Bispecific GPC3/PD-1 CAR-T cells for the treatment of HCC

Constantly stimulated by the tumor microenvironment (TME), programmed death 1 (PD-1) is elevated, and it interacts with PD ligand 1 (PD-L1), rendering chimeric antigen receptor (CAR)-T cells dysfunctional. Hence, CAR-T cells immune to PD-1-induced immunosuppression were constructed to improve the function of CAR-T cells in hepatocellular carcinoma (HCC). Double-target CAR-T cells, targeting glypican-3 (GPC3) [a tumour-associated antigen (TAA)] and hindering PD-1-PD-L1 binding, were established. The expression of GPC3, PD-L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR-T cells were determined using lactate dehydrogenase release assay, enzyme-linked immunosorbent assay, and flow cytometry, respectively. HCC cells were targeted and eliminated by double-target CAR-T cells. These double-target CAR-T cells limit PD-1-PD-L1 binding and sustain cytotoxicity to PD-L1(+) HCC cells. The relatively low IR expression and differentiation level in double-target CAR-T cells in tumour tissues induced tumour-suppression and extended survival in PD-L1(+) HCC TX models, as opposed to their single-target counterparts. The results of the present study suggested that the newly constructed double-target CAR-T cells exhibit stronger tumour-suppressing effects in HCC than their single-target counterparts, which are common, suggesting the potential of strengthening CAR-T cell activity in HCC treatment.