〔Abstract〕 Objective To construct a chimeric antigen receptor macrophage (CAR-M) targeting interleukin-13 receptor α (IL-13Rα) and intracellular domain integration, which can double activate the signal adaptor protein OX40 L of macrophages and T cells. Methods The CAR molecule targeting IL- 13Rα (IL-13Rα-OX40L-CAR) with OX40L as the intracellular signal domain was designed and synthesized, and it was cloned into the adenovirus expression vector. After THP-1 cells were induced to differentiate into macrophages (THP-1-M) by phorbol ester, the cells were infected with recombinant adenovirus to construct THP-1 macrophages stably expressing green fluorescent protein (GFP) labeled anti-IL- 13RαCAR. The expression efficiency of CAR molecules was monitored by flow cytometry. The empty vector group was used as the negative control, the CD3ζ group was used as the positive control, and the CD3ζ-OX40L group was used as the experimental group. The phagocytic function of macrophages in each group on IL- 13Rα high expression glioma cells (U251) and low expression glioma cells (T98G) was detected by in vitro co-culture experiments. Results CAR adenovirus was efficiently transfected into THP-1-M to obtain anti-IL- 13RαCAR-M. After co-cultured with glioma cells with different expression levels of IL- 13Rα, the results of flow cytometry showed that there was no significant difference among the three groups for T98G cells with low expression of IL- 13Rα . After co-culture with U251 cells with high expression of IL- 13Rα, the CD3ζ group showed enhanced phagocytosis compared with the empty vector group (P<0.05). Compared with the CD3ζ group, the phagocytosis ability of the CD3ζ-OX40L group was further enhanced (P<0.05). Conclusion Anti-IL13Rα-OX40L-CAR-M is successfully constructed by constructing IL- 13Rα-OX40L-CAR adenovirus and transfecting PMA-induced differentiated THP-1-M. It can engulf glioma cells with high expression of IL- 13Rα by specific targeting, and shows activation of intracellular domain superior to traditional CD3ζ .