Cytotoxicity-based High-throughput Screening System for CAR T Cell

ABSTRACT Some chimeric antigen receptor (CAR) T cell therapies have shown strong clinical efficacy, yet systematic screening of new CAR designs remains constrained by labor-intensive, low-throughput evaluation methods. To address this limitation, we developed a cytotoxicity-centered, high-throughput screening platform that integrates single-cell pooled screening with fully automated arrayed screening enabling both large-scale library handling and quantitative functional resolution for systematic CAR design exploration. Using a mutation-based CAR design approach guided by protein fitness prediction, we generated a 4-1BB–based CAR library with approximately 10⁸ theoretical variants while minimizing the prevalence of low-activity designs. In pooled screening, CAR T cells were evaluated at the single-cell level based on cytotoxicity and proliferation, enabling rapid enrichment of high-performing variants from a highly diverse library. Subsequent automated arrayed screening quantitatively measured cytotoxicity with high reproducibility, providing high-resolution functional data suitable for comparative ranking. Selected CAR variants demonstrated superior antitumor efficacy in a leukemia xenograft model compared with a template CAR. Furthermore, systematic analysis of mutation sites from an enhanced CAR variant identified essential mutation combinations underlying functional enhancement. Together, this study establishes a cytotoxicity-focused screening framework that provides a robust approach for optimizing CAR architectures and accelerating the development of CAR T-cell therapies. Competing Interest Statement A.O., Y.I., S.H., T.Y., T.K., D.I., and K.Y. are current employees of Hitachi, Ltd. S.M. is employed by Staff Service Co., Ltd. and is currently dispatched to Hitachi, Ltd. Y.N. is employed by PERSOL TEMPSTAFF CO., LDT. and is currently dispatched to Hitachi, Ltd. T.I. is employed by BREXA Advan Inc. and is currently dispatched to Hitachi, Ltd.