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.

基因工程“活药物”（包括嵌合抗原受体和 T 细胞受体 T 细胞）的抗肿瘤功效受其活化、增殖、抑制和耗竭的影响。共同反映这些功能的灵敏且可重复的细胞毒性测定是翻译这些细胞治疗剂的基本要求。在这里，我们比较了各种体外细胞毒性测定（包括铬释放、生物发光、阻抗和流式细胞术）的实验设置、适当的用途、优点和缺点，以及克服其局限性的措施。我们还重点介绍了美国食品和药物管理局 (FDA) 关于发布临床细胞治疗产品的效力测定的指令。此外，我们讨论了重复抗原暴露的高级测定以及免疫效应细胞、免疫调节抗体和具有可变抗原表达的靶标组合的同时测试。这篇综述文章应有助于使研究人员掌握必要的知识，以选择适当的细胞毒性试验来测试单独或联合使用免疫治疗剂的功效。