Although chimeric antigen receptor (CAR)-modified T cells have shown great success in the treatment of B cell malignancies, this approach has limited efficacy in patients with solid tumors. Various modifications in CAR structure have been explored to improve this efficacy, including the incorporation of two costimulatory domains. Because costimulatory signals are transduced together with T cell receptor signals during T cell activation, we engineered a type of CAR-T cells with a costimulatory signal that was activated independently from the tumor antigen to recapitulate physiological stimulation. We screened 12 costimulatory receptors to identify OX40 as the most effective CAR-T function enhancer. Our data indicated that these new CAR-T cells showed superior proliferation capability compared to current second-generation CAR-T cells. OX40 signaling reduced CAR-T cell apoptosis through up-regulation of genes encoding Bcl-2 family members and enhanced proliferation through increased activation of the NF-κB (nuclear factor κB), MAPK (mitogen-activated protein kinase), and PI3K-AKT (phosphoinositide 3-kinase to the kinase AKT) pathways. OX40 signaling not only enhanced the cytotoxicity of CAR-T cells but also reduced exhaustion markers, thereby maintaining their function in immunosuppressive tumor microenvironments. In mouse tumor models and in patients with metastatic lymphoma, these CAR-T cells exhibited robust amplification and antitumor activity. Our findings provide an alternative option for CAR-T optimization with the potential to overcome the challenge of treating solid tumors.
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