Abstract 4745: Quantitative single-cell functional characterization of CD19-specific CAR+ T Cells for immunotherapy.

Abstract

Abstract 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) that were considered untreatable, including the use of stem-cell transplantation. 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). Finally, automated time-lapse microscopy of hundreds of these effector target interactions allowed us to quantify the kinetics of effector mediated lysis and effector cell velocity profiles before and after lysis. These analyses demonstrate that individual CAR+ T cells demonstrate vastly different individual killing profiles, at least in vitro. 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 that correlate with clinical efficacy. Citation Format: Navin Varadarajan, Ivan Liadi, Gabrielle Romain, Harjeet Singh, Laurence J.N. Cooper. Quantitative single-cell functional characterization of CD19-specific CAR+ T Cells for immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4745. doi:10.1158/1538-7445.AM2013-4745