XCL1/Glypican-3 fusion protein induces potent CD8+ T-cell generation and enhances the anti-PD1 effect to suspend hepatocellular carcinoma development

Objective: To induce potent CD8+ T-cells against glypican-3(GPC3), which is overexpressed in hepatocellular carcinoma(HCC), to suspend tumor development. Methods: Since the chemokine receptor XCR1 is selectivelyexpressed on professional cross-presenting CD8α+ dendriticcells (DCs), which have been identified as the most potentcells to induce antigen-specific CD8+ T-cell generation, weconstructed a XCL1/GPC3 fusion protein as a potential cancervaccine. The fusion gene expression and targeting of the expressed fusion protein to CD8α+ DCs were examined inmouse lymph nodes. Protein function was further examined byusing freshly isolated human lymph node cells. We immunizedthe mice subcutaneously with the XCL1/GPC3 fusion gene andmonitored the tumor growth in a diethylnitrosamine-inducedautochthonous murine HCC model with a chronic hepatitis Bvirus (HBV) infection background. The effects of the XCL1/GPC3 fusion protein were further analyzed in human HCCtissue-derived xenografts in NSG immunodeficient mice. Results: The gene-transfected cells expressed and secretedextracellular XCL1/GPC3 fusion protein, which showed strongchemotactic activity for murine CD8α+ DCs and the humanequivalent, CD141+ DCs, in vitro. Compared with the GPC3-treated cells, XCL1/GPC3 fusion protein induced more potentGPC3-specific CD8+ T-cell generation that showed higherantigen-specific cytotoxicity to the GPC3144-152 peptide. Afterimmunization with the XCL1/GPC3 fusion gene, the productswere detected in the immunization sites of mice and in theirdraining lymph nodes. The fusion proteins were mainlyfound in the CD11c+ DCs in the T-cell zones of lymph nodes.Compared with the GPC3 gene alone, immunization with theXCL1/GPC3 fusion gene generated significantly more GPC3-specific CD8+ IFN-γ-producing T-cells in the mouse livers,which could specifically eliminate GPC3-expressing tumorcells. We immunized the diethylnitrosamine-treated mice thathave a chronic HBV infection background, starting at week 4,when HCC cells were detected in the liver by histology, or atweek 14, when small liver tumors were surgically observed. Byweek 22 after diethylnitrosamine injection when all the micewere sacrificed, significantly fewer HCC cells were found inthe XCL1/GPC3-immunized mice than in the GPC3 geneimmunizedmice (P u003c 0.01). T-cells isolated from XCL1/GPC3-immunized mice were able to eliminate subcutaneouslytransplanted GPC3-expressing hepatoma tumors. The effectswere further enhanced in combination with injection of 200 μgof anti-PD1 antibody. More activated NK cells, NKT cells, andtype 1 innate lymphoid cells (ILC1s) were also detected in theXCL1/GPC3-immunized mice livers. In NSG immunodeficientmice with human HCC tissue-derived xenografts, the XCL1/GPC3 fusion protein-stimulated autologous T-cells were able tosuspend HCC growth. One dose of anti-PD1 further enhancedthe effect on HCC growth inhibition. Conclusions: Immunization with the XCL1/GPC3 fusion gene couldinduce potent CD8+ T-cell generation. The XCL1/GPC3 serves as apromising cancer vaccine for intervention in HCC development. DOI: 10.20892/j.issn.2095-3941.2018.S056