AFP_158-166 Specific TCR-engineered T Cells For Adoptive Cellular Immunotherapy Of Hepatocellular Carcinoma

Part Ⅰ Construction of AFP158-166-specific TCR engineered T cellsObjective:To construct AFP158-166-specific TCR engineered T cells directed against hepatocellular carcinoma and evaluate their specific cytotoxicity in vitro.Method:Dendritic cells (DC) derived from peripheral blood mononuclear cells (PBMC) of HLA-A0201healthy donor were loaded with AFP158-166peptides and used to stimulate AFP158-166-specific T cell. Total RNAs were extracted from pentamer-sorted AFP158-166-specific T cells and reverse transcribed into cDNA. The coding sequence for the full-length of TCRa and TCRβ chains which linked by the sequence for the2A self-cleaving peptide was synthesized and cloned into a lentiviral vector. T cells were engineered with the AFP158-166-specific TCR gene and evaluated for their specific cytotoxicity of AFP+hepatocellular carcinoma.Results:Construction lentiviral vector expressing AFP158-166-specific TCR gene and preparating self-inactivating lentivirus. Construction AFP158-166-specific TCR engineered T cells by infecting T cells with lentivirus. FCM revealed the percentage of Pro5AFP158-166-MHC Pentamer+and CD8+T cells was1.8%. ELSPOT revealed the number of IFN-γ producing T cells in AFP158-166-specific TCR engineered T cells was raised80%than that of AFP158-166-specific T cells induced with autologous DCs pulsed with AFP158.166peptide. The specific cytotoxicity to AFP-expressing HLA-A0201hepatocellular carcinoma cells-HpeG2were raised from6.3±1.3%,17.9±5.2%, and39.2±3.7%to11.3±2.9%,27.9±7.8%, and62.7±10.4%.Conclusion:T cells engineered with the AFP158-166-specific TCR lentiviral vector were specific and efficient in killing AFP-expressing HLA-A0201restricted hepatocellular carcinoma cells.Part Ⅱ Construction of AFP158-166epitope and IL-15synergetic expression artificial antigen presenting cells Objective:To construct AFP158-166epitope and IL-15synergetic expression artificial antigen presenting cells and evaluate their function.Method:Construction lentiviral vector synergetic expressing AFP158-166epitope and IL-15and preparating self-inactivating lentivirus. Infection of BJAB B lymphoma cells with lentivirus, ELISA detection of IL-15expression, FCM detection of MHC, CD80, and CD86expression and liquid chromatography-mass spectrometry to detect the expression of AFP158-166-Stimulation of AFP-specific T cells with IL-15and AFP158-166epitope expressing BJAB lymphoma cells and evaluation their specific cytotoxicity of AFP+hepatocellular carcinoma.Results:Construction lentiviral vector synergetic expressing AFP158-166epitope and IL-15and preparating self-inactivating lentivirus. Construction IL-15and AFP158-166epitope expressing artificial APCs by infecting BJAB B lymphoma cells with lentivirus. Irradiation of20Gy gamma-ray could induce apoptosis and block proliferation but not kill the artificial APCs. Irradiation of20Gy gamma-ray had no impact on the expression of MHC, CD80, and CD86. The artificial APCs elicited strong stimulation and expansion of AFP158-166-specific T cells. The specific cytotoxicity of AFP158-166-specific T cells showed no difference between artificial APC group and DC group.Conclusion:IL-15and AFP158-166epitope expressing BJAB lymphoma cells served as artificial APCs and could stimulate AFP158-166-specific T cells proliferation. These T cells were specific and efficient in killing AFP-expressing HLA-A0201restricted hepatocellular carcinoma cells.Part Ⅲ AFP158-166specific TCR-engineered T cells for adoptive cellular immunotherapy of hepatocellular carcinomaObjective:To restimulate AFP158-166-specific TCR transgenic T cells by IL-15and AFP158-166epitope expressing BJAB lymphoma cells and evaluate the change of their specific cytotoxicity.Method:Infection of T cells from HLA-A0201healthy donor with lentivirus which expressed AFP158-166-specific TCR gene. Restimulation AFP158-166-specific TCR transgenic T cells by IL-15and AFP158-166epitope expressing BJAB lymphoma cells. The changes of percentage of Pro5AFP158-166-MHC Pentamer+and CD8+T cells and their specific cytotoxicity were evaluated. The model of NOD/SCID mice transplanted HepG2hepatocellular carcinoma cells was used to study the anti-tumor activity and tumor-infiltrating of AFP158-166specific TCR-engineered T cells in vivo.Results:The percentage of AFP158-166specific TCR-engineered T cells was increased from1.8%to4.2%by restimulation with the aAPCs. The ratio of CD8+T cells to CD4+T cells was increased from0.42to1.10. But the specific cytotoxicity of AFP158-166specific TCR-engineered T cells showed no change after the restimulation. The AFP158-166specific TCR-engineered T cells showed higher specific cytotoxicity of the model of NOD/SCID mice transplanted HepG2hepatocellular carcinoma cells.Conclusion:The percentage of AFP158-166specific TCR-engineered T cells was increased by restimulation with the aAPCs. The adoptive cellular immunotherapy of AFP158-166specific TCR-engineered T cells for hepatocellular carcinoma is effective.