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Archive > Volume 24 Issue 7 > 2017,24(7):748-755. DOI:10.3872/j.issn.1007-385X.2017.07.009 Prev Next
Basic Research
Construction of GPC3-CAR-T cell targeting hepatocellular carcinoma and identi-fication of its function
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Abstract:
Objective: To construct chimeric antigen receptor (CAR)-modified T cell (CAR-T) which targets GPC3 positive hepatoma cell, and to assess its killing efficacy to the GPC3 positive hepatoma cell. Methods: To enhance expression efficiency of the CAR molecule, technique for favored codon and modification of gene sequence were se-ected. CAR gene targeting GPC3 antigen was designed and lentiviral vector carrying GPC3-CAR gene, pCDH-GPC3-CAR, was constructed, which was transfected into T cell. Western blotting, flow cytometry, real-time cellanal-ysis (RTCA) and ELISA assays were used respectively to detect expression of GPC3-CAR molecule in the CAR-T cell, infection efficiency of the lentivirus to T cells, killing activity and specificity of the GPC3-CAR-T cell to the GPC3 positive hepatoma cell. Results: The gene segment in the pCDH-GPC3-CAR recombinant lentivirus plasmid,molecular mass of which is the same as the GPC3-CAR molecule, was found in a result of double enzyme digestion,indicating that the pCDH-GPC3-CAR lentivirus plasmid was successfully constructed. About 54.38% of the GPC3-T cell expressing GPC3-CAR molecule were celled as the GPC3-CAR-T cell. Killing efficacy of the GPC3-CAR-T cell to the GPC3 positive Huh-7 hepatoma cell was higher than that to the GPC3 negative SK-HEP-1 cell ([78.96±4.76]% vs [6.87±3.15]%). IFN-γ secretion efficiency of the GPC3-CAR-T cell co-cultured with the GPC3 positive Huh-7 hepatoma cell in the CAR-T group was higher than that in the Mock group ([21 371.4±1 808.3]pg/ml vs [152.8±12.5] pg/ml), which could be one of the mechanisms of high efficacy in killing hepatoma cells. Conclusion:The GPC3-CAR-T cell targeting hepatoma cells might have high efficacy ability to secrete cytokine IFN-γ and spe-cific, higher efficient ability to kill the GPC3 positive hepatoma cell, which would lay a foundation to carry further forward pre-clinical and clinical research on the GPC3-CAR-T cell.
Keywords:
glypican-3 （GPC3） ; chimeric antigen receptor T cells (CAR-T) ; immunotherapy ; hepatocellular carcinoma
Project Supported:
Project suported by the National Natural Science Foundation (No.81502434), and the Foundation for ProjectsAdministrated by the Third Military Medical University (No.2014YQN06)
