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Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity
Stefan Kiesgen1, John C Messinger1, Navin K Chintala1
1Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
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.
Insights
Selecting the right cytotoxicity assay is crucial for evaluating
Area of Science:
- Immunology and Cellular Therapy
- Biotechnology and Drug Development
Background:
- The efficacy of engineered T cell therapies (CAR-T and TCR-T) depends on their functional status, including activation, proliferation, inhibition, and exhaustion.
- Accurate assessment of these functions requires sensitive and reproducible in vitro cytotoxicity assays for clinical translation.
Purpose of the Study:
- To compare various in vitro cytotoxicity assays for their suitability in evaluating cellular therapeutics.
- To guide investigators in selecting appropriate assays for assessing immunotherapeutic efficacy.
Main Methods:
- Comparative analysis of chromium release, bioluminescence, impedance, and flow cytometry assays.
- Evaluation of experimental setup, advantages, disadvantages, and limitations of each assay.
- Discussion of advanced assays for repeated antigen exposure and combinatorial testing.
Main Results:
- Each assay type (chromium release, bioluminescence, impedance, flow cytometry) presents unique strengths and weaknesses for measuring T cell cytotoxicity.
- US Food and Drug Administration (FDA) guidelines for potency assays in cell therapy release were considered.
- Advanced assay designs can address complex scenarios like variable antigen expression and combination therapies.
Conclusions:
- The choice of cytotoxicity assay significantly impacts the assessment of 'living drug' efficacy.
- Understanding assay limitations and FDA requirements is essential for reliable evaluation of immunotherapeutic agents.
- This review provides a framework for selecting optimal assays for preclinical and clinical development.
