Figure 2.
The function of B7-H3 specific CAR-T cells. (a) Schematic structure of the B7-H3 targeted CARs. H3 CAR consisting of anti-B7-H3 scFv and following the CD8 hinge and transmembrane domain, 4-1BB intracellular domain and the CD3ζ intracellular domain. A control CAR was designed by replacing CD3ζ domain in the H3 CAR with a truncated form of CD3ζ. VH: variable heavy chain; VL: variable light chain; L: linker; T2A: 2A oligopeptides of Thosea asigna virus. (b) Expression of B7-H3-specific CAR or EGFP on human T cells was detected by flow cytometry using hB7-H3-mFc protein (black line); Flag-mFc protein served as an isotype control (gray). (c) Cytotoxicity of CAR-T cells was evaluated by incubating CAR-T cells with FarRed-labeled PG cells at different effector/target (E/T) ratios ranging from 1:1 to 15:1 for 12 h. The cells were stained with DAPI and the viability of the PG cells was analyzed by Flow cytometer. Cytotoxicity was then calculated as described in Materials and methods. (d) PG tumors were subcutaneously established in the right flanks of NCG mice. Control or H3 CAR-T cells were i.v. injected to treat the mice at day 5, 10, and 15 (red arrows). The mean tumor diameters in each group (n = 5) are shown. Error bars denote SD. P values by a two-way ANOVA. ***(P < .001). (e) IFN-gamma levels in the serum of the mice were measured by ELISA at day 15 after last treatment. All data are presented as mean ± standard deviation, and error bars denote SD (c-e). P values by a two-way ANOVA (d) or a two-tailed unpaired t-test (e). ***(P < .001). (f) Representative IHC staining using anti-B7-H3 mAb (clone 6A1, Abcam) for B7-H3 protein on the tumor tissues remained in the mice treated with H3 CAR-T cells.