Specific target cell killing by immune effector cells is a key functional endpoint for immunotherapy strategies including (i) T cell recruitment using bi-specific molecules, (ii) NK cell-mediated ADCC and (iii) cell therapy using CAR-T and/or CAR-NK cells. Common to these approaches is the need to quantitatively measure target cell killing in mixed cell cultures. Assays that measure total cell death are complicated by the presence of effector cells in the system, which can lead to high background and poor sensitivity. Assays that measure specific cell death of target cells often require radioactivity or other complex labeling protocols and are relatively low-throughput.
We have developed a highly sensitive bioluminescent assay to measure target cell-specific killing in mixed cell cultures. This assay involves target cell-specific expression of a cytosolic protein fused to HiBiT, an 11 a.a. protein tag. When the target cells are killed, HiBiT is released into the extracellular medium that contains LgBiT. Binding of HiBiT and LgBiT reconstitutes a bright, luminescent enzyme that is measured using a standard benchtop luminometer. The assay is sensitive and linear over a wide range of cell number (e.g. 100-500,000 cells). Here we demonstrate the target cell-specific expression of HaloTag-HiBiT, its release and binding to LgBiT following cell death, and quantitative measurement of bioluminescence at a single time-point or in real-time over several hours or days. The assay is compatible with a broad range of cell types expressing endogenous or exogenous cellular targets for antibody and/or cell therapies.