Mesothelin-directed protein-drug conjugates for mesothelin-low solid tumor therapy.

Mesothelin (MSLN) is an attractive therapeutic target for precision cancer treatments. However, MSLN can be cleaved and shed from tumor cells, resulting in the presence of soluble MSLN (sMSLN), which significantly hinders the efficacy of MSLN-targeted therapies. Here, we identify a MSLN-targeting designed ankyrin repeat protein (DARPin) M7, which specifically binds to the protease-sensitive C-terminal region of MSLN. Furthermore, we develop two auristatin-based DARPin-drug conjugates (DARPin-DCs), M7A-DC and M7GA-DC. We show that M7A-DC and M7GA-DC are effectively internalized by MSLN-positive cells, leading to the release of MMAE that induces lethal effects as well as bystander killing against MSLN-negative cells. Compared to ADCs, M7A-DC and M7GA-DC exhibit improved tumor spheroid penetration and cytotoxic activity in 3D models. Notably, M7GA-DC demonstrates enhanced tumor control and improved survival benefits in pancreatic cancer models with limited MSLN expression. Combination therapy with PD-1 blockade further promotes long-term immunological memory formation by activation of dendritic cells and reprogramming of the tumor microenvironment. These findings highlight the translational potential of DARPin-DC and its promising prospects for clinical combination with immunotherapies in the treatment of solid tumors, including refractory pancreatic cancer.