Designed triggering of endocytosis drives target degradation and signaling activation

Introduction:: The endocytosis of many cell surface receptors is triggered by binding of their endogenous ligands which shift the conformational state of the receptor1,2,3. However, existing ligands triggering endocytosis remain limited. Many native ligands could trigger off-target signaling and lacks potent endocytosis properties4,5. Synthetic ligands with higher potency usually requires chemical modification or tedious production for high multivalency, which is leveraged by Lysosome-targeting chimaeras (LYTAC) technology6,7. More generally, to date there does not exist a completely synthetic and systematic approach to design activators for endocytosis, and computational approach to rationally control ligand engagement with multi-domains of receptor is lacking.



Here we develop a generalizable protein design approach to rationally generate synthetic ligands that trigger endocytosis of native receptors such as IGF-2R. These endocytosis-targeted activating proteins, which we named EndoTag, are capable of inducing endocytosis to varying extents by controlling binding site, affinity, and rigidity. By fusing binding domains to both soluble and transmembrane targets with these EndoTags we show that this modular system can lead to lysosomal trafficking and efficient target degradation surpassing previously described chemically synthesized Lysosome-targeting chimeras (LYTACs); the designs thus function as protein based LYTAC (pLYTAC). The modularity and versatility of protein based EndoTags enabled seamless integration within protein networks to establish complex logic-based degradation operations and locally-secretable degraders. We further found that EndoTags can serve as excellent ligands to activate endosomal-based signaling particularly from a class of Notch-based synthetic receptors that require endosomal internalization for signaling.


Materials and Methods:: 1. Computational design of IGF-2R EndoTags

We sought to use de novo binder design approach integrating Rosetta Rifdock8,9 based on the structures of IGF-2R (PDB 6UM2). Next based on the binding proteins validated to bind at two distinct interfaces, we generated a rigid fusions between the two proteins by using RFInpainting10 . The designs were sequence optimized with ProteinMPNN11 and structure predicted with Alphafold212.

2. Cellular uptake and lysosomal trafficking assay in vitro

We validated the endocytosis ability with flow cytometry across multiple cell lines expressed IGF-2R. Confocal microscopy was applied to evaluate the lysosomal localization with the designed EndoTags

3. Degradation assay

We tested flow cytometry and Western Blot to quantify the degradation of cell surface receptors. We used Neo2 plate reader to measure the clearance of soluble proteins.

4. Signaling assay

We introduced SNIPER, a redesigned synthetic Notch (Syn-Notch) system that implemented de novo designed ligand LHDA and receptor LHDB, where LHDA and LHDB are designed heterodimer pairs that have binding affinity and selectivity to each other13. The intracellular domain is a transcription factor controlling the expression of blue fluorescence protein (BFP). So we can quantify the signaling activation of SNIPER via the fluorescence intensity of BFP. The transcription factor is only able to be released after the receptor gets endocytosis.


Results, Conclusions, and Discussions:: 1. Development IGF-2R EndoTags

The method described in the method section successfully generated high affinity binders separately bind to two distinct binding sites at IGF-2R, and fusion between these two proteins (EndoTags) were able to induce conformational change and triggered rapid endocytosis, reflected as increased cellular uptake in flow cytometry. Confocal imaging showed that the EndoTags were able to localize with lysosome right after 30min incubation.

2. Application of EndoTags as extracellular degraders

The genetical fusion of EndoTags with binding proteins against extracellular proteins functioned as potent degrader, named protein-LYTAC (pLYTAC). We showed the EndoTags significantly improve the degradation efficiency of the original glycan-based LYTAC in degrading EGFR and depleting IgG in vitro. Fusion of EndoTag with anti-PDL1 antibody also led to 90% depletion of PD-L1. To improve the selectivity of degradation, we plugged the EndoTags in a logic-gated system named co-LOCKR14, and the degrader was only able to degrade EGFR in the presence of HER2. To address the degrader efficiency issue, we applied the EndoTag in an adoptive cell therapy system. With remarkable production simplicity, we transfected the cells with plasmid expressing EGFR-EndoTag and the secreted EGFRn-EndoTag led to similar EGFR clearance as the exogenously added EGFR-EndoTag.

3. Application of EndoTags as signaling activator

Previously we developed SNIPR, a simplified version of Syn-Notch receptor where we used LHDA ligand to activate LHDB receptor (unpublished). However, the overall signal intensity was weak, due to the reliance on SNIPR endocytosis to release the intracellular transcription factor. We fused the LHDA ligand with EndoTag, the fusion ligand was able to induce more than 1000 fold increase in signal activation with the help of rapid endocytosis.



Our approach to design synthetic EndoTags offers a general strategy for inducing endocytosis through receptors in alternative to traditional ligands. The potent endocytosis of EndoTags enables broad applications including extracellular degraders and signaling activators, as well as improved drug/RNA delivery. With production simplicity and modularity, we showcase how it could achieve logic-gated degradation and secretable degradation, which are not accessible for existing degraders including LYTAC, KineTAC and AbTAC. Overall it holds extensive therapeutic promises for various diseases.

Acknowledgements (Optional): :

References (Optional): :

1. Oka, Y., Rozek, L. M. & Czech, M. P. Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process. J. Biol. Chem. 260, 9435–9442 (1985).


2. Brown, J., Jones, E. Y. & Forbes, B. E. Keeping IGF-II under control: Lessons from the IGF-II–IGF2R crystal structure. Trends Biochem. Sci. 34, 612–619 (2009).


3. Harris, L. K. & Westwood, M. Biology and significance of signalling pathways activated by IGF-II. Growth Factors 30, 1–12 (2012)


4. Byrd, J. C., Park, J. H., Schaffer, B. S., Garmroudi, F. & MacDonald, R. G. Dimerization of the insulin-like growth factor II/mannose 6-phosphate receptor. J. Biol. Chem. 275, 18647–18656 (2000).


5. Pance, K. et al. Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins. Nat. Biotechnol. 1–9 (2022)


6. Banik, S. M. et al. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 584, 291–297 (2020).


7. Ahn, G. et al. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat. Chem. Biol. 17, 937–946 (2021).


8. Cao, L. et al. Design of protein binding proteins from target structure alone. Nature 1–1 (2022)


9. Dou, J. et al. De novo design of a fluorescence-activating β-barrel. Nature 561, 485–491 (2018).


10. Wang, J. et al. Scaffolding protein functional sites using deep learning. Science 377, 387–394 (2022).


11. Dauparas, J. et al. Robust deep learning–based protein sequence design using ProteinMPNN. Science 378, 49–56 (2022).


12. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).


13. Sahtoe, D. D. et al. Reconfigurable asymmetric protein assemblies through implicit negative design. Science 375, eabj7662 (2022).


14. Lajoie, M. J. et al. Designed protein logic to target cells with precise combinations of surface antigens. Science 369, 1637–1643 (2020).