Abstract
The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein—for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins—the products of 40% of all protein-encoding genes7—are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified—but essential—component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.
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Data availability
CRISPRi screen results are provided in Supplementary Table 1, quantitative proteomics results are provided in Supplementary Table 2. The flow cytometry gating strategy is provided in the Supplementary Information. All data that supported the findings of this study are included and are also available from the corresponding author upon request. Source data are provided with this paper.
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Acknowledgements
We thank M. Bassik for the CRISPRi library and dCas9-KRAB-expressing cell lines; T. Waldmann for a gift of the HDLM-2 cell line and E. Appel for use of aqueous gel permeation chromatography equipment. This work was supported in part by National Institutes of Health (NIH) grant P30CA124435 using the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resource and by NIH grant R01CA227942 to C.R.B. S.M.B was supported by a National Institute of General Medical Sciences F32 Postdoctoral Fellowship. K.P. was supported by a National Science Foundation Graduate Research Fellowship, a Stanford Graduate Fellowship, and the Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program. S.W. was supported by a Banting Postdoctoral Fellowship from the Canadian Institutes of Health. G.A. was supported by a National Science Foundation Graduate Research Fellowship. N.M.R. was supported by NIH grant K00CA21245403.
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S.M.B., K.P. and C.R.B. conceived the project. S.M.B., K.P., S.W., G.A. and N.M.R. carried out experiments and interpreted data. S.M.B. and C.R.B. wrote the manuscript with input from all authors. C.R.B. provided supervision.
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C.R.B. is a co-founder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent) and InterVenn Biosciences, and a member of the board of directors of Eli Lilly & Company. S.M.B. is a consultant for Lycia Therapeutics. Stanford University has filed patent applications related to this work which are licensed to Lycia Therapeutics, listing S.M.B., K.P., G.A. and C.R.B. as inventors.
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Extended data figures and tables
Extended Data Fig. 1 Synthesis of M6Pn-NCA, poly(mannose-6-phosphate-co-Ala), poly(mannose-co-Ala) and poly(GalNAc-co-Ala).
a, Synthetic route to mannose-6-phosphonate-serine N-carboxyanhydride (NCA). b, Synthetic route to M6P-NCA, followed by Ni-catalysed polymerization. Polymerization reactions were carried out in a N2 glovebox for 48 h in tetrahydrofuran. c, d, General synthetic schemes for the polymerization of mannose-NCA (c) and GalNAc-NCA (d). DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; Fmoc, fluorenylmethyloxycarbonyl; mCPBA, meta-chloroperoxybenzoic acid; OTf, triflate.
Extended Data Fig. 2 Biotin-poly(M6Pn) LYTACs direct NA-647 to lysosomes.
a, General scheme for biotinylation of glycopolypeptides with sulfo-NHS biotin. Biotinylation reactions were performed in 1× PBS at room temperature overnight. b, Biotin–LYTAC-mediated NA-647 uptake is continuous over time in K562 cells. K562 cells were incubated at 37 °C in complete growth medium with 500 nM NA-647 or 500 nM NA-647 and 2 μM poly(M6Pn)short for the indicated time, then washed and analysed by live-cell flow cytometry. The MFI (mean fluorescence intensity) was measured relative to background fluorescence from untreated K562 cells. c, d, Biotinylated poly(M6Pn) LYTACs direct NA-647 to lysosomes in K562 cells (c) and Jurkat cells (d). Cells were incubated with PBS, 500 nM NA-647, or 500 nM NA-647 and 2 μM biotinylated poly(M6Pn)short for 0.5–1 h in complete growth medium. NA-647 (red) colocalized with acidic endosomes and lysosomes as labelled with LysoTracker Green (turquoise). Scale bar, 20 μm. Fluorescence intensity is normalized in the NA-647 channel for all images. For c, d, data are representative of two independent experiments. For b, data are mean ± s.d. of three independent experiments.
Extended Data Fig. 3 EGFR surface levels are unchanged upon EXOC1 and EXOC2 knockdown in HeLa cells.
