Harnessing tumor acidity: innovative lactic acid-responsive promoter enables precision control of CAR-T cell activity in solid tumors.

BACKGROUND: The acidic tumor microenvironment (TME) in solid tumors, driven by abnormal metabolism and lactic acid accumulation, suppresses chimeric antigen receptor-T (CAR-T) cell efficacy while posing safety risks from on-target, off-tumor toxicity (OTOT). This study aims to develop a novel CAR-T technology that leverages lactic acid as a tumor-specific trigger to achieve precise control of CAR activity. The objective is to enable adaptation to the acidic TME while maintaining robust anti-tumor efficacy and mitigating OTOT. METHODS: We engineered a lactic acid-responsive promoter (LARP) using RNA sequencing-identified lactic acid-sensitive genes. This promoter was integrated into HER2-targeting CAR to construct LAR CAR-T cells. CAR expression dynamics under acidic vs neutral conditions were quantified via flow cytometry. Phenotypic profiling (memory markers), in vitro cytotoxicity, and cytokine secretion were assessed. In vivo OTOT was evaluated in our previously constructed humanized HER2 mice, while anti-tumor efficacy and OTOT were further tested in this mouse model bearing tumors. RESULTS: Our findings demonstrate that the LARP responds to lactic acid, leading to increased CAR expression in acidic conditions. The ex vivo-expanded LAR CAR-T cells exhibited an enhanced memory phenotype and superior tumor-killing capacity in vitro under acidity. In vivo, LAR CAR-T cells achieved tumor eradication comparable to conventional CAR-T cells and exhibited significantly enhanced safety profiles, characterized by the absence of acute hepatotoxicity and minimal off-target organ toxicity. CONCLUSIONS: Our LARP strategy exploits tumor acidity as a precise low/high switch for CAR-T cells. By restricting potent CAR expression to the acidic TME while minimizing activity in normal tissues, LAR CAR-T overcomes key barriers of efficacy and OTOT in solid tumors. This lactic acid-sensing paradigm offers a clinically translatable platform for precise immunotherapy.