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LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation

Nature Chemical Biology volume 17, pages 937–946 (2021)Cite this article

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Abstract

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.

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Fig. 1: LYTACs can hijack the ASPGR for targeted and cell-specific protein degradation.
Fig. 2: GalNAc-LYTACs promote degradation of EGFR in HCC cell lines.
Fig. 3: GalNAc-LYTACs operate via an endo-lysosomal mechanism and attenuate EGFR-driven signaling.
Fig. 4: Ctx-GalNAc mediates selective degradation of EGFR in ASGPR-expressing cells.
Fig. 5: GalNAc-LYTAC degrades the membrane proteins HER2 and integrins and induces antiproliferative effects in HEPG2 cells.
Fig. 6: Site-specific conjugation improves pharmacokinetics of antibody-based GalNAc-LYTACs.

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Data availability

All data that supported the findings of this manuscript are included and are also available from the corresponding author upon request. The flow cytometry gating strategy is provided in the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank M.A. Gray (Stanford University) for providing FGE-expressing Expi293 cells and her expertise in site-specific antibody conjugations. We thank M.A. Gray and J. Tanzo (Stanford University) for providing HIPS-azide and the Cochran laboratory for providing PIP–Fc (mIgG2a). We thank S. Pitteri and K. Lau (Canary Center at Stanford) for performing MALDI–TOF–MS characterization and analysis. We thank J. Vilches-Moure (Stanford University) and the Stanford AHS for performing liver histopathology. We thank the Stanford VSC Diagnostics Lab for liver biochemistry testing. We thank K. Pedram (Stanford University) for helpful discussions. We thank T. McLaughlin and Stanford University Mass Spectrometry for HRMS characterization. This work was supported, in part, by a National Institutes of Health grant R01GM058867 (C.R.B.) and a St. Baldrick’s/Stand Up 2 Cancer Pediatric Dream Team Translational Cancer Research Grant (J.R.C.). Researchers were supported by National Science Foundation Graduate Research Fellowship (G.A. and C.L.M.), a National Institute of General Medical Sciences F32 Postdoctoral Fellowship (S.M.B.) and National Institutes of Health grant K00CA21245403 (N.M.R.).

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Authors and Affiliations

  1. Department of Chemistry and Stanford ChEM-H, Stanford University, Stanford, CA, USA

    Green Ahn, Steven M. Banik, Nicholas M. Riley & Carolyn R. Bertozzi

  2. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Caitlyn L. Miller & Jennifer R. Cochran

  3. Howard Hughes Medical Institute, Stanford, CA, USA

    Carolyn R. Bertozzi

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Contributions

G.A., S.M.B. and C.R.B. conceived the project. G.A., S.M.B., C.L.M. and N.M.R. carried out the experiments and interpreted data. G.A. and C.L.M. synthesized and purified LYTAC conjugates. G.A., C.L.M. and S.M.B. carried out and analyzed in vitro degradation experiments. N.M.R. characterized and analyzed LYTAC conjugates by MS. G.A., S.M.B. and C.L.M. conceived and performed experiments to analyze the pharmacokinetic profiles and toxicity of LYTACs in vivo. J.R.C. oversaw and provided insights and materials such as PIP–Fc. G.A. and C.R.B. wrote the manuscript with input from all authors. C.R.B. provided supervision.

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Correspondence to Carolyn R. Bertozzi.

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Competing interests

Stanford University has filed patent applications relating to lysosome-targeting chimeras, which are licensed to Lycia Therapeutics, listing G.A., S.M.B. and C.R.B. as co-inventors. G.A., S.M.B., C.L.M., J.R.C. and C.R.B. are co-inventors on a patent application relating to PIP-LYTACs filed by Stanford University (docket number STAN-1780PRV). 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. J.R.C. is a founder of xCella Biosciences and Combangio Inc. and co-founder and director of Trapeze Therapeutics.

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Peer review information Nature Chemical Biology thanks Padma Devarajan, Lindy Durrant and Joshiawa Paulk for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 HER2 degradation by Ptz-GalNAc is inhibited by exogenous tri-GalNAc ligand.

Degradation of HER2 in HEPG2 cells determined by live-cell flow cytometry following co-treatment with DMSO or 5 mM of exogenous tri-GalNAc ligand (10) and 10 nM Ptz conjugates for 48 h. Data are the mean of three independent experiments ± SEM. P values were determined by unpaired two-tailed t-tests.

Source data

Extended Data Fig. 2 Ptz-GalNAc internalizes membrane HER2 within 2 hours.

a, Visualization of HER2 in HEPG2 cells by confocal microscopy after 10 nM pertuzumab conjugate treatments for 2 h. EEA1 is included as an early endosomal marker. b, Visualization of HER2 in HEPG2 cells by confocal microscopy after 10 nM pertuzumab conjugate treatments for 48 h. EEA1 is included as an early endosomal marker. Images are representative of two independent experiments. Scale bar, 30 µm.

Extended Data Fig. 3 Ctx-GalNAcs show similar lysosomal health as untreated cells.

a, Visualization and quantification of Lysotracker by confocal microscopy imaging of HEP3B cells treated with 10 nM cetuximab conjugates for 48 h or 1 mM LLOMe for 1 hour. b, Visualization and quantification of Cathepsin B activity using Magic Red in HEP3B cells treated with 10 nM cetuximab conjugates for 48 h or 1 mM LLOMe for 1 h. c, Visualization and quantification of ALIX in HEP3B cells treated with 10 nM cetuximab conjugates for 48 h or 1 mM LLOMe for 1 h. Scale bar, 30 µm. Values are the average ± SEM of three separate images from confocal microscopy. P values were determined by unpaired two-tailed t-tests.

