Lysosome-targeting chimaeras for degradation of extracellular proteins

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.

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-M6Pn_long (lane 5) or ctx-M6Pn_short (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.

Fig. 1 ∣. LYTACs using CI-M6PR traffic proteins to lysosomes.

a, The concept of LYTACs, in which a glycopolypeptide ligand for CI-M6PR is conjugated to an antibody to traffic secreted and membrane-associated proteins to lysosomes. b, Synthesis of M6Pn glycopolypeptide ligands for CI-M6PR. bpy, bipyridyl; COD, cyclooctadiene; RT, room temperature; THF, tetrahydrofuran; TMS, trimethylsilyl. c, Assay for the internalization of NA-647 by biotin-based LYTACs. d, Panel of synthetic M6P and M6Pn glycopolypeptides and controls. L, long; S, short. e, Fold changes in mean fluorescence intensity (MFI) for K562 cells incubated at 37 °C for 1 h with 500 nM NA-647 or 500 nM NA-647 and 2 μM biotinylated glycopolypeptide in complete growth media. MFI was determined by live-cell flow cytometry. f, Live-cell confocal microscopy images of K562 cells treated as in e, then labelled with LysoTracker Green for 30 min. Scale bar, 10 μm. g, Panel of cell lines for NA-647 uptake experiments, performed as in e. For e, g, data are mean ± s.d. of three independent experiments. For f, images are representative of two independent experiments. P values were determined by unpaired two-tailed t-tests; fold changes are reported relative to incubation with protein targets alone. NS, not significant.

Fig. 2 ∣. CRISPRi screen identifies key cellular machinery for LYTACs.

a, Schematic of a CRISPRi screen in K562 cells stably expressing dCas9-KRAB and a library of sgRNAs with genome-wide coverage. b, Selected gene hits for regulation of NA-647 internalization by LYTACs. c, Gene ontology (GO) annotation for significant hits (<5% FDR). d, e, Cell surface expression levels of CI-M6PR in dCas9-KRAB K562 cells (d) or dCas9-KRAB HeLa cells (e) transfected with control sgRNA, IGF2R-targeting sgRNA, EXOC1-targeting sgRNA or EXOC2-targeting sgRNA. Cells were stained for CI-M6PR and subjected to live-cell flow cytometry. f, g, Western blot analysis of EXOC1 and CI-M6PR in K562 (f) and HeLa (g) CRISPRi knockdown lines. For c, analysis is representative of two replicates; d, f, g are representative of two independent experiments. K562 cells rapidly lost the exocyst knockdown phenotype after two passages. For e, data are mean ± s.d. of three independent experiments in HeLa and normalized to non-targeting sgRNA. P values were determined by unpaired two-tailed t-tests.

Fig. 3 ∣. LYTACs target soluble proteins to lysosomes for degradation.

a, Synthetic scheme for antibody-based LYTACs with a goat anti-mouse IgG. b, Native gel (with Coomassie staining) of polyclonal goat anti-mouse IgG conjugates. c–f, Uptake of mCherry antigen and its antibody by a LYTAC, into K562 cells (d), HEK-293 cells (e) or Jurkat cells (f) with 50 nM mCherry, and 25 nM mouse anti-mCherry or 25 nM mouse anti-mCherry with 25 nM goat anti-mouse or conjugates after 1 h at 37 °C. Mean fluorescence intensity (MFI) was measured by live-cell flow cytometry. g, Uptake of ApoE4-647 into K562 cells; experiments were performed analogously to d. Median fluorescence intensity (MFI) was measured by live-cell flow cytometry. h, Cells were incubated as in g for 8 h in the presence or absence of 0.1 mg ml⁻¹ leupeptin (LPT), then lysed. Lysates were separated by SDS–PAGE, and ApoE4-647 was detected via in-gel fluorescence. Results are representative of two (h) or three (b) independent experiments. Data are mean ± s.d. of three independent experiments (d–g). P values were determined by unpaired two-tailed t-tests.; fold changes are reported relative to incubation with protein targets alone.

Fig. 4 ∣. LYTACs accelerate degradation of membrane proteins.

a, Schematic of EGFR degradation using LYTACs. b, EGFR levels after treatment with 10 nM Ab-2 for 24 h in dCas9-KRAB HeLa cells expressing a control sgRNA or sgRNA targeting IGF2R. c, EGFR levels after treatment with 10 nM Ab-2 for 48 h in dCas9-KRAB HeLa cells expressing a control sgRNA in the presence of 5 mM mannose-6-phosphate (M6P) or 200 μM chloroquine. d, EGFR levels after treatment with Fab-1 in dCas9-KRAB HeLa cells expressing a control sgRNA or sgRNA targeting IGF2R. e, Fold change in the abundance of 3,877 HeLa proteins detected by quantitative proteomics analysis after 24-h treatment with either 10 nM Ab-2 (left) or ctx (right) relative to untreated cells, data are the mean of three biological replicates. f, Cellular localization of EGFR after treatment with ctx or Ab-2 for 48 h. Scale bar, 20 μm. g, Levels of pEGFR in dCas9-KRAB HeLa cells expressing a control sgRNA after 24-h incubation with 10 nM ctx or Ab-2, then incubation with EGF for 10 min or 60 min. h, Levels of EGFR after treatment with 10 nM Ab-2 in Hep3B cells for 48 h. i, Levels of pEGFR, pAkt and pERK1/2 in Hep3B cells after 48 h incubation with 10 nM ctx or Ab-2, then incubation with EGF for 30 min. j, Degradation of CD71 mediated by a primary antibody and Ab-1. k, CD71 levels after treatment with 50 nM anti-CD71 or anti-CD71 and Ab-1 in Jurkat cells after 24 h. l, Uptake of transferrin-647 in Jurkat cells treated with anti-CD71 or anti-CD71 and Ab-1 for 24 h. m, PD-L1 levels in HDLM-2 cells after treatment with 25 nM atezolizumab (atz) or atz-LYTAC for 48 h. n, Levels of PD-L1 degradation with 25 nM atz-LYTAC after 48 h in HDLM-2 cells in the presence of 5 mM M6P. o, Serum levels of ctx or ctx-LYTAC in BALB/c mice injected intraperitoneally at 5 mg kg⁻¹. p, Quantification of serum ctx-M6Pn relative to ctx after intraperitoneal injection. Data are representative of two (c, d, f, g, l, n) or three (b, h, i, k, o) independent experiments or mice (o). Data are mean ± s.d. (m) or mean ± s.e.m. (p) of three independent experiments (m, one of which is shown in n) or mice (p). P values were determined by unpaired two-tailed t-tests. Relative values were calculated via densitometry and normalized to loading control (b–d, h, k, m, n) or relative to ctx levels (p).