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Chimeric antigen receptor (CAR)-modified T (CAR-T) cell therapy has been proven to be a powerful tool for the treatment of cancer, however, the limits are obvious, especially for solid tumors. Therefore, constantly optimizing the structure of CAR to improve its therapeutic effect is necessary. In this study, we generated three different third-generation CARs targeting IL13Rα2, with the same scFv, but different transmembrane domains (TMDs) from CD4, CD8 or CD28 (IL13-CD4TM-28.BB.ζ, IL13-CD8TM-28.BB.ζ and IL13-CD28TM-28.BB.ζ). CARs were transduced into primary T cells using retroviruses. The anti-GBM efficacy of CAR-T cells was monitored by flow cytometry and real-time cell analysis (RTCA) in vitro and examined in two xenograft mouse models. The differentially expressed genes related to different anti-GBM activity were screened by high throughput RNA sequencing. We observed that T cells transduced with these three CARs have similar anti-tumor activity when co-cultured with U373 cells which expressed higher IL13Rα2 but exhibited different anti-tumor activity when co-cultured with U251 cells that expressed lower IL13Rα2. All the three groups of CAR-T cells can be activated by U373 cells, but only IL13-CD28TM-28.BB.ζ CAR-T cells could be activated and expressed increased IFN-γ after co-culturing with U251 cells. IL13-CD28TM-28.BB.ζ CAR-T cells exhibited the best anti-tumor activity in xenograft mouse models which can infiltrate into the tumors. The superior anti-tumor efficacy of IL13-CD28TM-28.BB.ζ CAR-T cells was partially owing to differentially expressed extracellular assembly, extracellular matrix, cell migration and adhesion-related genes which contribute to the lower activation threshold, increased cell proliferation, and elevated migration capacity. [ABSTRACT FROM AUTHOR]