Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation

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

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

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.

References

  1. Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

    Article  PubMed  CAS  Google Scholar 

  2. Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Sakamoto, K. M. et al. Protacs: chimeric molecules that target proteins to the Skp1–Cullin–F box complex for ubiquitination and degradation. Proc. Natl Acad. Sci. 98, 8554–8559 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Winter, G. E. et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nabet, B. et al. The dTAG system for immediate and target-specific protein degradation. Nat. Chem. Biol. 14, 431–441 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nabet, B. et al Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat. Commun. 11, 4687 (2020).

  7. Clift, D. et al. A method for the acute and rapid degradation of endogenous proteins. Cell 171, 1692–1706 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Naito, M., Ohoka, N. & Shibata, N. SNIPERs—hijacking IAP activity to induce protein degradation. Drug Discov. Today Technol. 31, 35–42 (2019).

    Article  PubMed  Google Scholar 

  9. Takahashi, D. et al. AUTACs: cargo-specific degraders using selective autophagy. Mol. Cell. 76, 797–810 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Li, Z. et al. Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds. Nature 575, 203–209 (2019).

    Article  CAS  PubMed  Google Scholar 

  11. Li, Z., Zhu, C., Ding, Y., Fei, Y. & Lu, B. ATTEC: a potential new approach to target proteinopathies. Autophagy 16, 185–187 (2020).

    Article  CAS  PubMed  Google Scholar 

  12. Mullard, A. First targeted protein degrader hits the clinic. Nat. Rev. Drug Discov. 18, 237–239 (2019).

    Google Scholar 

  13. Banik, S. M. et al. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 584, 291–297 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ghosh, P., Dahms, N. M. & Kornfeld, S. Mannose 6-phosphate receptors: new twists in the tale. Nat. Rev. Mol. Cell Biol. 4, 202–213 (2003).

    Article  CAS  PubMed  Google Scholar 

  15. Zimmermann, T. S. et al. Clinical proof of concept for a novel hepatocyte-targeting GalNAc-siRNA conjugate. Mol. Ther. 25, 71–78 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Spiess, M. The asialoglycoprotein receptor: a model for endocytic transport receptors. Biochemistry 29, 10009–10018 (1990).

    Article  CAS  PubMed  Google Scholar 

  17. Park, E. I., Mi, Y., Unverzagt, C., Gabius, H.-J. & Baenziger, J. U. The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acidα2,6GalNAc. Proc. Natl Acad. Sci. 102, 17125–17129 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Stockert, R. J. The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol. Rev. 75, 591–609 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Matsuda, S. et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chem. Biol. 10, 1181–1187 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. Springer, A. D. & Dowdy, S. F. GalNAc-siRNA conjugates: leading the way for delivery of RNAi therapeutics. Nucleic Acid Ther. 28, 109–118 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Prakash, T. P. et al. Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res. 42, 8796–8807 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kanasty, R., Dorkin, J. R., Vegas, A. & Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 12, 967–977 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Lee, Y. C. et al. Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J. Biol. Chem. 258, 199–202 (1983).

    Article  CAS  PubMed  Google Scholar 

  24. Nair, J. K. et al. Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J. Am. Chem. Soc. 136, 16958–16961 (2014).

    Article  CAS  PubMed  Google Scholar 

  25. Huang, X., Leroux, J.-C. & Castagner, B. Well-defined multivalent ligands for hepatocytes targeting via asialoglycoprotein receptor. Bioconjug. Chem. 28, 283–295 (2017).

    Article  CAS  PubMed  Google Scholar 

  26. Huang, Y. Preclinical and clinical advances of GalNAc-decorated nucleic acid therapeutics. Mol. Ther. Nucleic Acids 6, 116–132 (2017).

    Article  CAS  PubMed  Google Scholar 

  27. Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Probert, M. A., Zhang, J. & Bundle, D. R. Synthesis of α- and β-linked tyvelose epitopes of the Trichinella spiralis glycan: 2-acetamido-2-deoxy-3-O-(3,6-dideoxy-d-arabino-hexopyranosyl)-β-d-galactopyranosides. Carbohydr. Res. 296, 149–170 (1996).

    Article  CAS  PubMed  Google Scholar 

  29. Ezzoukhry, Z. et al. EGFR activation is a potential determinant of primary resistance of hepatocellular carcinoma cells to sorafenib. Int. J. Cancer 131, 2961–2969 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. Blivet-Van Eggelpoël, M.-J. et al. Epidermal growth factor receptor and HER-3 restrict cell response to sorafenib in hepatocellular carcinoma cells. J. Hepatol. 57, 108–115 (2012).

