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Designed endocytosis-inducing proteins degrade targets and amplify signals

Medicine and Health

Designed endocytosis-inducing proteins degrade targets and amplify signals

B. Huang, M. Abedi, et al.

Discover how researchers including Buwei Huang and Mohamad Abedi designed EndoTags that promote targeted degradation of proteins, overcoming the limitations of traditional therapeutic approaches. These innovative binding proteins enhance endocytosis and tissue-specific targeting, showing great therapeutic potential in cancer treatment and beyond.

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Playback language: English
Introduction
The endocytosis of many cell surface receptors is triggered by binding of their endogenous ligands, which can shift the conformational or oligomerization state of the receptor and induce receptor clustering and recruitment of adaptor proteins. Native endocytosis-inducing ligands have been utilized to target extracellular and membrane proteins to the lysosome for degradation. Although powerful, these approaches have the limitations that native ligands can trigger off-target signalling, their binding sites may be occupied by existing ligands, and instability and—in some cases—the need for modification can complicate manufacturing. Bio-orthogonal inducers of endocytosis could have therapeutic utility for targeted degradation or for initiating signalling through pathways involving endocytosis, and could provide powerful tools for investigating the association between cellular trafficking and receptor conformational and oligomerization state. Antibodies have been identified that stimulate endocytosis, but this can require considerable empirical screening for any target receptor. We reasoned that de novo protein design could enable the creation of bio-orthogonal endocytosis-inducing proteins that avoid the above limitations using strategies customized for the target receptor. To enable tissue-specific control over endocytosis for downstream applications, we selected target receptors with distinct tissue expression profiles: IGF2R is expressed in most tissues, asialoglycoprotein receptor (ASGPR) is expressed primarily in the liver, transferrin receptor (TfR) is expressed in the brain, liver and muscles, and sortilin is expressed in the brain and spinal cord. For receptors such as sortilin and TfR that constitutively traffic between the cell surface and the endosome-lysosome, binding to the receptor at a site that does not compete for the natural ligand could be sufficient. For receptors such as IGF2R, for which conformational change triggers endocytosis, binding must induce rearrangement of receptor extracellular domains, whereas for others, such as ASGPR, for which endocytosis is stimulated by clustering, binding should induce oligomerization. Fusion of designed proteins with these properties to a second target-binding protein could promote endocytosis and lysosomal trafficking of the target. We set out to design such endocytosis-targeting proteins, which we call EndoTags, for all four receptor systems (IGF2R, ASGPR, sortilin and TfR), and to explore their utility for modulating protein degradation and cellular signalling.
Literature Review
Existing methods for triggering receptor endocytosis, such as using native ligands or chemically modified ligands, have limitations including off-target signaling, competition with endogenous ligands, and complex manufacturing processes. The use of antibodies to stimulate endocytosis also requires extensive empirical screening. The paper cites several previous studies involving LYTACs (Lysosome-Targeting Chimeras) and KineTACs (Cytokine Receptor-Targeting Chimeras) that utilize this principle, but acknowledge their limitations. The authors also refer to previous research on the mechanisms of endocytosis in various receptors, including IGF2R, ASGPR, TfR, and sortilin, providing a foundation for their design strategies. Studies on the use of antibodies to stimulate endocytosis and their challenges are noted. The literature review highlights the need for a more efficient and versatile method for inducing receptor endocytosis, paving the way for their proposed EndoTag approach.
Methodology
The study employed computational protein design to create novel endocytosis-inducing proteins, termed EndoTags. The researchers targeted four receptors with distinct tissue expression profiles: IGF2R (ubiquitous), ASGPR (liver-specific), TfR (brain, liver, muscle), and sortilin (brain, spinal cord). Different design strategies were used based on the endocytosis mechanism of each receptor. For constitutively cycling receptors (TfR and sortilin), the goal was to design binders that target sites not overlapping with native ligand binding sites, preventing competition and off-target effects. Rosetta de novo binder design and yeast display were used for screening. Binding affinities were measured using biolayer interferometry (BLI). For IGF2R, which undergoes conformational changes upon ligand binding, the strategy was to design proteins that mimic this conformational change by binding