An in situ generated CAR-M with IFNg and negative dominance Sirpa isoform augments hepatocellular carcinoma immunotherapy

Background and AimsChimeric 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 ResultsBuilding on first-generation CD3{zeta}-based CAR-M, we developed a novel CAR targeting human GPC3, incorporating IFN-{gamma} and the extracellular domain of SIRP (SIRPECD) to improve CAR-M persistence and block the CD47-SIRP immune checkpoint. Following delivery via lipid nanoparticle-encapsulated mRNA (LNP-mRNA), the self-secreted IFN-{gamma} sustained M1 polarization through phospho-STAT1 activation. Meanwhile, the ectopically expressed SIRPECD competitively bound to CD47 on tumor cells, thereby blocking the endogenous SIRP-SHP2 interaction in a dominant-negative manner. This design enhanced pro-inflammatory activity and anti-tumor efficacy compared to CD3{zeta}-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 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. ConclusionsCollectively, 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.