Robust and highly efficient generation of functional chimeric antigen receptor engineered murine macrophages that alleviate cardiac fibrosis

To investigate the therapeutic potential of chimeric antigen receptor engineered macrophages (CAR-Ms), studies in immunocompetent mouse models are essential. However, due to the low editing efficiency and limited proliferative capacity of primary murine macrophages, efficient generation of functional CAR-Ms remains a technical challenge, hampering the advancement of this promising immunotherapeutic approach. Here, we established immortalized mouse monocytes using the DHFR-HOXB8 system, in which the presence of overexpressed HOXB8 protein is controlled by trimethoprim (TMP), allowing for conditional immortalization and subsequent macrophage differentiation. Transcriptomic and functional analyses confirmed that DHFR-HOXB8-derived cells retain bona fide monocyte and macrophage identity, closely resembling primary cells. Using this platform, we achieved CAR editing efficiencies exceeding 95% in monocytes/macrophages and showed that CAR expression conferred potent phagocytic and cytotoxic activity upon DHFR-HOXB8-derived macrophages against target cells. Adoptive transfer of FAP-targeted CAR-Ms in mouse models of myocardial infarction (MI) and transverse aortic constriction (TAC) attenuated cardiac fibrosis and improved cardiac function, demonstrating their in vivo activity and underscoring their therapeutic potential across diverse cardiac pathologies. This study thus provides a potent platform for generating genetically engineered macrophages suitable for preclinical murine studies.