Abstract
BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapy faces significant challenges in treating solid tumors, primarily due to the immunosuppressive tumor microenvironment (TME) and rapid T-cell exhaustion mediated by cytokines such as transforming growth factor-β (TGF-β). Developing strategies to remodel the TME and sustain T-cell function is critical. In this study, we investigated a pharmacological strategy using Asiaticoside (AC), a natural compound, as an adjuvant to enhance the efficacy of mesothelin (MSLN)-targeting CAR-T cells in ovarian cancer. METHODS: We engineered MSLN-specific CAR-T cells and evaluated their therapeutic efficacy in combination with AC using in vitro co-culture assays and in vivo xenograft models. Transcriptional changes were analyzed via RNA sequencing (RNA-seq), while the underlying molecular mechanism was investigated by focusing on the TGF-β/SMAD signaling axis. In vivo efficacy and safety were evaluated in NCG mice bearing subcutaneous or intraperitoneal metastatic SKOV-3-luc ovarian tumors, treated with the combination of CAR-T cells and AC. RESULTS: AC treatment significantly potentiated CAR-T cell cytotoxicity and reduced the expression of exhaustion markers (PD-1, TIM-3, and LAG-3) upon continuous antigen exposure. Mechanistically, AC functioned as an inhibitor of TGF-β signaling, effectively suppressing TGF-β1-induced phosphorylation of SMAD2/3. In mouse models, the combination of AC and CAR-T therapy exerted superior antitumor activity compared to CAR-T monotherapy, significantly suppressing tumor growth without inducing systemic toxicity or organ damage. CONCLUSION: Our findings demonstrate that AC alleviates CAR-T cell exhaustion and antagonizes TGF-β-mediated immunosuppression. AC represents a promising, clinically translatable pharmacological adjuvant to overcome the bottlenecks of CAR-T cell therapy in solid tumors.