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81 Optimizing ex vivo CAR-T cell­­­-mediated cytotoxicity assay through multimodality imaging
  1. John G Foulke1,
  2. Luping Chen2,
  3. Hyeyoun Chang1,
  4. Kevin M Tyo2,
  5. Fang Tian2 and
  6. Zhizhan Gu2
  1. 1American Type Culture Collection, Manassas, VA, USA
  2. 2ATCC (American Type Culture Collection), Gaithersburg, MD, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background CAR-T cell-based therapies have demonstrated remarkable efficacy in treating malignant cancers, especially liquid tumors, and are increasingly being evaluated in clinical trials for solid tumors. With FDA’s initiative for advancing alternative methods for drug discovery and development, full human ex vivo assays are increasingly essential for precision CAR-T development. However, prevailing ex vivo CAR-T cell-mediated cytotoxicity assays are limited by their use of radioactive materials, lack of real-time measurement, low throughput, and automatability, among others.

Methods To address these limitations, we optimized the assay using multimodality imaging methods, including bioluminescence, impedance tracking, phase contrast, and fluorescence, to track CAR-T cells cocultured with CD19, CD20, and HER2 luciferase reporter cancer cells in real-time. Additionally, we varied the ratio of CAR-T cells to cancer cells to determine optimal cytotoxicity readouts.

Results Our findings demonstrated that the CAR-T cell group effectively attacked cancer cells and the optimized assay provided superior temporal and spatial precision measurements of ex vivo CAR-T killing of cancer cells.

Conclusions These results confirm the reliability, consistency, and high throughput of the optimized assay.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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