Source data

Extended Data Fig. 4 Ptz-GalNAcs do not affect lysosomal health.

a. Visualization and quantification of Cathepsin B activity using Magic Red in HEPG2 cells treated with 100 nM Ptz conjugates for 48 h or 1 mM LLOMe for 1 h. b, Visualization and quantification of ALIX in HEPG2 cells treated with 100 nM Ptz conjugates for 48 h or 1 mM LLOMe for 1 h. Scale bar, 30 µm. Values are the average ± SEM of three separate images from confocal microscopy. P values were determined by unpaired two-tailed t-tests.

Source data

Extended Data Fig. 5 PIP-GalNAc conjugate and ASGPR are required for enhanced anti-proliferative effect.

a, Time-course percent proliferation of HEPG2 cells during 44 h of treatment with 50 or 100 nM PIP or PIP-GalNAc. b, Percent proliferation of HEPG2 cells over 48 h with 100 nM exogenous tri-GalNAc, 100 nM PIP, 100 nM PIP + 100 nM exogenous tri-GalNAc, or 100 nM PIP-GalNAc conjugate. c, Percent proliferation of HEPG2 cells at 48 h following co-incubation of 100 nM of PIP or PIP-GalNAc with or without 10 mg/ml asialofetuin (ASF). Data are three independent experiments in b. For c, values are the average of three independent experiments ± SEM. Ordinary two-way ANOVA with adjusted P values shown from Tukey’s multiple comparisons.

Source data

Extended Data Fig. 6 Analysis of site-specific conjugation of the tri-GalNAc ligand to three different locations on cetuximab.

a, Reducing SDS-PAGE gel of Ctx and Ctx with aldehyde tag at C-terminus, Hinge, and CH1 Heavy chain. b, The proportion of signal seen between tri-GalNAc-modified (blue) peptides and peptides from the sequence that should have harbored the tri-GalNAc ligand but were seen unmodified (gray). Due to the dimer nature of the antibody, 50% of signal as modified indicates one site of modification per antibody molecule while 100% of signal as modified shows two ligands per antibody molecule. c, EThcD spectra of peptides showing site-specific localization of the tri-GalNAc ligand in the SMARTag sequence. Note, ‘M’ represents the intact mass of the modified peptide, ‘GalNAc’ shows the oxonium ion of a GalNAc residue, and the ‘M-GalNAc(x)’ annotations show the intact mass minus x number of GalNAc moieties. a is a representative data from two independent experiments.

Source data

Extended Data Fig. 7 Analysis of site-specific conjugation of the tri-GalNAc ligand to three different locations on pertuzumab.

a, Reducing SDS-PAGE gel of Ptz with aldehyde tag at C-terminus, Hinge, and CH1 Heavy chain.b, The proportion of signal seen between tri-GalNAc-modified (blue) peptides and peptides from the sequence that should have harbored the tri-GalNAc ligand but were seen unmodified (gray). Due to the dimer nature of the antibody, 50% of signal as modified indicates one site of modification per antibody molecule while 100% of signal as modified shows two ligands per antibody molecule. c, EThcD spectra of peptides showing site-specific localization of the tri-GalNAc ligand in the SMARTag sequence. Note, ‘M’ represents the intact mass of the modified peptide, ‘GalNAc’ shows the oxonium ion of a GalNAc residue, and the ‘M-GalNAc(x)’ annotations show the intact mass minus x number of GalNAc moieties. a is a representative data from two independent experiments.

Source data

Extended Data Fig. 8 Non-specific Ctx-GalNAc conjugates show enhanced uptake in vitro compared to site-specific Ctx conjugates.

a, Binding of Ctx conjugates in HEPG2 cells measured by live-cell flow cytometry following 1 h incubation on ice. b, Mean fluorescence intensity (MFI) relative to the control (human IgG-647 only) for HEPG2 cells incubated at 37 °C for 1 h with 50 nM human IgG-647 and 25 nM Ctx, Ctx-(tri-GalNAc)10, Ctx-C-term-(tri-GalNAc)1, or Ctx-CH1-(tri-GalNAc)1. MFI was determined by live cell flow cytometry. Values are the average ± SEM of three independent experiments. P values were determined by unpaired two-tailed t-tests.

Source data

Extended Data Fig. 9 Durability of LYTAC-mediated degradation in HEP3B cells.

a, HEP3B cells were treated with 10 nM Ctx conjugates, then washed with PBS 3 times, and were incubated in fresh media for 6, 24, 48 h. EGFR levels were measured by western blot. 100 ng/ml of EGF was included as a control. b, Quantification of EGFR levels with and without wash-off following treatment with Ctx conjugates. Values are the average of three independent experiments ± SEM. P values were determined by unpaired two-tailed t-tests.

Source data

Extended Data Fig. 10 GalNAc-LYTACs do not cause hepatic toxicity in mice.

a, Balb/c mice were intraperitoneally injected with 5 mg/kg of Ctx or Ctx-(tri-GalNAc)10 every 2 days or 5 mg/kg Ctx or Ctx-C-term-(tri-GalNAc)1 every 4 days for a week. Plasma and liver were harvested on day 8, and levels of liver enzymes (b – alanine transaminase (ALT); c – aspartate transaminase (AST), d, alkaline phosphatase (ALP), e – total bilirubin) from plasma were measured. Values in b-e are the average of three independent mice ± SEM and were evaluated using Ordinary one-way ANOVA with Tukey’s multiple comparisons. f, Representative H&E staining of the liver from three independent experiments. Scale bar, 40 µm.

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Supplementary Information

Supplementary Table 1, Figs. 1–13 and Notes 1 and 2.

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Ahn, G., Banik, S.M., Miller, C.L. et al. LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat Chem Biol 17, 937–946 (2021). https://doi.org/10.1038/s41589-021-00770-1

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