Abstract 3184: Development of novel anti-mesothelin CAR-T cells for the treatment of solid tumors

Abstract Introduction: Mesothelin (MSLN) is overexpressed in many human solid tumors and absent from most healthy tissues. Despite multiple clinical trials, little success has been achieved by MSLN CARs. Shed MSLN accumulates in patient sera and tumor draining fluids, potentially blocking MSLN CAR function (1, 2). Alpaca-derived VHHs are an attractive option for CAR design that offer exquisite epitope targeting and the potential to circumvent the challenges of cleaved and shed MSLN. Methods: Eight alpaca-derived VHHs targeting the MSLN membrane-proximal region were incorporated into a second-generation CD28-CD3ζ CAR framework. A previously developed scFv-based MSLN CAR with either CD28 or 4-1BB co-stimulation was used as comparison. MSLN CAR-T cells from healthy donors were tested in vitro for CAR surface expression, overnight killing and real-time consecutive tumor re-challenge. Cytokine production by CAR-T cells was measured after overnight co-culture with MSLN+ tumor cells. In vivo CAR-T cell function was evaluated in an AsPC1 pancreatic tumor NSG mouse xenograft model. Persistence of CAR-T cells in vivo was investigated by measuring T cells in peripheral blood (PB) and at tumor site. Additionally, shed MSLN in plasma samples was quantified by ELISA. We also tested the effect of soluble MSLN on the potency of CAR-T cells as analyzed by spiking MSLN into in vitro killing assays, modeling in vivo conditions. Results: VHH CAR D0492 and scFv CAR D0405 were selected for their high surface expression, in vitro cytotoxicity and expansion. In vitro, scFv-based CARs with a CD28 co-stimulatory domain demonstrated higher anti-tumor activity, in comparison to 4-1BB. CAR-T cell production of TNFα, IL2 and IFNγ in response to MSLN+ A431 tumor cells was consistently stronger in CD28 CARs. The VHH CAR showed moderate cytokine release without target stimulation, assumed to be via self-association on the T cell surface. In vivo, tumor-bearing mice treated with MSLN CAR-T cells each delayed tumor progression without adverse effects on body weight. In contrast to in vitro studies, the scFv-based 4-1BB CAR D0181 showed the highest expansion and persistence in PB. IHC staining of tumor samples demonstrated the presence of T cells within tumors in mice treated with anti-MSLN CARs. Shed MSLN plasma concentration in vivo at the end of study take down (TD) correlated with tumor size. Importantly, spiking with equivalent or greater MSLN concentrations did not impede CAR-T cell activity in an in vitro cytotoxicity assay. Conclusions: A series of novel MSLN CARs demonstrated potent in vitro and in vivo anti-tumor activity, were well-tolerated and insensitive to inhibition by soluble MSLN. Second generation scFv-based CARs with 4-1BB co-stimulation demonstrated greater CAR expansion in vivo. VHH CARs demonstrated equivalent in vivo tumor control to scFv, highlighting the importance in both formats to targeting MSLN membrane-proximal domains. Citation Format: Sayyed Hamid Zarkesh Esfahani, Kun Luo, Michael Wagner, Ngoc Tran, Brittany Steimle, Rimas Orentas, Peirong Hu, Dina Schneider. Development of novel anti-mesothelin CAR-T cells for the treatment of solid tumors [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 3184.