Optimized cytotoxicity assay for co-suspended effector and target cells

In recent years, adoptive cell therapy of immune effector cells, such as chimeric antigen receptor-T (CAR-T) cells, natural killer (NK) cells, and epitope-specific cytotoxic T lymphocyte (CTL) cells have been employed in clinical trials. In addition, CD19 CAR-T cells have been approved by the FDA for treatment of non-Hodgkin lymphoma and diffuse large B-cell lymphoma. In this context, it is vital to detect cellular cytotoxicity and monitor the quality of ex vivo expanded immune cells before product release and patient infusion. Target cells could proliferate in parallel with effector cells during the cytotoxicity assay, making it difficult to estimate the death ratio using conventional approaches. Meanwhile, non-specific dyes or non-homogeneous biomarkers for target cells may interfere with the final readout post addition of effector cells. Here, we modified a component of the coincubation medium to suppress the spontaneous release of bis(acetoxymethyl)2,2':6',2″-terpyridine-6,6″-dicarboxylate and sustained the window at a stable range (~70%). Further, the optimized Eu-TDA method presented reliable outcomes compared with lactate dehydrogenase detection and was compatible with cytotoxicity tests for NK cells and specific CTLs. Finally, the reported assay can accurately detect death of target cells depending on the amount of hydrophilic complex and can be reliably applied in quality control and cell activity evaluation tests on co-suspended effector and target cells. SUMMARY: A medium component for cellular coincubations (and associated protocols) have been optimized and validated for cytotoxicity assays, which can reliably evaluate the potency of engineered CD19 CAR-T cells, NK cells, and specific CTLs. In particular, the reported method can be applied widely in routine assays for bi-suspended effector and target cells with a stable window.