Project description:CAR T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction is a common cause of treatment failure. Here we present CELLFIE, a CRISPR screening platform for enhancing CAR T cells across multiple clinical objectives. We performed genome-wide screens in human primary CAR T cells with readouts capturing key aspects of T cell biology, including proliferation, target cell recognition, activation, apoptosis and fratricide, and exhaustion. Screening hits were prioritized using a new in vivo CROP-seq method in a xenograft model of human leukemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered RHOG knockout as a potent and unexpected CAR T cell enhancer, both individually and together with FAS knockout, which was validated across multiple in vivo models, CAR designs, and patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify effective knockout pairs, and saturation base editing screens to characterize RHOG variants. In summary, we discovered, validated, and biologically characterized CRISPR-boosted CAR T cells that outperform standard CAR T cells in widely used benchmarks, establishing a foundational resource for optimizing cell-based immunotherapies.
Project description:Chimeric antigen receptor-engineered macrophages (CAR-Ms) hold great promise for solid tumor immunotherapy. The intracellular domains ICDs of CARs determine the phenotypic output of therapeutic macrophages but remain largely unexplored. Here, we constructed a library of CARs containing 131 unique signaling domains derived from native immune receptors and revealed 17 ICDs that enhanced macrophage phagocytosis, the inflammatory response and tumor infiltration in vitro and in vivo. We further developed a scalable 3’ barcode technology, CARode, to uniquely label and trace ICD variants in a large-scale combinatorial signaling domain CAR library and applied it to perform single-cell RNA sequencing and single-cell CAR analysis to measure the synergetic effects of ICD combinations in promoting macrophage activation. This platform includes a novel CD40-LY9-FCRL1 chimeric receptor that modulates the tumor microenvironment and improves solid tumor clearance. In conclusion, pooled screening facilitates the discovery of complex ICD constructs to program macrophage functions for therapeutic applications.
Project description:Chimeric antigen receptor-engineered macrophages (CAR-Ms) hold great promise for solid tumor immunotherapy. The intracellular domains ICDs of CARs determine the phenotypic output of therapeutic macrophages but remain largely unexplored. Here, we constructed a library of CARs containing 131 unique signaling domains derived from native immune receptors and revealed 17 ICDs that enhanced macrophage phagocytosis, the inflammatory response and tumor infiltration in vitro and in vivo. We further developed a scalable 3’ barcode technology, CARode, to uniquely label and trace ICD variants in a large-scale combinatorial signaling domain CAR library and applied it to perform single-cell RNA sequencing and single-cell CAR analysis to measure the synergetic effects of ICD combinations in promoting macrophage activation. This platform includes a novel CD40-LY9-FCRL1 chimeric receptor that modulates the tumor microenvironment and improves solid tumor clearance. In conclusion, pooled screening facilitates the discovery of complex ICD constructs to program macrophage functions for therapeutic applications.
Project description:Chimeric antigen receptor-engineered macrophages (CAR-Ms) hold great promise for solid tumor immunotherapy. The intracellular domains ICDs of CARs determine the phenotypic output of therapeutic macrophages but remain largely unexplored. Here, we constructed a library of CARs containing 131 unique signaling domains derived from native immune receptors and revealed 17 ICDs that enhanced macrophage phagocytosis, the inflammatory response and tumor infiltration in vitro and in vivo. We further developed a scalable 3’ barcode technology, CARode, to uniquely label and trace ICD variants in a large-scale combinatorial signaling domain CAR library and applied it to perform single-cell RNA sequencing and single-cell CAR analysis to measure the synergetic effects of ICD combinations in promoting macrophage activation. This platform includes a novel CD40-LY9-FCRL1 chimeric receptor that modulates the tumor microenvironment and improves solid tumor clearance. In conclusion, pooled screening facilitates the discovery of complex ICD constructs to program macrophage functions for therapeutic applications.