An in situ generated CAR-M with IFN-γ and negative dominance Sirpα isoform augments hepatocellular carcinoma immunotherapy

Abstract Background and aims Chimeric antigen receptor (CAR) T cells have shown strong efficacy in hematological cancers but limited success in solid tumors. Macrophages, with their natural ability to infiltrate tumors, modulate immunity, and phagocytose cancer cells, offer a promising alternative when engineered with CARs. While studies have demonstrated the feasibility and anti-tumor activity of CAR macrophages (CAR-M), enhancing their persistence and phagocytic capacity remains a key challenge. Methods and results Building on first-generation CD3ζ-based CAR-M, we developed a novel CAR targeting human GPC3, incorporating IFN-γ and the extracellular domain of SIRPα (SIRPα ECD ) to improve CAR-M persistence and block the CD47–SIRPα immune checkpoint. Following delivery via lipid nanoparticle-encapsulated mRNA (LNP-mRNA), the self-secreted IFN-γ sustained M1 polarization through phospho-STAT1 activation. Meanwhile, the ectopically expressed SIRPα ECD competitively bound to CD47 on tumor cells, thereby blocking the endogenous SIRPα–SHP2 interaction in a dominant-negative manner. This design enhanced pro-inflammatory activity and anti-tumor efficacy compared to CD3ζ-only CAR-M. Single-cell RNA sequencing and cellular analysis showed that in situ programmed CAR-M reprogrammed the tumor microenvironment toward inflammation in a murine hepatocellular carcinoma (HCC) model. Moreover, CAR-M derived from human peripheral blood mononuclear cells (PBMCs) effectively phagocytosed human HCC organoids while sparing healthy tissues, indicating clinical potential. Conclusions Collectively, our work presents a novel CAR design that enhances phagocytic function and sustains anti-tumor activity, offering a promising strategy for human solid tumor immunotherapy.