Cetuximab binds equally to dCas9-KRAB HeLa cells transfected with non-targeting sgRNA and cells transfected with sgRNA targeting IGF2R, EXOC1 or EXOC2, indicating no change in EGFR surface levels. Cells were subjected to live-cell flow cytometry using cetuximab followed by an anti-human Alexa Fluor-647-conjugated anti-human (anti-human 647) secondary antibody. Data are representative of two independent experiments.
Extended Data Fig. 4 Ab-1 mediates uptake of soluble proteins to lysosomes.
a, Uptake of an Alexa Fluor-488 (AF488)-labelled mouse IgG (m-IgG-488) into cells using antibody LYTACs. b, Uptake of m-IgG-488 using Ab-1. Mean fluorescence intensity (MFI) fold change over background uptake measured by live-cell flow cytometry. K562 cells were incubated at 37 °C for 1 h with 50 nM IgG-488 or 50 nM IgG and 25 nM of anti-mouse or Ab-1. c, AF488 signal (green) colocalized with acidic endosomes and lysosomes as labelled with LysoTracker Red (magenta). Expanded view shows a cell containing IgG-488 and LysoTracker Red. Scale bar, 20 μm. d, ApoE4-647 uptake over time. K562 cells were incubated with 50 nM ApoE4-647 in the presence or absence of 25 nM anti-ApoE4 and Ab-1. At the indicated time point, cells were aliquoted and median fold intensity (MFI) measurements were measured by live-cell flow cytometry. e, Total protein levels for leupeptin inhibition of apoE4 degradation in K562 cells, corresponding to lanes shown in Fig. 3h. Total protein was visualized by Coomassie stain. f, Flow cytometry plots of ApoE4-647 uptake over time, with or without leupeptin inhibition. g, Uptake of ApoE4-647 to lysosomes. K562 cells were incubated with PBS, 50 nM ApoE4-647, 25 nM anti-ApoE4 and 25 nM Ab-1 for 1 h or 24 h in complete growth media at 37 °C. Alexa Fluor-647 signal (red) colocalizes with acidic endosomes and lysosomes as labelled with LysoTracker Green (turquoise). Data are representative of two (c, e–g) independent experiments. Data are mean ± s.d. of three independent experiments (b, d). P values were determined by unpaired two-tailed t-tests; fold changes are reported relative to incubation with protein targets alone (b) or background fluorescence (d).
Extended Data Fig. 5 Optimization of LYTAC-mediated EGFR degradation.
a, Native gel of cetuximab (ctx)-based LYTACs. b, Levels of EGFR in HeLa cells treated with 100 nM ctx (lane 3), ctx-GalNAc (lane 4), ctx-M6Pnlong (lane 5) or ctx-M6Pnshort (lane 6) for 24 h in complete growth medium. EGF stimulation is a positive control for EGFR downregulation. c, Synthesis of linker-swapped Ab-2. Ctx was labelled with NHS-PEG4-N3, then incubated with BCN-functionalized poly(M6Pn)short for 3 days at room temperature. Reaction progress was monitored by native gel electrophoresis and visualized with Coomassie stain. d, Native gel of ctx-Fab-based LYTACs. e, EGFR levels in dCas9-KRAB HeLa cells transfected with non-targeting sgRNA against GAL4 after incubation with 100 nM, 10 nM,1 nM, or 0.1 nM conjugates for 36 h in complete growth medium. f, EGFR levels in dCas9-KRAB HeLa cells transfected with non-targeting GAL4 sgRNA incubated with Ab-2 or ctx for the indicated time. g, Quantification of LYTAC or ctx-mediated EGFR degradation in dCas9-KRAB HeLa expressing GAL4 sgRNA over time as read out by western blot relative to untreated cells. h, Levels of pEGFR in dCas9-KRAB HeLa cells expressing an sgRNA targeting IGF2R after 24 h incubation with 10 nM ctx or Ab-2, then incubation with EGF for 10 or 60 min. Data are representative of two (a–e, h) or three (f) independent experiments. For g, data are mean ± s.d. of three independent experiments, one of which is shown in f. Per cent control was calculated by densitometry and normalized to loading control (b, e, f).
