Abstract: Chimeric antigen receptor (CAR) T cell therapy has emerged as a key new tool in the treatment of human cancers, achieving unprecedented patient outcomes in difficult to treat cancers such as acute lymphoblastic leukemia. To extend this treatment modality to new cancer types and improve its efficacy, a variety of enhancements have been proposed. ArsenalBio ICT (Integrated Circuit T Cell) cells include genetic perturbations to alter cell state, receptor strategies to target multiple tumor antigens, engineered expression of cytokines and chemokines, and variations in the chimeric antigen receptor (CAR) binding or signaling domains.
To effectively survey this complex design space, we have developed a massively parallel, in vitro platform for novel cell therapy discovery. This highly automated platform enables parallel characterization of thousands of cellular designs with diverse readouts, including T cell proliferation, cytotoxicity, cytokine secretion, and flow cytometry. Whole transcriptome profiling is an additional, key readout enabled by a cost-effective, multiplexed 3’ digital RNA-seq protocol. As a comprehensive, global assessment of cell states, this readout allows for an in-depth characterization of phenotypic differences between engineered T cell designs and provides insight into underlying mechanisms.
Here we describe the use of this platform to optimize ICT cell design with a focus on a transcriptional profiling pipeline and analysis. Tens of thousands of variations of genetic perturbations, logic gate components, and CAR signaling domains were initially filtered using pooled screens with bulk and single-cell sequencing readouts. The resulting designs were evaluated using our automated, arrayed platform to identify individual components and their combinations with enhanced function. The ICT cell components identified using this approach promote superior potency, expansion, and persistence and induce resistance to tumor microenvironment-associated suppression. Our high-throughput, data-driven approach to cell design and optimization provides a scalable platform that greatly accelerates the development of safe and efficacious ICT cell therapies.