N-deglycosylation targeting chimera (DGlyTAC): a strategy for immune checkpoint proteins inactivation by specifically removing N-glycan

• Published in: Signal transduction and targeted therapy Vol. 10; no. 1; pp. 139 - 15
• Main Authors: Li, Li, Wu, Jiajia, Cao, Weiqian, Zhang, Wei, Wu, Qi, Li, Yaxu, Yang, Yanrong, Shan, Zezhi, Zheng, Zening, Ge, Xin, Lin, Liang, Wang, Ping
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 28.04.2025; Nature Publishing Group
• Subjects: 631/1647/664; 631/67/1059/153; Animals; B7-H1 Antigen; Cancer; Cancer immunotherapy; Cancer Research; Cell Biology; Cell Line, Tumor; Cell surface; Chimeras; Deglycosylation; Glycosylation; Humans; Immune checkpoint; Immune Checkpoint Proteins - genetics; Immune Checkpoint Proteins - immunology; Internal Medicine; Medicine; Medicine & Public Health; Membrane proteins; Mice; N-glycans; N-glycosidase; Nanobodies; Neoplasms - genetics; Neoplasms - immunology; Neoplasms - therapy; Oncology; Pathology; PD-L1 protein; Polysaccharides; Polysaccharides - chemistry; Polysaccharides - genetics; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - pharmacology
• ISSN: 2059-3635; 2095-9907; 2059-3635
• DOI: 10.1038/s41392-025-02219-6

Among the leading methods for triggering therapeutic anti-cancer immunity is the inhibition of immune checkpoint pathways. N-glycosylation is found to be essential for the function of various immune checkpoint proteins, playing a critical role in their stability and interaction with immune cells. Removing the N-glycans of these proteins seems to be an alternative therapy, but there is a lack of a de-N-glycosylation technique for target protein specificity, which limits its clinical application. Here, we developed a novel technique for specifically removing N-glycans from a target protein on the cell surface, named deglycosylation targeting chimera (DGlyTAC), which employs a fusing protein consisting of Peptide-N-glycosidase F (PNGF) and target-specific nanobody/affibody (Nb/Af). The DGlyTAC technique was developed to target a range of glycosylated surface proteins, especially these immune checkpoints—CD24, CD47, and PD-L1, which minimally affected the overall N-glycosylation landscape and the N-glycosylation of other representative membrane proteins, ensuring high specificity and minimal off-target effects. Importantly, DGlyTAC technique was successfully applied to lead inactivation of these immune checkpoints, especially PD-L1, and showed more potential in cancer immunotherapy than inhibitors. Finally, PD-L1 targeted DGlyTAC showed therapeutic effects on several tumors in vivo, even better than PD-L1 antibody. Overall, we created a novel target-specific N-glysocylation erasing technique that establishes a modular strategy for directing membrane proteins inactivation, with broad implications on tumor immune therapeutics.