Bispecific c-Met/PD-L1 CAR-T Cells Have Enhanced Therapeutic Effects on Hepatocellular Carcinoma

Abstract

T cells expressing chimeric antigen receptors, especially CD19 CAR-T cells have exhibited effective antitumor activities in B cell malignancies, but due to several factors such as antigen escape effects and tumor microenvironment, their curative potential in hepatocellular carcinoma has not been encouraging. To reduce the antigen escape risk of hepatocellular carcinoma, this study was to design and construct a bispecific CAR targeting c-Met and PD-L1. c-Met/PD-L1 CAR-T cells were obtained by lentiviral transfection, and the transfection efficiency was monitored by flow cytometry analysis. LDH release assays were used to elucidate the efficacy of c-Met/PD-L1 CAR-T cells on hepatocellular carcinoma cells. In addition, xenograft models bearing human hepatocellular carcinoma were constructed to detect the antitumor effect of c-Met/PD-L1 CAR-T cells in vivo. The results shown that this bispecific CAR was manufactured successfully, T cells modified with this bispecific CAR demonstrated improved antitumor activities against c-Met and PD-L1 positive hepatocellular carcinoma cells when compared with those of monovalent c-Met CAR-T cells or PD-L1 CAR-T cells but shown no distinct cytotoxicity on hepatocytes in vitro. In vivo experiments shown that c-Met/PD-L1 CAR-T cells significantly inhibited tumor growth and improve survival persistence compared with other groups. These results suggested that the design of single-chain, bi-specific c-Met/PD-L1 CAR-T is more effective than that of monovalent c-Met CAR-T for the treatment of hepatocellular carcinoma., and this bi-specific c-Met/PD-L1 CAR is rational and implementable with current T-cell engineering technology.

Keywords: CAR-T cell; PD-L1; antigen relapse effects; c-Met; hepatocellular carcinoma.

Figure 1

Construction and identification of CP CAR-T cells. (A) Schematic illustration of a lentiviral vector encoding CARs. (B) Expression of CD3ζ in 293T cells transfected with different CARs was analyzed by Western blot analysis. (C) In vitro proliferation rate of CAR-T cells. (D) Transduction efficiency of chimeric receptor expression on T cells was detected using flow cytometry. (E) The phenotype of CAR-expressing T cells was detected by flow cytometry after staining with anti-human CD4-FITC and anti-human CD8-APC. Data are shown as mean ± SD.

Figure 2

In vitro cytotoxicity of CP CAR-T cells against HCC cells. (A) Human CAR- T transduced cells or activated T cells were co-incubated with human MHCC97, HepG2 HCC cell lines, human normal liver epithelial cells LO2 and c-Met knockdown HepG2 cells (HepG2^lo) for 12 h. The cell ratios of effector: target (E: T) was 10:1, 5:1, and 2:1, specific lysis was tested by LDH release assay. (B) At the cell ratios of E: T was 10:1, the specific cytotoxicity of different effector cells against various target cells. Data are representative of three independent experiments. Data are shown as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001. (*CP CAR-T cells group compared with activated T cells, *marked with red color means CP CAR-T cells group compared with monovalent c-Met CAR-T cells group).

Figure 3

In vitro more specific cytokines produced by c-Met/PD-L1 CAR-T cells against HCC cells. At the cell ratios of E: T was 10:1, c-Met/PD-L1, c-Met, PD-L1, Unrelated CAR-T cells or activated T cells were co-incubated with human MHCC97 cells, HepG2 HCC cells, human normal liver epithelial cells LO2 and c-Met knockdown HepG2 cells (HepG2^lo), for 24 h. (A) IFN-γ production was measured by ELISA after the incubation of the CAR- T cells or activated T cells and target cells. (B) IL-2 -production was measured by ELISA after the incubation of the CAR- T cells or activated T cells and target cells. Data are representative of three independent experiments. Data are shown as mean ± SD. *p <0.05, ** p <0.01 and *** p <0.001.

Figure 4

In vivo antitumor activities of c-Met/PD-L1 CAR-T cells against c-Met/PD-L1 positive subcutaneous HCC tumors. (A) Schematic representation the subcutaneous implantation of HepG2-fLuc cells and treatment of CAR-T cells. (B) Mice bearing HepG2-fLuc subcutaneous xenografts progression was assessed by bioluminescence assay on day 0, 3, 6, 9, and 13(n=5). (C) Quantification of tumor burden was depicted according to the bioluminescence signal. (D) Representative H&E images of tumor xenografts were taken with a microscope under 100x and 200x magnification. (E) Kaplan–Meier survival curves of mice treated with CP CAR-T cells, c-Met CAR-T cells and activated T cells (n=5). (F) Representative H&E images of normal organ of mice treating with CP CAR-T cells, c-Met CAR-T cells and activated T cells were taken with a microscope under 100x magnification. Data are shown as mean ± SD. *p < 0.05.

Figure 5

CP CAR-T cells specifically inhibiting c-Met/PD-L1 expression in subcutaneous HCC tumors. (A) Schematic showing how to establish a bilateral subcutaneous tumor. (B) Tumor progression was assessed by bioluminescence assay on day 0 and 9 after CAR-T cells injection(n=5). (C) Quantification of tumor burden was depicted according to the bioluminescence signal. (D, E) Immunohistochemical (IHC) staining for anti-human c-Met were performed on tumor samples, representative images were taken with a microscope under 100x and 400x magnification. (F) Immunohistochemical (IHC) staining for anti-human Ki-67 were performed on tumor samples, representative images were taken with a microscope under 400x. Data are shown as mean ± SD. **p < 0.01.