Figure 3.

In vivo persistence and safety analysis of ErbB2CAR-T cells. (A) The mice were injected with 2 × 10⁶ cells IP NAR-LUC cells. After 20 days, they were treated with ErbB2CAR (2.5 × 10⁶ IP or 10 × 10⁶ IP or 10 × 10⁶ IV cells), UNT, or were left untreated. They were euthanized after 45 days. Cells from the spleen, ovary, and blood were collected and stained with anti-human CD3 and analyzed by flow cytometry. Each group included an average of 8–11 mice ± SEM. Data were analyzed by one-way ANOVA test * p < 0.05. (B) The leftmost pie diagram depicts the percentage of CCR7 and/or CD45RA expression out of CD3/ErbB2CAR-GFP-positive T cells (average of four independent transductions). The mice were then euthanized 45 days post-NAR-LUC injection. Splenic single-cell suspension was prepared and stained with anti-human CD3/CCR7/CD45RA and analyzed by flow cytometry. The three right pies depict the percentage of CCR7 and/or CD45RA expression out of CD3-positive cells. EM—effector memory cells, CM—central memory, TN—naïve T cells, and EFF—effector T cells. Each group included an average of 5–11 mice. (C) The mice were euthanized 45 days post-NAR-LUC inoculation, and RNA levels from the splenic cells were measured for PD-1 and TIM-3. The relative expressions were normalized to the housekeeping gene GAPDH, and UNT-treated mice were considered as 1. Each group included an average of 4–5 mice ± SEM. Data were analyzed by 1-way ANOVA test * p < 0.05. (D) Primary cells from different tissues of healthy human donors were co-cultured with lymphocyte T cells transduced for 24 h with ErbB2CAR at an E:T ratio of 2:1. Supernatants were collected and secreted IFNγ was measured by ELISA. The tested target cells included: SKOV3 and OVCAR8 cells (positive control), KEpC—Kidney Epithelial Cells, LEpC—Lung Epithelial Cells, PC—Pancreatic Cells, CMEnC—Cardiac Microvascular Endothelial Cells, RBC—Red Blood Cells, and NT—No Target.