural killer (NK) cells function by

Meeting abstracts T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR) have shown considerable promise in clinical trials even in tumors refractory to all other treatment methods. In particular, the use of CAR T cells rendered specific for CD19 demonstrated