(795f) Quantitative Single-Cell Functional Characterization of CD19-Specific Chimeric Antigen Receptor T Cells

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 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, by employing a novel methodology: single-cell nanowell screening (SNS). 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 within the 6 hour window of observation. Hierarchical clustering indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity of T cells. CAR+ T cells that secreted IFNγ upon target ligation did not undergo activation-induced cell death (AICD) whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04). In summary, our SNS-based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to determine in vitro functions of CAR+ T cells.