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

Abstract

Constantly stimulated by the tumor microenvironment (TME), programmed death1 (PD‑1) is elevated, and it interacts with PD ligand1 (PD‑L1), rendering chimeric antigen receptor (CAR)‑Tcells dysfunctional. Hence, CAR‑Tcells immune to PD‑1‑induced immunosuppression were constructed to improve the function of CAR‑Tcells in hepatocellular carcinoma (HCC). Double‑target CAR‑Tcells, 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‑Tcells 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‑Tcells. These double‑target CAR‑Tcells 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‑Tcells 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.