SUMMARY
Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers, but have shown limited efficacy against solid tumors. Here, we introduce ‘CAR Pooling’, a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with diverse immune costimulatory domains were assessed in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several non-native domains from the TNF receptor family exhibited enhanced proliferation (CD40) or cytotoxicity (BAFF-R and TACI) relative to clinical benchmarks, and fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic and NK-cell-like innate phenotype previously associated with positive clinical outcomes. ‘CAR Pooling’ enables efficient exploration of how CAR design affects cell activity and can be applied to optimize receptors across a range of applications and cell types.
Competing Interest Statement
K.T.R. is a cofounder, consultant, SAB member, and stockholder of Arsenal Biosciences. He was a founding scientist/consultant and stockholder in Cell Design Labs, now a Gilead Company. K.T.R. holds stock in Gilead. K.T.R. is on the SAB of Ziopharm Oncology and an Advisor to Venrock. D.B.G. is a stockholder and consultant of Arsenal Biosciences. D.B.G is a scientific advisor and stockholder of Manifold Bio, Gordian Biotechnology, and NExTNet. J.A.B is a co-founder, CEO and a Board member of Sonoma Biotherapeutics. He is a co-founder of Celsius Therapeutics; a member of the Board of Directors of Gilead and Provention Bio, and a member of the scientific advisory boards of Arcus Biosciences, Solid Biosciences, and Vir Biotechnology. J.A.B is the A.W. and Mary Margaret Clausen Distinguished Professor in Metabolism and Endocrinology in the Diabetes Center at the University of California, San Francisco. A.M. is a compensated co-founder, member of the boards of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. was a compensated member of the scientific advisory board at PACT Pharma and was a compensated advisor to Juno Therapeutics and Trizell. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, and PACT Pharma. A.M. has received fees from Merck and Vertex. The Marson lab has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead, and Anthem. C.J.Y. is a Scientific Advisory Board member for and holds equity in Related Sciences and ImmunAI, is a consultant for and holds equity in Maze Therapeutics, and is a consultant for TReX Bio. C.J.Y. has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. K.T.R, D.B.G, C.S.A., J.A.B., and A.M. are listed as inventors on a patent application related to this work, which has been licensed.
