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.