Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins

Abstract

Targeted protein degradation (TPD) technology has drawn significant attention from researchers in both academia and industry. It is rapidly evolved as a new therapeutic modality and also a useful chemical tool in selectively depleting various protein targets. As most efforts focus on cytosolic proteins using PROteolysis TArgeting Chimera (PROTAC), LYsosome TArgeting Chimera (LYTAC) recently emerged as a promising technology to deliver extracellular protein targets to lysosome for degradation through the cation-independent mannose-6-phosphate receptor (CI-M6PR). In this study, we exploited the potential of the asialoglycoprotein receptor (ASGPR), a lysosomal targeting receptor specifically expressed on liver cells, for the degradation of extracellular proteins including membrane proteins. The ligand of ASGPR, triantennary N -acetylgalactosamine (tri-GalNAc), was conjugated to biotin, antibodies, or fragments of antibodies to generate a new class of degraders. We demonstrated that the extracellular protein targets could be successfully internalized and delivered into lysosome for degradation in liver cell lines specifically by these degraders. This work will add a new dimension to TPD with cell type specificity.

Degradation from the outside in: Targeting extracellular and membrane proteins for degradation through the endolysosomal pathway

“…Contemporaneously, Tang and coworkers also demonstrated that biotin or antibodies linked to a tri-GalNAc ligand can mediate uptake of soluble cargos such as NeutrAvidin and IgG in HEPG2 cells. This work further assessed the effects of cargo and LYTAC size with results suggesting that smaller complexes are more efficiently internalized by ASGPR in HEPG2 cells (Zhou et al, 2021).…”

Major advances in targeted protein degradation: PROTACs, LYTACs, and MADTACs

“…The Tang and Bertozzi groups have similarly utilized ASGPR as the internalizing receptor for lysosomal induced degradation ( 159 , 160 ). The Bertozzi group observed a higher internalization efficiency with ASGPR as compared with CI-M6PR, with decreases in surface EGFR occurring at tenfold lower concentrations.…”

“…Lastly, they observed that a single site-specific tri-GalNAc ligand conjugated to their PIP protein caused its degradation. The Tang group also showed both small molecules (biotin) and antibodies (anti-biotin IgG-647) coupled to a tri-GalNAC can successfully induce internalization and degradation by the lysosome ( 159 ). In addition, their study showed that small proteins are more readily internalized than larger proteins.…”

Emerging protein degradation strategies: expanding the scope to extracellular and membrane proteins

“…After being converted to Tri-GalNAc-NHS (N-hydroxysuccinimide) ester, Tri-GalNAc ligands are conjugated with the lysine residues on the antibody 24 . Compared to M6Pn, selectively delivering undesired proteins to liver by tri-GalNAc ligands is much safer than ubiquitously delivery of POIs to various types of cells 24 . We compare the advantages and disadvantages of polyM6Pn and tri-GalNAc in Table 2 .…”