Figure 2.

DARPin CAR-T cells bind to Her2 and release cytokines in response to Her2-expressing target cells in vitro. (A) CAR-T cells were bound to soluble Her2 in triplicate by incubating with various concentrations of soluble Her2-Fc chimera followed by a PE-conjugated anti-human Fc antibody (n = 3, mean ± standard error of the mean [SEM]; NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (B) Her2 expression on target cells was detected by labeling with PE-conjugated anti-human Her2 antibody. (C) On day 8 of ex vivo expansion, CAR-T cells were co-cultured with MDA.MB.468, MDA.MB.231, SKOV3, or MDA.MB.468.Her2 cells with Brefeldin A protein transport inhibitor for 6 h to detect interferon gamma (IFN-γ) release. Unstimulated CAR-T cells served as a negative control, while CAR-T cells stimulated with anti-human CD3/CD28 were used as positive controls. IFN-γ was measured with intracellular staining. CD8⁺IFN-γ⁺ cells are shown in each gate. Representative data from one donor are shown herein. (D) Summarized data from IFN-γ assay performed in triplicate are displayed in a bar graph (n = 3, mean ± SEM; NS, not significant; *p < 0.05; **p < 0.01.