Conditionally activated immunotoxins with prolonged half-life can enhance the anti-tumor activity.

Immunotoxin has become a highly promising therapy for treating cancer and has achieved good results in preclinical trials targeting various cancers. However, there are still some issues that limit the development and application of immunotoxins, such as short half-life and toxic side effects on healthy tissues. In this study, we designed a tumor-conditional immunotoxin called Nb-uPA-A1-PE24. Anti-HSA nanobody (Nb) was fused to the N-terminus of A1-PE24 (immunotoxin targeting mesothelin) via a linker cleavable by tumor-associated proteases, urokinase-type plasminogen activator (uPA). Nb binds to HSA (human serum albumin) in the blood circulation, which not only prolongs the half-life of immunotoxins, but also creates a certain spatial barrier between A1 and mesothelin, thereby reducing the toxicity of Nb-uPA-A1-PE24 to healthy tissues expressing mesothelin. Moreover, uPA cleavable element rendered the immunotoxin conditional activation specifically in tumor microenvironment. In animal experiments, the half-life of the newly designed immunotoxins was increased dramatically. Noted, Nb-uPA-A1-PE24 has better enrichment at tumor, and shows robust anti-tumor effects in multiple preclinical models, such as pancreatic cancer and gastric cancer models. The results indicate that this strategy has greater potential and higher safety for application in tumor treatment, providing new ideas and strategies for the development of immunotoxins for cancer patients.