Chemically Masked aPD-L1-Sialidase Conjugate for Tumor-Specific Desialylation and Immunotherapy

Immune checkpoint inhibitors (ICIs) have revolutionized cancer immunotherapy, yet their clinical efficacy remains limited by low response rates and frequent immune-related adverse events caused by on-target off-tumor toxicity. In this study, we developed a chemically masked anti-PD-L1 nanobody-sialidase conjugate (CAPS) to achieve precise control of protein function. The chemical mask is installed via site-specific PEGylation following the incorporation of a genetically encoded non-canonical amino acid, and is selectively removed by MMP2 within the tumor microenvironment to restore function locally. This design achieves "one construct, three functions" by enabling simultaneous control over nanobody affinity, desialylation activity, and tumor tissue penetration, thereby enhancing tumor specificity and concurrently disrupting the PD-1/PD-L1 and Sialoglycan/Siglec axes. Compared to the unmasked control (anti-PD-L1 nanobody-sialidase, APS), CAPS exhibited a 144-fold extension in systemic half-life and a 9.3-fold increase in tumor-site accumulation. Collectively, CAPS demonstrated superior antitumor efficacy, enhanced immune activation, and an improved safety profile in mouse models of ICI-resistant colorectal cancer. These findings support CAPS as a new class of glyco-ICIs and offer a promising therapeutic strategy to overcome the limitations of current ICIs by enhancing therapeutic efficacy while minimizing on-target off-tumor toxicity.