B7-H3-mediated reversal of CAR-T cell exhaustion induces a notable antitumour response in ovarian cancer models.

BACKGROUND: Functional CAR-T cell exhaustion in the immunosuppressive tumour microenvironment remains the main barrier to the success of CAR-T cell therapy for treating solid tumours. Mesothelin (MSLN) has emerged as an attractive target for CAR-T cell therapy for several solid malignancies, including ovarian cancer. In this study, we aimed to investigate the role and mechanism of lipid metabolites in anti-MSLN CAR-T cell exhaustion in ovarian cancer cells. METHODS: We engineered anti-MSLN CAR-T cells targeting ovarian cancer cells with high MSLN expression as a pivotal tool for in vitro and in vivo experiments. Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the critical role of oxylipin 12-HETE in the exhaustion of CAR-T cells. By employing structure-based high-throughput virtual screening (HTVS), we identified the inhibitor targeting B7-H3. FINDINGS: We demonstrated that GPR31-dependent 12-HETE accumulation in the ovarian cancer microenvironment drives CAR-T cell exhaustion via lipid peroxidation, impairing their antitumour efficacy. Genetic or pharmacological inhibition of the 12-HETE/GPR31 axis restored CAR-T cell cytotoxicity and proliferation, leading to significant tumour regression in murine models. Silencing B7-H3 relieved repression of FOXO3, leading to reduced 12-LOX expression and lower 12-HETE levels, which places B7-H3 upstream of this metabolic checkpoint. Through structure-based screening, we identified HI-TOPK-032 as a potent B7-H3 inhibitor that synergised with CAR-T cell therapy by reversing exhaustion markers (e.g., PD-1, TIM-3) and enhancing cytokine polyfunctionality. Combined HI-TOPK-032 and anti-PD-1 treatment achieved superior tumour control compared to monotherapies, particularly in B7-H3/12-LOX-high patient-derived xenografts, underscoring its precision therapeutic potential. INTERPRETATION: CAR-T cell therapy combined with HI-TOPK-032 is a promising novel strategy for treating MSLN-expressing solid tumours. FUNDING: This study was funded by the National Natural Science Foundation of China (Grant number: 82503173), Beijing Hospitals Authority's Ascent Plan (Grant number: DFL20221201), Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support (Grant number: ZYLX202120), Beijing Natural Science Foundation (Grant number: 7162063), Capital Medical University Laboratory for Clinical Medicine and Gynecological Tumour Precise Diagnosis and Treatment Innovation Studio.