c-Met is a hepatocyte growth factor receptor overexpressed in many tumors such as hepatocellular carcinoma(HCC).Therefore,c-Met may serve as a promising target for HCC immunotherapy.Modifying T cells to express c-Met-specific chimeric antigen receptor(CAR)is an attractive strategy in treating c-Met-positive HCC.This study aimed to systematically evaluate the inhibitory effects of 2nd-and 3rd-generation c-Met CAR-T cells on hepatocellular carcinoma(HCC)cells.Here,2nd-and 3rd-generation c-Met CARs containing an anti-c-Met single-chain variable fragment(scFv)as well as the CD28 signaling domain and CD3ζ(c-Met-28-3ζ),the CD 137 signaling domain and CD3ζ(c-Met-137-3ζ),or the CD28 and CD137 signaling domains and CD3ζ(c-Met-28-137-3ζ)were constructed,and their abilities to target c-Met-positive HCC cells were evaluated in vitro and in vivo.All c-Met CARs were stably expressed on T cell membrane,and c-Met CAR-T cells aggregated around c-Met-positive HCC cells and specifically killed them in vitro.c-Met-28-137-3ζ CAR-T cells secreted more interferon-gamma(IFN-γ)and interleukin 2(IL-2)than c-Met-28-3ζ CAR-T cells and c-Met-137-3ζ CAR-T cells.Compared with c-Met low-expressed cells,c-Met CAR-T cells secreted more cytokines when co-cultured with c-Met high-expressed cells.Moreover,c-Met-28-137-3ζ CAR-T cells eradicated HCC more effectively in xenograft tumor models compared with the control groups.This study suggests that 3rd-generation c-Met CAR-T cells are more effective in inhibiting c-Met-positive HCC cells than 2nd-generation c-Met CAR-T cells,thereby providing a promising therapeutic intervention for c-Met-positive HCC.