HiBiT mRNA delivery: a versatile platform for cell therapy and cytotoxicity assays in diverse cell lines 4504

Abstract Description The HiBiT Target Cell Killing Assay (HiBiT TCK Assay) is a streamlined, sensitive method for selectively quantifying target cell lysis in coculture experiments with effector cells. High BiT (HiBiT) is an 11 a.a. peptide that binds spontaneously to Large BiT (LgBiT), forming a bright, luminescent enzyme. For HiBiT TCK, target cells are engineered to express a HiBiT-tagged protein, and release of this protein by dead/dying cells leads to complementation with cell impermeable LgBiT. This gain-of-signal approach enables precise measurements of target cell killing induced by various effectors, including CAR-T cells and cell killing mediated by bispecific antibodies. Here we demonstrate use of mRNA transfection to transiently express HaloTag-HiBiT in target cells, allowing for the rapid and uniform labeling of both dividing and non-dividing cells. This approach extends the HiBiT TCK Assay beyond use of stable cell lines alone. We also demonstrate use of mRNA transfection to modulate target receptor expression levels in cells stably expressing a HiBiT-tagged protein. Topic Categories Technological Innovations in Immunology (TECH)