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Archive > Volume 33 Issue 3 > 2026,33(3):233-242. DOI:10.3872/j.issn.1007-385X.2026.03.002 Prev Next
Basic Research
Ectopic expression of hemoglobin subunits enhances the in vitro cytotoxicity of CAR-T cells against tumor cells under hypoxic conditions
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Abstract:
[Abstract] Objective: To investigate whether ectopic expression of hemoglobin subunits (HBA/HBB) improves chimeric antigen receptor T cell (CAR-T cell) function and enhances cytotoxicity against tumor cells under hypoxic conditions. Methods: The CAR sequence targeting HER2 was synthesized by full gene synthesis technology, and the CAR lentiviral vectors co-expressing HBA or HBB were constructed. After packaging the lentivirus, human primary T lymphocytes were infected to prepare HBA CAR-T and HBB CAR-T. Hypoxia in mouse solid tumors was detected by hypoxia probes. The proportion of CAR-T cells in the tumor, the percentage of CAR-T cells expressing hemoglobin, and the levels of reactive oxygen species and apoptosis of CAR-T cells under different conditions were detected by flow cytometry. The expression of related hemoglobin subunits in CAR-T cells was detected by WB assay. The proliferation level of cells was detected by hemocytometer, the cytotoxicity of CAR-T cells against tumor cells was detected by luciferase reporter assay, the expression level of HIF-1α in CAR-T cells was detected by qPCR, the oxygen content in T cells was detected by MitoXpress Intra kit. Results: The solid tumor models constructed from different cell lines all exhibited significant hypoxia, and the infiltration level of CAR-T cells was significantly negatively correlated with the degree of hypoxia (P < 0.000 1). HBA CAR-T and HBB CAR-T were successfully constructed (positive rate > 60%), and the corresponding hemoglobin subunits were stably expressed. Under hypoxic conditions, the ROS level and apoptosis level of HBA CAR-T and HBB CAR-T significantly decreased, and their proliferation and cytotoxicity against tumor cells were significantly stronger than those of conventional CAR-T cells (all P < 0.05). HBA CAR-T and HBB CAR-T showed decreased HIF-1α expression (all P < 0.001), and their level of hypoxia significantly decreased (all P < 0.001). Conclusion: The ectopic expression of hemoglobin can reverse the functional impairment of CAR-T cells under hypoxic conditions and enhance their cytotoxicity against tumor cells in vitro.
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