Pooled screening identifies combinatorial CAR signaling domains for next-generation CAR-M immunotherapies [scRNA-seq]

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. Overall design: PSMA-positive prostate tumors were bilaterally inoculated into the subcutaneous flanks of NCG mice. After confirmation of tumor engraftment, human peripheral blood mononuclear cells (PBMCs) were transplanted to reconstitute the immune system. UTD or COMB7 CAR-Ms were injected intratumorally into either the right or left flank. Four days later, the tumors were harvested and the human CD45-positive cells were sorted and subjected to scRNA-seq.