Extended Data Fig. 6 Mixed-cell assay demonstrates that binding specificity is comparable between ctx-M6Pn and ctx.
a, Scheme for mixed cell assay. HeLa cells were lifted and labelled with CellTracker Deep Red, then mixed in a 1:1 ratio with Jurkat cells. The mixed cell sample was stained with either 10 nM ctx or ctx-M6Pn conjugate, followed by anti-human 488, then subjected to live-cell flow cytometry. b, Cell surface CI-M6PR levels on HeLa cells (CIM6PR+EGFR+) and Jurkat cells (CIM6PR+EGFR−) were measured by live-cell flow cytometry. HeLa and Jurkat cells exhibited similar levels of cell-surface CI-M6PR. c, Ctx and ctx-M6Pn exhibit equivalent binding to HeLa cells, and ctx-M6Pn exhibits minimal increased binding to Jurkat cells relative to ctx. Data are representative of two independent experiments (b) or two experimental replicates (c).
Extended Data Fig. 7 LYTACs mediate EGFR degradation in multiple cell lines.
a, EGFR levels in BT-474, MDA-MB-361, or HepG2 cells after incubation with 10–20 nM conjugates. b, Proliferation of HepG2 cells in the presence of EGF (200 ng ml−1) and 50 nM cetuximab or Ab-2. Cells were incubated with EGF and antibodies for 48 h, and proliferation measured using an MTS assay. Data are representative of three independent experiments (a). For b, data are mean ± s.e.m. of three independent experiments. P values were determined by unpaired two-tailed t-tests. Per cent control was calculated by densitometry and normalized to loading control.
Extended Data Fig. 8 Synthesis of anti PD-L1 glycopolypeptide conjugates, PD-L1 degradation, and CD71 degradation depends on M6P binding.
a, Anti-PD-L1 was non-specifically labelled with BCN, then incubated with poly(M6Pn)short for 3 days at room temperature. Reaction progress was monitored by native gel electrophoresis and visualized by Coomassie stain. b, Cell-surface PD-L1 determined by live-cell flow cytometry after incubation with anti-PD-L1 or conjugates (50 nM). At each time point, cells were washed, lifted, brought to 4 °C, then stained for PD-L1 using excess unconjugated anti-PD-L1 (1 μM). c, PD-L1 levels in MDA-MB-231 cells after 48-h incubation with anti-PD-L1 or Ab-3. d, PD-L1 levels in HDLM-2 cells after 36 h incubation with anti-PD-L1 or Ab-3. e, Quantification of PD-L1 degradation in HDLM-2 cells with Ab-3. f, Atezolizumab was non-specifically labelled with NHS-(PEG)4-N3, then incubated with poly(M6Pn)short-BCN for 3 days at room temperature. Reaction progress was monitored by native gel electrophoresis and visualized by Coomassie stain. g, Levels of CD71 in Jurkat cells after 24 h in the presence of 5 mM M6P. Data are representative of two (a, c, f, g) independent experiments. For b, data are mean ± s.d. of three independent experiments, and cell surface levels are relative to untreated cells. For e, data are mean ± s.d. of three independent experiments, one of which is shown in d. Per cent control was calculated by densitometry and normalized to loading control.
Extended Data Fig. 9 Human IgG in select mouse tissues.
Livers and spleens were collected from mice 72 h after intraperitoneal injection of ctx or ctx-M6Pn. Data are representative of three independent groups, one mouse per treatment per group.
Supplementary information
Supplementary Information
This file contains Supplemental Methods: All synthetic chemistry procedures and characterization data are described, as well as additional methods for cellular assays. Flow Cytometry Gating Strategy: A representative diagram of the flow cytometry gating strategy used to analyze all flow cytometry data. NMR Spectra: 1H, 13C, and where necessary, 31P, NMR spectra of all new compounds and glycopolypeptides. MALDI-MS Spectra: Representative MALDI-MS spectra of cetuximab and cetuximab non-specifically labeled with azides.
Supplementary Figure
Full Gels and Blots: Uncropped Western blots, SDS-PAGE gels, and native gels.
Supplementary Table
Supplementary Data Table 1: The full CRISPRi screen dataset.
Supplementary Table
Supplementary Data Table 2: The quantitative proteomics dataset comparing protein levels in untreated and treated cells.
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Banik, S.M., Pedram, K., Wisnovsky, S. et al. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 584, 291–297 (2020). https://doi.org/10.1038/s41586-020-2545-9
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DOI: https://doi.org/10.1038/s41586-020-2545-9
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