Designed endocytosis-inducing proteins degrade targets and amplify signals.

1. Nature. 2025 Feb;638(8051):796-804. doi: 10.1038/s41586-024-07948-2. Epub 2024 Sep 25. Designed endocytosis-inducing proteins degrade targets and amplify signals. Huang B(#)(1)(2)(3), Abedi M(#)(1)(2), Ahn G(#)(4), Coventry B(#)(1)(2)(5), Sappington I(1)(2), Tang C(6), Wang R(7), Schlichthaerle T(1)(2), Zhang JZ(1)(2), Wang Y(2), Goreshnik I(1)(2)(5), Chiu CW(2), Chazin-Gray A(1)(2), Chan S(2), Gerben S(2), Murray A(2), Wang S(1)(2), O'Neill J(8), Yi L(8), Yeh R(8), Misquith A(8), Wolf A(8), Tomasovic LM(9)(10), Piraner DI(11), Duran Gonzalez MJ(11), Bennett NR(1)(2), Venkatesh P(1)(2), Ahlrichs M(2), Dobbins C(2), Yang W(1)(2), Wang X(1)(2), Sahtoe DD(12), Vafeados D(2), Mout R(13), Shivaei S(14), Cao L(15), Carter L(2), Stewart L(2), Spangler JB(8), Roybal KT(11), Greisen PJ(8), Li X(7), Bernardes GJL(6)(16), Bertozzi CR(4)(17)(18), Baker D(19)(20)(21). Author information: (1)Department of Biochemistry, University of Washington, Seattle, WA, USA. (2)Institute for Protein Design, University of Washington, Seattle, WA, USA. (3)Department of Bioengineering, University of Washington, Seattle, WA, USA. (4)Department of Chemistry, Stanford University, Stanford, CA, USA. (5)Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. (6)Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal. (7)Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA. (8)Novo Nordisk, Måløv, Denmark. (9)Departments of Biomedical Engineering and Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA. (10)Medical Scientist Training Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA. (11)Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA, USA. (12)Hubrecht Institute, Utrecht, The Netherlands. (13)Harvard Medical School, Harvard University, Boston, MA, USA. (14)Division of Biology and Bioengineering, California Institute of Technology, Pasadena, CA, USA. (15)School of Life Sciences, Westlake University, Hangzhou, China. (16)Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, UK. (17)Howard Hughes Medical Institute, Stanford, CA, USA. (18)Sarafan ChEM-H, Stanford University, Stanford, CA, USA. (19)Department of Biochemistry, University of Washington, Seattle, WA, USA. dabaker@uw.edu. (20)Institute for Protein Design, University of Washington, Seattle, WA, USA. dabaker@uw.edu. (21)Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA. dabaker@uw.edu. (#)Contributed equally Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (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. © 2024. The Author(s). DOI: 10.1038/s41586-024-07948-2 PMCID: PMC11839401 PMID: 39322662 [Indexed for MEDLINE] Conflict of interest statement: Competing interests: B.H., M. Abedi, G.A., I.S., L.S. and D.B. are co-inventors on a provisional patent application that incorporates discoveries described in this manuscript. B.C., I.G., J.O., P.G., L.S. and D.B. are co-inventors on a provisional patent application that incorpor