Characterization of T Cell Engaging Bispecific Antibodies and Combination Therapies with a High-Throughput...

T cell dependent bispecific antibodies (TCBs) are a potent class of therapeutic molecules that redirect the cytotoxic activity of CD8+ T cells against target cells by engagement of CD3 and a tumor antigen. In vitro functional screening of TCBs often relies on end point readouts of activity that fail to capture kinetics of T cell activation. The lower throughput of traditional T cell killing assays also poses challenges to assess TCB potency and efficacy against tumor cell lines varying in target expression level, and across a range of effector to target cell ratios. Here, we describe the development of an automated, high throughput T cell dependent in vitro cytotoxicity assay that enables kinetic and multiparametric assessment of T cell killing and cytokine production. We applied this assay to determine the functional activity of TCBs against hematological and solid tumor targets, including molecules that bind to different tumor antigens and CD3 epitopes. Moreover, we incorporated a set of data analysis and visualization tools to integrate multiple parameters of T cell activity and classify TCBs into distinct subsets. We expanded the platform to screen co-stimulatory receptor agonist antibodies that combine with TCBs to enhance antitumor activity. Taken together, our approach comprises a novel combination of biological, technical, and analytical methods to characterize T cell functional activity. This method enables robust and high throughput assessment of TCBs and combination therapies and enhances selection of lead candidates with distinct characteristics for further development.