Designed Endocytosis-Triggering Proteins mediate Targeted Degradation and amplify signaling

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.