A quadrivalent in vivo CAR-macrophage repress solid tumors and overcome tumor heterogeneity through antigen spreading

We designed a quadrivalent in vivo CAR-engineered macrophage based on the LNP-mRNA system, delivering FAP CAR-ΔTGFβRII and GPC3 CAR-Super IL2 (LNP-GF CAR) via LNP for the treatment of primary hepatocellular carcinoma. LNP-GF CAR demonstrated significant anti-tumor efficacy in a primary hepatocellular carcinoma mouse model. To further dissect the anti-tumor mechanism of LNP-GF CAR, we performed single-cell transcriptomic sequencing on tumor regions of the liver from treated model mice. Based on the single-cell transcriptomic data, we found that exogenous CAR mRNA was highly enriched in tissue-resident macrophages within the liver tumor regions. CAR-expressing macrophages showed significant upregulation of genes related to antigen cross-presentation and promotion of T cell activation and proliferation. Consistently, the numbers of proliferative and activated CD8 T cells were significantly increased. These findings provide further evidence that in situ CAR macrophages can activate CD8 T cells through antigen cross-presentation, and deepen our understanding of the anti-tumor mechanism of LNP-GF CAR.