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Fig. 1 | Journal of Translational Medicine

Fig. 1

From: CRISPR/Cas9-mediated TGFβRII disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells in vitro

Fig. 1

MSLN and CLDN6 are highly expressed on tumor cell lines and CAR CLDN6 or MSLN recognize and kill them efficiently. A A 2nd Gen. CAR CLDN6 or MSLN construct based on chimerisation to the hinge, transmembrane, and cytosolic region of CD8α, the cytosolic region of 4-1BB and CD3ς for costimulatory and stimulatory signaling, respectively. B MSLN expression on i) Hela and ii) OVCAR-3 cell line, iii) exogenous MSLN expression on iDCs after electroporation of 10 μg MSLN IVT-RNA, and non-expressing tumor cell line iv) SK-OV-3 and v) Colo-699 N. Endogenous CLDN6 expression on vi) PA-1 and vii) OVCAR-3 cell surface viii) exogenous CLDN6 expression on MDA-MB-231 after transduction with CLDN6 encoding virus particles, and ix) CLDN6 expression on iDCs after electroporation of 2 μg CLDN6 IVT-RNA, respectively. C: i) CAR MSLN expression on human CD8 T cells one day after electroporation with 10 μg CAR MSLN IVT-RNA, ii) IFN-γ secretion assay proved that MSLN-specific CAR recognizes MSLN positive cells (HeLa and OVCAR-3), while they do not react with antigen negative cell lines (SK-OV-3 and COLO-699 N), iii) Spheroid killing assay demonstrated that CAR MSLN T-cells recognize and kill HeLa tumor spheroids at also a very low E:T-ratio. D: i) CAR CLDN6 expression on human CD8 T cells one day after electroporation with 10 μg CAR CLDN6 IVT-RNA, ii) IFN-γ secretion assay proved that CAR CLDN6 can specifically recognize CLDN6 electroporated iDCs in a dose dependent manner while they do not react with antigen negative cells, iii) Xcelligence based killing assay showed that CAR CLDN6 T-cells recognize and kill exogenously CLDN6 expressing SK-OV-3 cell lines, while mock T-cells remained inactive. vi) CFSE based proliferation assay showed that CAR CLDN6 T cells divided strongly up to a frequency of 80%, not only in the presence of a high dose of heterologously expressed CLDN6 in iDCs (2 μg), but also at a dose of as low as 0.02 ug CLDN6. P values were determined by one-way Anova using multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. In all experiments, mean ± SD of three technical replicates are given and experiments, involving T cells, are repeated for at least three donors

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