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Published on: November 9, 2020
Designed endocytosis-inducing proteins degrade targets and amplify signals
Buwei Huang1,2,3, Mohamad Abedi1,2, Green Ahn4
1Department of Biochemistry, University of Washington, Seattle, WA, USA.
Abstract:
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.
Insights
Researchers developed novel computational design approaches for endocytosis-triggering binding proteins (EndoTags). These EndoTags facilitate targeted protein degradation and signaling activation, offering significant therapeutic potential for various diseases.
Area of Science:
- Biotechnology and Molecular Engineering
- Drug Discovery and Development
- Cell Biology and Receptor Trafficking
Background:
- Endocytosis and lysosomal trafficking of cell surface receptors are crucial biological processes.
- Existing therapeutic strategies like LYTACs and KineTACs target proteins for degradation but face limitations such as ligand competition and chemical modification requirements.
- A need exists for versatile and genetically encodable methods to induce receptor-mediated endocytosis for therapeutic applications.
Purpose of the Study:
- To computationally design novel endocytosis-triggering binding proteins (EndoTags) that overcome limitations of existing protein degradation technologies.
- To demonstrate the efficacy of EndoTags in mediating lysosomal trafficking and targeted protein degradation.
- To explore the therapeutic potential of EndoTags as targeted degradation inducers, signaling activators, and cellular uptake enhancers.
Main Methods:
- Computational design of endocytosis-triggering binding proteins (EndoTags).
- Fusion of designed EndoTags to soluble or transmembrane protein binders targeting specific receptors (IGF2R, ASGPR, sortilin, transferrin receptors).
- In vitro and in vivo validation of EndoTag-mediated lysosomal trafficking, target degradation, and therapeutic efficacy in a mouse tumor model (PD-L1 antibody fusion).
Main Results:
- Successfully designed and validated EndoTags for multiple receptors, demonstrating lysosomal trafficking and target degradation upon fusion to binders.
- EndoTag fusion to a PD-L1 antibody significantly enhanced therapeutic efficacy in a mouse tumor model compared to the antibody alone.
- EndoTag technology enables modularity, genetic encodability, AND gate control for specificity, localized secretion, and enhanced signaling (nearly 100-fold increase).
Conclusions:
- EndoTags represent a powerful and versatile platform for targeted protein degradation, overcoming limitations of previous approaches.
- The modularity and genetic encodability of EndoTags offer significant advantages for therapeutic development, including precise control and manufacturing.
- EndoTags hold considerable therapeutic promise for targeted degradation, activating endocytosis-dependent pathways, and enhancing cellular uptake for drug and RNA conjugates.
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