Bioluminescent assay platforms for the discovery and development of engineered T cell therapies for cancer 3787

Description T cell therapies including CAR-T are a promising approach for the treatment of cancer. Development of these therapies relies on the ability to rigorously evaluate candidate TCRs and CARs, characterize the effector functions of cell therapy products, and reproducibly measure the potency for development and lot release. We have developed novel bioluminescent bioassays to address these bottlenecks. To facilitate the characterization of transgenic TCRs, we developed two TCRαβ-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR α and β chains into TCRαβ-null reporter T cel llines results in peptide-dependent TCR activation and luciferate reporter expression. The select expression of CD4 or CD8 enables the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. To quantitate the functions of engineered T cells, we have used split NanoBiT luciferase technology to develop two homogenous assays: a HiBiT target cell killing assay and cytokine detection immunoassays. Incubation of CAR-T with target cells stably expressing HiBiT results in lysis of the target cells and induction of a luminescent signal proportional to cell death. Cytokine production can be quantitatively measured in the homogenous immunoassays. These luminescent assays are homogenous, fast, highly sensitive, and have robust assay windows. They represent a new set of tools for the discovery and development of T cell-based immunotherapies.