The Killing Effect Of MUC1Chimeric Antigen Receptor-engineered Jurkat T Cells On Hepatocellular Carcinoma

Chimeric antigen receptors (CARs) usually consist of an antigen binding moiety ofa monoclonal antibody, an extracelluar hinge, a transmembrane region and the signalingendomain derived from ITAM of CD3ζ/FcεRIγ and costimulation region ofCD28/4-1BB/OX40/ICOS/DAP10/LCK. Chimeric antigen receptor engineered T cells(CAR-T) can efficiently target tumor cells that have downregulated HLA expression orproteasomal antigen processing,2mechanisms that contribute to tumor escape fromTCR-mediated immunity.MUC1(Mucin1) is a tumor-associated marker that is over-expressed in breast,ovarian,lung, prostate, colon, and pancreatic cancer tissues. The major extracellulardomain of this tumor-associated transmembrane molecule is composed of tandem repeatunits of20amino acids (PDTRPAPGSTAPPAHGVTSA) and its core protein is alsoaberrantly glycosylated, making the tumor-associated mucin antigenically distinct fromthe normal mucin. Overexpression and hypoglycosylation make MUC1a highlyattractive target for cancer immunotherapy.In this study, the expression cassettes of both the first and the third generationMUC1-specific CAR gene (i.e. MUC1-CAR and G3MUC1-CAR) were constructed andcloned into lentivirus transfer plasmids, respectively. Then, the obtained recombinantlentiviruses carrying the MUC1-specific CAR gene and hrGFP reporter genes were usedto infect Jurkat T cells in vitro to establish MUC1-sepcific CAR-engineered Jurkat cells(i.e. CAR-T cells). Subsequently, the assays of CCK-8, ELISA, and LDH were used todetect the cell proliferation, IL-2secretion and killing effect of the CAR-T cells,respectively. We successfully constructed the expression cassettes of MUC1-sepcific CAR geneand the corresponding recombinant lentivirus, established the MUC1-specificCAR-engineered Jurkat T cells. Both MUC1-specific CARs-engineered Jurkat T cellscould recognize MUC1molecule and specifically kill MUC1over-expression HCCcells while leave normal hepatic cells almost undamaged. In addition, the cellproliferation, the secretion of IL-2and killing effect of the G3MUC1-CAR-engineeredJurkat T cells was significantly superior to the MUC1-CAR-engineered counterpart.To conclusion, the MUC1-sepcific CAR-engineered Jurkat T cells can specificallykill MUC1over-expression HCC cell. The reseach provided a reliable experimentalbasis for the future application of MUC1-CAR modified primary T cells for theadoptive immunotherapy of hepatocellular carcinoma.