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Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity

Nature Protocols volume 16, pages 1331–1342 (2021)Cite this article

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Abstract

The antitumor efficacy of genetically engineered ‘living drugs’, including chimeric antigen receptor and T-cell receptor T cells, is influenced by their activation, proliferation, inhibition, and exhaustion. A sensitive and reproducible cytotoxicity assay that collectively reflects these functions is an essential requirement for translation of these cellular therapeutic agents. Here, we compare various in vitro cytotoxicity assays (including chromium release, bioluminescence, impedance, and flow cytometry) with respect to their experimental setup, appropriate uses, advantages, and disadvantages, and measures to overcome their limitations. We also highlight the US Food and Drug Administration (FDA) directives for a potency assay for release of clinical cell therapy products. In addition, we discuss advanced assays of repeated antigen exposure and simultaneous testing of combinations of immune effector cells, immunomodulatory antibodies, and targets with variable antigen expression. This review article should help to equip investigators with the necessary knowledge to select appropriate cytotoxicity assays to test the efficacy of immunotherapeutic agents alone or in combination.

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Fig. 1: Interassay comparison of cell-mediated cytotoxicity assessment.

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Acknowledgements

P.S.A.’s laboratory work is supported by grants from the National Institutes of Health (P30 CA008748, R01 CA236615-01, and R01 CA235667), the US Department of Defense (BC132124, LC160212, CA170630, and CA180889), the Batishwa Fellowship, the Comedy vs Cancer Award, the Dalle Pezze Foundation, the Derfner Foundation, the Esophageal Cancer Education Fund, the Geoffrey Beene Foundation, the Memorial Sloan Kettering Technology Development Fund, the Miner Fund for Mesothelioma Research, the Mr. William H. Goodwin and Alice Goodwin, the Commonwealth Foundation for Cancer Research, and the Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center.

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  1. Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Stefan Kiesgen, John C. Messinger, Navin K. Chintala, Zachary Tano & Prasad S. Adusumilli

  2. Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Prasad S. Adusumilli

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P.S.A. has received research funding from ATARA Biotherapeutics and Acea Biosciences, has served on the Scientific Advisory Board or as consultant to ATARA Biotherapeutics, Bayer, Carisma Therapeutics, Imugene, and Takeda Therapeutics, and has patents, royalties, and intellectual property on mesothelin-targeted CARs and other T-cell therapies, and method for detection of cancer cells using virus, and pending patent applications on T-cell therapies.

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Kiesgen, S., Messinger, J.C., Chintala, N.K. et al. Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity. Nat Protoc 16, 1331–1342 (2021). https://doi.org/10.1038/s41596-020-00467-0

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