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 therapeutic success. We implemented Timelapse Imaging Microscopy In Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multi-killing via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that while CAR4 cells can participate in killing and multi-killing, they do so at slower rates, likely due to the lower Granzyme B content. Significantly, in both sets of T cells, a minor sub-population of individual T cells identified by their high motility, demonstrated efficient killing of single tumor cells. By comparing both the multi-killer and single killer CAR+ T cells it appears that the propensity and kinetics of T-cell apoptosis was modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multi-killing should be evaluated in the context of their ability to resist activation induced cell death (AICD). We anticipate that TIMING may be utilized to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy.

• Fc-engineered mAb promotes NK cell ADCC via better activation, serial killing, and kinetic boosting at higher target cell densities.• Enhanced target killing also increased frequency of NK cell apoptosis, but this effect is donor-dependent.The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effectormediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cellmediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. (Blood. 2014;124(22):3241-3249) Introduction Therapeutic monoclonal antibodies (mAbs) elicit functional responses through many different mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity, antibody-dependent cell-mediated phagocytosis (ADCP), and direct induction of apoptosis in tumor cells.1 By using the principles of glycoengineering and mutagenesis, Fc variants have been isolated that show either increased affinity for the activating receptors or altered selectivity for the activating/inhibitory receptors. [2][3][4] Preliminary clinical data with such antibodies Fc-engineered to improve the ADCC/ADCP potential and targeting CD19, CD20, Her2, or CD40 have shown reasonable promise in improving the therapeutic potential of mAb.5-8 Natural killer (NK) cells occupy a pivotal role in immunity: not only can they exert direct cytotoxicity toward infected or tumor cells but they also participate in shaping the adaptive response. 9,10 In the context of mAb treatment, NK cells are unique in that they express only the low-affinity activating FcgR CD16 (FcgRIIIa), and no inhibitory antibody receptors, underscoring a significant role in ADCC.11-13 Several studies using mouse tumor models have established a link between ...

Supplementary data are available at Bioinformatics online.

Supplementary data are available at Bioinformatics online.