Granzyme B-based CAR-T cells targeting membrane-bound HSP70 suppress solid tumor growth and metastasis

Utilizing CAR-T cells to eliminate circulating tumor cells (CTCs) and inhibit metastasis is a promising strategy. However, this approach is hindered by the lack of specific antigens. Membrane-bound HSP70 (mHSP70) is commonly expressed on the cell membrane of numerous tumor types, notably on CTCs, making it an ideal target for CAR-T therapy to treat these malignancies and prevent metastasis. Here, we generated CAR T cells based on natural ligand granzyme B (GrB-CAR T) targeting mHSP70. GrB-CAR T cells exhibited potent cytotoxicity against a broad spectrum of cancer cell lines and stem-like cancer cells in vitro and effectively inhibited xenograft tumor growth in vivo. Importantly, CTCs maintain mHSP70 expression in xenograft models, and GrB-CAR T cells markedly decreased the number of CTCs, thereby preventing cancer metastasis. Moreover, despite human granzyme B exhibits cross-reactivity with mouse and macaque mHSP70-particularly given the complete homology between macaque and human mHSP70-no obvious adverse effects were observed in the animals treated with GrB-CAR T cells. These results demonstrate GrB-CAR T cells as a safe and effective approach with broad-spectrum anticancer activity and provide compelling experimental evidence for CAR T cell-mediated metastasis inhibition through targeting CTCs.