Designed endocytosis-inducing proteins degrade targets and amplify signals

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras (LYTACs) and cytokine receptor-targeting chimeras (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders 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. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. 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. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates.

Buwei Huang, Mohamad Abedi, Green Ahn, Brian Coventry, Isaac Sappington, Cong Tang, Rong Wang, Thomas Schlichthaerel, Jason Z. Zhang, Yujia Wang, Inna Goreshnik, Ching Wen Chiu, Adam Chazin-Gray, Sidney Chan, Stacey Gerben, Analisa Murray, Shunzhi Wang, Jason O'Neill, Li Yi, Ronald Yeh, Ayesha Misquith, Anitra Wolf, Luke M. Tomasovic, Dan I. Piraner, Maria J. Duran Gonzalez, Nathaniel R. Bennett, Preetham Venkatesh, Maggie Ahlrichs, Craig Dobbins, Wei Yang, Xinru Wang, Danny D. Sahtoe, Dionne Vafeados, Rubul Mout, Shirin Shivaei, Longxing Cao, Lauren Carter, Lance Stewart, Jamie B. Spangler, Kole T. Roybal, Per Jr Greisen, Xiaochun Li, Gonçalo J. L. Bernardes, Carolyn R. Bertozzi, David Baker