Epitope selection overcomes antigen shedding to enable effective CAR-M therapy in solid tumors

Chimeric antigen receptor-macrophages (CAR-Ms) represent a promising platform for solid tumors, but barriers limiting durable responses remain incompletely defined. Antigen shedding may be a key barrier because, unlike T cells, macrophages require sustained antigen engagement for effective phagocytosis. However, whether shed antigen directly impairs CAR-M function has not been established. Using mesothelin (MSLN) as a model target, we generated CAR-Ms targeting either an N-terminal epitope (SS1) or a membrane-proximal, non-shed epitope (15B6). Shed MSLN markedly impaired the phagocytic and cytotoxic activity of SS1 CAR-Ms, establishing antigen shedding as a direct functional barrier. In contrast, 15B6 CAR-Ms remained largely resistant and showed stronger antigen-specific activity across multiple MSLN⁺ tumor cells, demonstrating that rational epitope selection can overcome this barrier. In immunocompetent, MSLN-tolerant transgenic mouse models of colorectal and pancreatic cancer, 15B6 CAR-Ms demonstrated superior anti-tumor activity. In colorectal tumor-bearing mice, a single infusion of 15B6 CAR-Ms significantly prolonged survival, with 35% achieving complete tumor regression. Rechallenge of cured mice with MSLN^- tumor cells resulted in complete tumor rejection, suggesting the development of durable systemic anti-tumor immunity. Together, these findings identify antigen shedding as a critical barrier and support epitope selection as an important design strategy for CAR-M therapies targeting shed tumor antigens.