Panoply™ Human KDR Over-expressing Stable Cell Line

CAR-T cell therapy is a revolutionary cancer immunotherapy that involves transfecting or transducing T cells with chimeric antigen receptors (CARs). Although CAR-T cell therapy has achieved remarkable success in the treatment of hematological malignancies, its efficacy against solid tumors is limited. Here, researchers aimed to explore whether CAR-modified T cells targeting vascular endothelial growth factor receptor 2/kinase insert domain receptor (KDR) could disrupt tumors and their vasculature. Computational modeling studies suggested that this CAR construct might be effective in treating lung cancer. The researchers validated this finding through in vitro and in vivo experiments. KDR-CAR-T cells efficiently targeted and killed the KDR-overexpressing A549 cell line (KDR-A549 cell) by expressing IFN-γ and releasing granzyme B. In vivo studies showed that KDR-CAR-T cells significantly inhibited the growth of KDR-A549 xenograft tumors in BALB/c-nu mice by day 10. Characterization of KDR-CAR-modified T cells through computational biology and wet lab experiments suggests that it holds promise as a novel therapeutic strategy for lung cancer and may be applicable to the treatment of other vascularized solid tumors.

Here, researchers used the CCK-8 assay to quantitatively detect the proliferation of functional KDR-CAR-J cells. The results showed no statistically significant difference between 24 and 48 hours. However, compared to Jurkat cells, KDR-CAR-J cells showed slower growth at 72 hours (Figure 1a). To measure the cytotoxicity of CAR-transduced T cells, researchers co-cultured KDR-CAR-J cells with KDR-A549 cells at different effector cell to target cell ratios (E:T ratios), namely 5:1, 10:1, and 20:1. All effector cell to target cell ratios had similar effects on the cytotoxicity of KDR-A549 cells (Figure 1b). After Hoechst33258 staining, target cell apoptosis was observed (Figure 1c). The results showed that the apoptosis rate of target cells (KDR-A549) in the KDR-CAR-J group was significantly higher compared to the control group. In this study, 293T cells were used as a normal control to assess the non-toxicity of KDR-CAR-J cells to normal cells. Statistical analysis was performed on the number of cell deaths (Figure 1d). Cytokine secretion was assessed by co-culturing KDR-CAR-J cells and KDR-A549 cells as described above, then staining with PE-labeled CD69 antibody and analyzing by flow cytometry. CD69 is an early marker of T cell activation. The results showed that KDR-CAR-J cells expressed CD69 at effector cell to target cell ratios of 5:1, 10:1, and 20:1 (44.41%, 35.43%, and 38.39%, respectively) (Figure 1e), confirming that KDR-CAR-J cells expressed CD69 on their surface after recognizing target cells, compared to Jurkat cells as a control. There were no significant differences between the groups, so a 5:1 ratio was chosen for subsequent experiments.

Figure 1. KDR-CAR-J cells targeted KDR-A549 cells. (Zhong M, et al., 2023)