Designed endocytosis-inducing proteins degrade targets and amplify signals

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Acknowledgement

The project or effort depicted was or is sponsored by the Department of Defense, Defense Threat Reduction Agency grant HDTRA1-21-1-0007 (B.H. and L.S.); DARPA Synergistic Discovery and Design (SD2) HR0011835403 contract FA8750-17-C-0219 (W.Y.) and Defense Threat Reduction Agency Grant HDTRA1-21-1-0038 (I.G. and W.Y.). This research was supported by the National Institutes of Health’s National Institute on Aging, grant R01AG063845 (B.H., B.C., I.G. and W.Y.); the National Institutes of Health’s National Cancer Institute, grant R01CA240339 (I.G.); the Audacious Project at the Institute for Protein Design (L.S., X.W., T.S., I.S. and S.W.); the Nordstrom Barrier Institute for Protein Design Directors Fund (B.H., I.G. and M. Abedi); AMGEN Donation to the Institute for Protein Design (S.W. and X.W.); The Open Philanthropy Project Improving Protein Design Fund (B.C. and I.G.); E. Schmidt, W. Schmidt and Schmidt Futures funding from E. Schmidt and W. Schmidt by recommendation of the Schmidt Futures programme (I.G.); the European Molecular Biology Organization via ALTF191-2021 (T.S.); NIH grant GM058867 (C.R.B.); and the Jane Coffin Childs Memorial Fund for Medical Research (M. Abedi). This research was also funded by National Science Foundation Graduate Research Fellowship and Stanford Center for Molecular Analysis and Design (G.A.). We acknowledge the excellent support from the Biology Imaging Facility at the University of Washington during the confocal imaging experiments. The authors thank M. Gloegl for help with the SPR experiment setup; M. Exposit for support with the OT-2 liquid handler; X. Li for help with mass spectrometry analysis of proteins; B. Wicky, L. Milles and R. Ragotte for optimizing the golden gate assembly protocol; A.Coubet for the analysis of preliminary cryo-electron microscopy (cryo-EM) data; D. Lee for transfection of secretable EGFR-IGF_EndoTag; S. Thompson for bridging the connection of the main collaborators in this project; the Banik laboratory for providing the IGF2R-knockout cell line; and I. Haydon for help with graphics. Figures 2b–g and 4a,c include elements created with BioRender.com.

Data Availability

The raw data for the flow cytometry, next-generation sequencing, designed binder models and sequences are available at https://doi.org/10.5281/zenodo.11002950 (ref. ⁵²). Source data are provided with this paper.

• Source Data Fig. 2

Code Availability

The Rosetta modelling suite (https://www.rosettacommons.org) is available to academic and non-commercial users for free. Commercial licences for the suite are also available through the University of Washington Technology Transfer Office. The source code for RIF docking is available at https://github.com/rifdock/rifdock. The source code for protein inpainting is available at https://github.com/RosettaCommons/RFDesign. The source code for ProteinMPNN is available at https://github.com/dauparas/ProteinMPNN. The scripts used in this paper for binder design applying the above codes, customized image processing and data processing are available at https://doi.org/10.5281/zenodo.11002950

Conflict of Interest

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 incorporates discoveries described in this manuscript.

Supplemental Material

• Supplementary Information: This file contains Supplementary Figs. 1–23 and Supplementary Tables 1–5.

Contributions

B.H., M. Abedi, G.A. and D.B. designed the research. B.H. designed, screened and optimized the binders for IGF2R and ASGPR. B.H. and I.S. designed and screened the rigid IGF_EndoTags. P.V. ran partial diffusion for ASGPR binders. B.C. designed and optimized the binders for sortilin. I.G. screened and optimized the binders for sortilin. J.O., Y.L., R.Y., Y.L., A. Misquith, A.W. and P.G. modelled, characterized and produced the N-glycan variants for sortilin. B.C. and L. Cao developed the Rifdock binder design pipeline. N.R.B. set up the PPI MPNN and AlphaFold2 pipeline for computational binder design. B.H. and M. Abedi studied the endocytosis enhancement effect in vitro. B.H., M. Abedi and G.A. evaluated the protein-degradation function of all EndoTags. M. Abedi, J.Z.Z. and T.S. performed the imaging experiments. M. Abedi, D.I.P., A.C.-G., M.J.D.G. and K.T.R. developed the SNIPR platform. W.Y. developed the CTLA4mb. W.Y. and X.W. contributed to binder design, library preparation and assay development. M. Abedi evaluated the application of EndoTag for signalling activation. M. Abedi evaluated the co-LOCKR AND gate degrader and secretable degrader. R.W. prepared the IGF2R target protein with advice from X.L. R.W. and S.W. performed the cryo-EM experiment and collected the cryo-EM density map. S.S. wrote the image analysis script used for imaging. Y.W. performed the proteomics analysis for whole-cell protein degradation. L.M.T. and J.B.S. characterized the antibody fusion of pLYTACs. D.D.S. generated the design basis of the TfR_EndoTag. C.T. and G.J.L.B. designed the in vivo anti-tumour experiments. C.T. conducted the in vivo experiments and data collection. B.H., C.W.C, S.C. and S.G produced and purified the protein used in the research. D.D.S. optimized the TfR_EndoTag. G.A., M. Abedi, M. Ahlrichs and C.D. prepared the cells used for this study. L. Carter and L.S. coordinated the resources and funding required for the research. C.R.B. and D.B. supervised this research. B.H., M. Abedi, G.A. and D.B. wrote the manuscript with the input from the other authors. All authors analysed data and revised the manuscript.