Development of Homogeneous Dual-cell Based T-cell Dependent Cellular Cytotoxicity Assays

Protein-based therapeutics have undergone an evolution since the first products were marketed over 30 years ago. These early modalities were directed towards interaction with a single target while currently, many protein-based therapeutics are being designed to encompass more than one functional domain and are engineered to interact with more than one target. The Bispecific T-cell Engager (BiTE®) therapeutic class are comprised of two single chain variable fragments (scFv) from two distinct monoclonal antibodies which are connected by a short linker. One scFv is directed against cluster of differentiation 3 (CD3) on T cells, while the other is directed against a cell-surface tumor antigen. In vivo, the engagement of the BiTE® molecule with the T cell and the tumor cell induces the formation of a cytolytic synapse resulting in specific lysis of the tumor cells. In vitro, T cell dependent cellular cytotoxicity (TDCC) assays are used to demonstrate this mechanism of action. Traditional dual-cell based cytotoxicity assays have included labelling target cells with either dyes or radioactivity rendering the assay format to be non-homogeneous. We have developed and implemented homogeneous dual cell-based TDCC assays by employing the use of target cells that constitutively express luciferase as a surrogate marker of viable cell number. By utilizing this format for target cells, the change in viable target cell number can be readily detected by addition of a reagent that contains detergent to lyse the cells, and luciferin, a substrate for luciferase. This talk will highlight some of the important elements to consider during assay development.