Fig. 2

Cytotoxicity of anti-CAIX CAR-T cells in vitro. (A) Tet-On CAIX inducible skrc-59 cell line is engineered to utilize the Tet-op promoter to sense different concentrations of Dox to control CAIX expression. (B) CAIX expression on the Tet-On cells in the presence of different Dox concentrations. (C) Cytotoxicity of anti-CAIX CAR-T cells on CAIX high skrc-59 tumor cells. CD8 CAR-T cells with E:T ratio of 2:1. The variants of CAR-T cells are arranged in descending order of affinity from left to right indicated by increasing KD values. P values are defined by unpaired two-tailed t-tests between each CAR-T to untransduced T cell (UNT) (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (D) Cytotoxicity of anti-CAIX CAR-T cells on CAIX low MMNK-1 cholangiocytes. CD8 CAR-T cells with E:T ratio of 2:1. From left to right, within the group of CAIX targeted CAR-T cells, the KD value of each scFv is increasing, meaning the affinity is decreasing. P values are defined by unpaired two-tailed t-tests between each CAR-T to untransduced T cell (UNT) (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). Specificity index is defined by using the cytotoxicity on skrc-59 tumor cells divided by the cytotoxicity on MMNK-1 cells. (E) CAR constructs of G9-41BB, G36-41BB and G250-CD3 are shown. Cytotoxicity of (F) G36, (G) G9 and (H) G250 on Tet-On inducible skrc-59 cells. The variants of CAR-T cells are arranged in escalating order of CAIX density on the cell surface. All data with error bars are presented as mean ± SD. P values are defined by unpaired two-tailed t-tests (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). Only significant differences are shown in the plot