Cited by 138CAR-T cell therapy researchImmunotherapy and Immune ResponsesT-cell and B-cell Immunology

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

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Kiesgen, Stefan, Messinger, John C., Chintala, Navin K., Tano, Zachary, Adusumilli, Prasad S.·2021·Nature Protocols

DOI 10.1038/s41596-020-00467-0PMC PMC8064272

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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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See all 138

Authors: Kiesgen, S., Messinger, J. C., Chintala, N. K., Tano, Z., & Adusumilli, P. S.
Affiliations: Memorial Sloan Kettering Cancer Center (US)
Corresponding author: Adusumilli, Prasad S.
ORCID: Chintala, Navin K.
Adusumilli, Prasad S.
Venue: Nature Protocols
Publisher: Springer Science and Business Media LLC
Published: 2021-02-15
Volume: 16
Issue: 3
Pages: 1331-1342
ISSN: 1750-2799 (ISSN-L)
1754-2189
DOI: 10.1038/s41596-020-00467-0
PMC: PMC8064272
OpenAlex: W3132904428
Cited by: 138
References: 100
Citations by year: 2026: 24 · 2025: 37 · 2024: 32 · 2023: 22 · 2022: 18 · 2021: 5
FWCI: 2.33
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: www.springer.com/tdm (tdm)
www.springer.com/tdm (vor)
Locations: Nature Protocols (publishedVersion)
PubMed (publishedVersion)
PubMed Central (acceptedVersion)
Funders: National Cancer Institute (R01CA236615, R01CA235667, P30CA008748, T32CA009501)
Topics: Health Sciences › Medicine › Oncology › CAR-T cell therapy research
Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords: chimeric antigen receptor T cells, T cell cytotoxicity, chromium release assay, bioluminescence assay, flow cytometry, potency assay, T cell exhaustion, T cell proliferation, combination immunotherapy, immunomodulatory antibodies, T cell receptor T cells
MeSH terms: Receptors, Chimeric Antigen, Animals, Cytotoxicity Tests, Immunologic, Flow Cytometry, Humans, Receptors, Antigen, T-Cell, T-Lymphocytes, Immunotherapy, Adoptive, Xenograft Model Antitumor Assays

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

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