Abstract 4799: MSLN and MUC1 dual-targeting CAR-T with conditional activation and tumor specificity to improve clinical safety and efficacy

Abstract Background: MSLN and MUC1 are highly expressed in numerous solid tumors, presenting them as potential targets for solid tumor treatment. Nevertheless, these two targets also display varying degrees of expression in important human tissues, at risk of triggering the "on-target off-tumor" effect in CAR-T therapy. Additionally, the tumor immune microenvironment (TME) has a strong immunosuppressive impact on CAR-T cells, making them prone to exhaustion and consequently difficult to exert a long-lasting anti-tumor effect. Methods: In this study, a dual-targeting CAR-T (designated as BZE2203) for MUC1 and MSLN was generated, validated in pre-clinical and clinical studies. The anti-MSLN VHH was engineered with a pH-dependent characteristic, endowing CAR-T cells with cytotoxicity only at acidic pH in the TME. The anti-MUC1 VHH was selected based on antigen-density dependence to ensure that it has no cytotoxic effect on cells with low MUC1 expression in normal tissues. Meanwhile, the CAR-T was armed with secreting anti-PD1 VHH to alleviate the inhibitory effect of TME on CAR-T cells and tumor infiltrating lymphocytes. Cao-V3 xenograft animal model was utilized to evaluate the safety and efficacy of BZE2203 CAR-T in vivo. In an investigator-initiated trial (IIT), patients including two with platinum-resistant ovarian cancer were recruited and infused with a low-dose of BZE2203 CAR-T cells to preliminarily assess the safety and efficacy of this product. Results: In vitro experiments showed that BZE2203 CAR-T cells exerted cytotoxicity on MSLN-positive target cells only in the acidic pH but not neutral pH, and on MUC1-positive tumor cells in a density-dependent manner, but no cytotoxic effect on normal human primary lung cells with low MUC1 expression. After being activated by target cells, CAR-T cells secrete high level of anti-PD1 VHHs. BZE2203 CAR-T showed substantial anti-tumor effect in the Cao-V3 animal model. In the IIT clinical trial, patients did not experience severe safety issues such as cytokine storm and neurotoxicity caused by CAR-T treatment, and two ovarian cancer patients achieved partial remission. Conclusions: Our results indicate that pH-dependent and antigen-density-dependent CAR-T can mitigate on-target off-tumor toxicities, demonstrating significant potential for improved safety and efficacy in clinical applications of solid tumors. Citation Format: Jiaguo Li, Yan Sun, Yong Xia, Jinxing Lou, Jun Guo, Lijie Rong, Dan Sun, Zhicai Lin, Yi Liu, Xinhao Wang, Wenfeng Xu, Qijun Qian. MSLN and MUC1 dual-targeting CAR-T with conditional activation and tumor specificity to improve clinical safety and efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4799.