Development of Highly Efficient Lysosome‐Targeting Peptide Ligands for the Internalization of Extracellular Proteins

Lysosome-targeting chimeras (LYTACs) induce the internalization of extracellular and membrane-associated proteins by routing them to lysosomes via endogenous receptors, such as the insulin-like growth factor-2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor. While current IGF2R ligands are effective, expanding the LYTAC toolkit with structurally simple and synthetically accessible ligands remains a practical necessity. Here, we first validated that previously reported dimeric and monomeric peptides induce robust internalization, establishing them as effective IGF2R-targeting LYTAC ligands. To further optimize the peptide architecture, we performed systematic mutational scanning on its monomeric sequence to simplify the peptide architecture. We identified the L4 variant, which features a single leucine substitution at the fourth position. This optimized monomer induces greater internalization efficiency than the previously reported wild-type monomeric peptide ligand. To demonstrate its utility, we conjugated the L4 recruiter to a specific PCSK9 binder. The resulting bifunctional molecule successfully induced the dose- and time-dependent internalization of PCSK9. Collectively, these findings establish several peptide variants as practical and highly effective building blocks for the development of extracellular protein internalization platforms.