Development of a tumor-region-selective activation monoclonal antibody targeting the 4-1BB receptor for enhanced therapeutic efficacy and safety

4-1BB is a co-stimulatory immune checkpoint receptor that triggers CD8⁺ T cell activation, leading to robust anti-tumor responses. Although antibodies targeting 4-1BB show promise in preclinical studies, systemic 4-1BB over-activation can cause severe hepatotoxicity, limiting their clinical use. In this study, we developed Pro-Urelumab, an engineered version of the clinical anti-4-1BB antibody (Urelumab), utilizing an autologous hinge as a spatial hindrance-based antibody lock, linked the antibody and antibody lock with a matrix metalloproteinase-2/9 (MMP-2/9) substrate. This design selectively reactivates Pro-Urelumab within the tumor microenvironment, reducing systemic toxicity. Our results demonstrated that Pro-Urelumab exhibited a 389-fold reduction in binding ability toward the 4-1BB receptor compared to Urelumab, effectively preventing pro-inflammatory cytokine secretion from T cells. After MMP-2/9 cleavage, its agonist activity was fully restored. In a human T-cell-transfer mouse model, Pro-Urelumab avoided the 4-1BB antigen sink effect without causing organ damage. Mice treated with Pro-Urelumab exhibited 100 % survival over 14 days, while all Urelumab-treated mice succumbed to treatment-related toxicity. Additionally, Pro-Urelumab achieved 77 % tumor growth inhibition (TGI), compared to 45 % with Urelumab, and significantly increased T cell activation within the tumor. This study underscores the potential of tumor-selective 4-1BB activation for enhancing both the efficacy and safety of immuno-oncology therapies.