In vitro immunotherapy potency assays using real-time cell analysis

Abstract

A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time it takes to kill 50% of the target tumor cells) and % cytolysis are calculated and used for comparing the relative efficacy of different reagents. In summary, our results demonstrate the xCELLigence platform to be well suited for potency assays, providing quantitative assessment with high reproducibility and a greatly simplified work flow.

Co-SLD suppressed the growth of oral squamous cell carcinoma via disrupting mitochondrial function

“…As described in Figure 5(B), there was a dose-or time-dependent suppression of survival of CAL27 cells induced by Co-SLD varying from 5 to 20 lM. KT50 (the amount of time it takes to kill 50% of the target tumour cells) was selected as a quantitative parameter for evaluating the efficient killing kinetic of Co-SLD [68]. KT 50 values were roughly 63, 41 and 13 h, in CAL27 cells exposed to 5, 10, and 20 lM, respectively.…”

Production and quality testing of multipotent mesenchymal stromal cell therapeutics for clinical use

“…In an elegant approach, Lee et al started their search for a potency marker to predict BM‐MSCs’ efficacy to treat sterile inflammations comparing more efficacious to less efficacious MSC preparations together with gain‐of‐function experiments, which led to the discovery of TNF‐α–stimulated gene‐6 as a biomarker that identifies MSC preparations with potent anti‐inflammatory properties. A cytolytic immunopotency assay using impedance to measure the viability of target tumor cells affected by immunotherapeutic interventions was introduced . Killer et al reported that MSCs cocultured with immune cells increased their glycolytic and respiratory activity, and this enhanced cell metabolism was accompanied by higher T‐cell suppressive capacities of MSCs.…”

IL‐18R‐dependent and independent pathways account for IL‐18‐enhanced antitumor ability of CAR‐T cells

“…24 After culturing SKOV3 cells or HER2tumor cells (10 4 cells per well) in E-plate 96 (ACEA Bioscience) for approximately 24 hours, pretreated CAR-T cells with or without 100 ng/mL IL-18 were added to the plates (E:T = 5). This cell detection technology is based on the microelectronic impedance technology, and the size of impedance depends on the number, size and shape of the adherent cells, and the quality of the cell-substrate attachment.…”

“…This cell detection technology is based on the microelectronic impedance technology, and the size of impedance depends on the number, size and shape of the adherent cells, and the quality of the cell-substrate attachment. 24 After culturing SKOV3 cells or HER2tumor cells (10 4 cells per well) in E-plate 96 (ACEA Bioscience) for approximately 24 hours, pretreated CAR-T cells with or without 100 ng/mL IL-18 were added to the plates (E:T = 5). Data were acquired and analyzed according to the protocols specified by the manufacturers (ACEA Bioscience, Inc. RTCA Software 2.1).…”