CAR-T: How Carl June's Team evaluates Cytotoxic Activity

from the 4th Immunogenicity & Immunotoxicity Conference Jan 25-26, San Diego, CA, USA:

Discovery and Pre-Clinical Evaluations of CAR T Cell Cytotoxic Activity Using the xCELLigence Real Time Cell Analyzer

Shannon McGettigan (presenting Author, Research Specialist, University of Pennsylvania), Yanping Luo, Keisuke Watanabe, John Scholler, Carl H June

Cytotoxicity assays are an important characterization in the development of anti-cancer therapeutics.Chromium release assays are considered the gold standard for evaluating lymphocyte cytotoxic activity but requires the burden of using radioactive materials, is time consuming, and is limited to a single time point.Our lab and others have developed flow cytometric and luciferase based cytotoxcity assays for screening and evaluating novel therapeutic CAR T cells against a wide range of cancer cell lines and primary tumor cells; however, these can also be time consuming and limited to a single snapshot.

In order to monitor the overall killing activity of our CAR T cell therapies as a function of time and more rapidly drive our understandings in early development of therapeutic T cells, we have started to utilize the xCELLigence real time cell analyzer (RTCA). Our studies have compared how measurement of changes in adherent cell’s electrical impedance compares to our standard cytotoxicity measurements by the remaining viable cell numbers in flow based assays or relative changes in luciferase activity. We found that a correlation exists between the platforms for measuring cytotoxicity.

Real time cellular impedance analysis reveals kinetic differences that cannot be captured practically with conventional fixed end point platforms. We have found that using the xCELLigence platform has many benefits beyond just real time monitoring:

The assay requires minimal number of cells which can be retrieved for further analysis, saves time, provides a kinetic readout of combination therapies, and is a quick quality control cytotoxicity assay for therapeutic T cells used in in vivo experiments.

Together the xCelligence Real time platform shows valuable utility for screening gene modified T cells cytotoxic function, characterizing the kinetics of their activity, evaluating the dosage and timing of combination therapies in vitro and providing a quick stable platform for quality control of therapeutic T cells.

Example: PBMC-Mediated Cytolysis of BT474 Cells in Presence and Absence of Trastuzumab

ADCC - Cytotoxicity -Trastuzumab

Cell index (CI) values are proportionally reduced with increasing effector to target (E/T) ratios in the presence of trastuzumab. BT474 clone 5 cells were maintained for 26 h and then were treated with media alone (control) or with media plus mononuclear cells isolated from human blood (panel A). Cells were treated in an identical fashion in Panel B except for the inclusion of 0.1 ug/ml of trastuzumab. Cell index values were normalized at the time of addition. Blue represents growth with no mononuclear cells (control) while green, orange, purple and red represents growth in the presence of MNCs at E/T ratios of 0.5/1, 1/1, 2:1, and 6/1, respectively. The vertical dashed lines indicate the 16 h window of time after treatment used to determine AUC values. Normalized cell index values are plotted in 15 min increments as the average of three replicate with the standard deviation.

Oncoimmunology. 2012 Sep 1;1(6):810-821. Understanding key assay parameters that affect measurements of trastuzumab-mediated ADCC against Her2 positive breast cancer cells.Kute T, Stehle Jr JR, Ornelles D, Walker N, Delbono O, Vaughn JP. Wake Forest University School of Medicine; USA.


xCELLigence & CAR-T cytotoxicity screening: Find out more!






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