Rapid CAR Screening and circRNA-driven CAR-NK Cells for Persistent Shed-resistant Immunotherapy

Chimeric antigen receptor (CAR)-based therapeutics offer great potential against cancer and autoimmune diseases. This study presents a high-throughput mRNA-based platform to rapidly screen and optimize CAR-NK cells targeting shed-resistant membrane-proximal epitopes of mesothelin (MSLN)-an antigen frequently overexpressed in solid tumors. Using a yeast surface-display library, we identified single-chain variable fragments that selectively bind membrane-immobilized mature MSLN, minimizing inhibition by soluble MSLN (solMSLN) and enhancing cell-cell avidity. Linear mRNA (linRNA) encoding CAR was electroporated into human primary NK cells for live-cell phenotyping screening of antigen-specific cytotoxicity. Final optimization included co-stimulatory domain (OX40ζ) and cytokine (IL21) co-transfection. The finalized circular mRNA (circRNA)-based CAR-MS10-NK cells exhibited prolonged CAR and superior anti-tumor activities in MSLN-positive models, including solMSLN-secreting pancreatic tumors. In vivo, circCAR-MS10-NK cells significantly reduced tumor burden, outperforming lentivirus-based CAR-NK counterparts in both potency and manufacturability. This platform enables rapid non-viral CAR design and functional screening, for the scalable cost-effective development of shed-resistant antigen-targeting immunotherapies.