to distinct domains (domain 6 and domain 11) of the receptor. The Rosetta RIFdock method was used for designing minibinders to these domains. Different linker lengths and domain arrangements were tested to optimize endocytosis efficiency. Both flexible and rigid fusions of domain 6 and domain 11 binders were designed and screened. For ASGPR, which is stimulated by receptor clustering, the approach was to design multivalent binders (ASGPR EndoTags) to induce receptor oligomerization. An updated Rosetta design approach, incorporating ProteinMPNN for sequence design and AlphaFold for evaluation, was used. Yeast display and FACS were employed for screening. Following design and screening, the EndoTags were fused with target protein binders (e.g., EGFR minibinders, PD-L1 antibody) to create protein-LYTACs (pLYTACs). Cellular uptake and target protein degradation were assessed using flow cytometry, confocal microscopy, western blotting, and quantitative proteomics. In vivo efficacy was evaluated using a mouse tumor model. A synthetic signaling system (ortho-SNIPR) was used to investigate the effect of EndoTags on signal amplification. The study also explored logic-gated targeted degradation using the Co-LOCKR system and the possibility of localized secretion of EndoTags from engineered cells.
Key Findings
The researchers successfully designed and characterized EndoTags for four different receptors: IGF2R, ASGPR, sortilin, and TfR. These EndoTags showed significant enhancement of cellular uptake when fused to target protein binders. For example, fusion of EndoTags with an EGFR minibinder resulted in efficient EGFR degradation in various cell lines (HeLa, H1975, Hep3B), demonstrating tissue-specific targeting via the choice of receptor. The effectiveness of the EndoTags was confirmed by receptor knockout experiments. Fusion with a PD-L1 antibody (atezolizumab) significantly improved anti-tumor efficacy in a mouse model, compared to the antibody alone. The study also demonstrated that EndoTags can enhance signaling through engineered ligand-receptor systems, achieving nearly a 100-fold increase in signal activation in a synthetic Notch-derived system (ortho-SNIPR). Furthermore, the modularity of EndoTags enabled the creation of logic-gated targeted degradation systems (using Co-LOCKR) that only degrade the target protein in the presence of a specific marker, enhancing specificity. The EndoTags were shown to be functional even when secreted from engineered cells, which opens up possibilities for adoptive cell therapies and more targeted protein degradation strategies.
Discussion
The findings of this study demonstrate the successful application of computational protein design to create effective bio-orthogonal inducers of endocytosis (EndoTags). The EndoTags overcome several limitations of existing targeted protein degradation approaches, offering improved specificity, genetic encodability, and simplified manufacturing. The ability to target different receptors with distinct tissue distributions enables tissue-specific protein degradation. The significant increase in anti-tumor efficacy observed in the mouse model highlights the therapeutic potential of EndoTags. The enhancement of signaling in the synthetic system suggests that EndoTags can be used not only for protein degradation but also for modulating cellular signaling pathways. The successful demonstration of logic-gated degradation and localized secretion from cells significantly expands the applicability of this technology.
Conclusion
This study presents a novel approach for targeted protein degradation and signal amplification using computationally designed EndoTags. These all-protein, modular, and genetically encodable molecules overcome limitations of existing methods by avoiding competition with native ligands, simplifying manufacturing, and enabling tissue-specific targeting. The enhanced efficacy in a mouse tumor model and the demonstration of signal amplification suggest significant therapeutic potential. Future research should focus on exploring the applicability of EndoTags to a broader range of receptors and signaling pathways and investigating the potential of EndoTags for improving the delivery of other therapeutics, such as nucleic acids and small molecules.
Limitations
While the study demonstrates significant promise, some limitations should be considered. The in vivo studies were conducted using a single tumor model, and further investigation in other model systems is needed to validate the generalizability of the findings. The immunogenicity of the de novo designed proteins remains to be fully assessed, although the authors note previous work suggesting low immunogenicity of a related protein design. Although the EndoTags were shown to be functional when secreted from cells, the long-term effects and potential toxicity of prolonged exposure to secreted EndoTags need further investigation. The ortho-SNIPR signaling system is a synthetic system and the relevance of the signal enhancement findings to endogenous signaling pathways warrants further exploration.
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