    Article  PubMed  CAS  Google Scholar 

  31. Baselga, J. et al. Phase II multicenter study of the antiepidermal growth factor receptor monoclonal antibody cetuximab in combination with platinum-based chemotherapy in patients with platinum-refractory metastatic and/or recurrent squamous cell carcinoma of the head and neck. J. Clin. Oncol. 23, 5568–5577 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Vermorken, J. B. et al. Open-label, uncontrolled, multicenter phase II study to evaluate the efficacy and toxicity of cetuximab as a single agent in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck who failed to respond to platinum-based therapy. J. Clin. Oncol. 25, 2171–2177 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Bondeson, D. P. et al. Lessons in PROTAC design from selective degradation with a promiscuous warhead. Cell Chem. Biol. 25, 78–87 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Shi, J.-H. et al. Recognition of HER2 expression in hepatocellular carcinoma and its significance in postoperative tumor recurrence. Cancer Med. 8, 1269–1278 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kimura, R. H., Levin, A. M., Cochran, F. V. & Cochran, J. R. Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low-nanomolar affinity. Proteins 77, 359–369 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Cox, N., Kintzing, J. R., Smith, M., Grant, G. A. & Cochran, J. R. Integrin-targeting knottin peptide–drug conjugates are potent inhibitors of tumor cell proliferation. Angew. Chem. Int. Ed. Engl. 55, 9894–9897 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kwan, B. H. et al. Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses. J. Exp. Med. 214, 1679–1690 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Arangoa, M. A., Düzgüneş, N. & Tros de Ilarduya, C. Increased receptor-mediated gene delivery to the liver by protamine-enhanced-asialofetuin-lipoplexes. Gene Ther. 10, 5–14 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Treichel, U., Meyer zum Büschenfelde, K. H., Dienes, H. P. & Gerken, G. Receptor-mediated entry of hepatitis B virus particles into liver cells. Arch. Virol. 142, 493–498 (1997).

    Article  CAS  PubMed  Google Scholar 

  41. Jin, Y. et al. OPN and αvβ3 expression are predictors of disease severity and worse prognosis in hepatocellular carcinoma. PLoS ONE 9, e87930 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Li, J. et al. Antisense integrin αv and β3 gene therapy suppresses subcutaneously implanted hepatocellular carcinomas. Dig. Liver Dis. 39, 557–565 (2007).

    Article  CAS  PubMed  Google Scholar 

  43. Rabuka, D., Rush, J. S., deHart, G. W., Wu, P. & Bertozzi, C. R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat. Protoc. 7, 1052–1067 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Agarwal, P. et al. Hydrazino-Pictet–Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjug. Chem. 24, 846–851 (2013).

    Article  CAS  PubMed  Google Scholar 

  45. Gray, M. et al. Targeted glycan degradation potentiates the anticancer immune response in vivo. Nat. Chem. Bio 16, 1376–1374 (2020).

    Article  CAS  Google Scholar 

  46. Maneiro, M. et al Antibody-PROTAC conjugates enable HER2–dependent targeted protein degradation of BRD4. ACS Chem. Biol. 15, 1306–1312 (2020).

  47. Lai, A. C. & Crews, C. M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101–114 (2017).

    Article  CAS  PubMed  Google Scholar 

  48. Schapira, M., Calabrese, M. F., Bullock, A. N. & Crews, C. M. Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug Discov. 18, 949–963 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Stahl, P. D. The macrophage mannose receptor: current status. Am. J. Respir. Cell Mol. Biol. 2, 317–318 (1990).

    Article  CAS  PubMed  Google Scholar 

  50. O’Reilly, M. K., Tian, H. & Paulson, J. C. CD22 is a recycling receptor that can shuttle cargo between the cell surface and endosomal compartments of B cells. J. Immunol. 186, 1554–1563 (2011).

    Article  PubMed  CAS  Google Scholar 

  51. Rose, C. M. et al. A calibration routine for efficient ETD in large-scale proteomics. J. Am. Soc. Mass. Spectrom. 26, 1848–1857 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Solntsev, S. K., Shortreed, M. R., Frey, B. L. & Smith, L. M. Enhanced global post-translational modification discovery with MetaMorpheus. J. Proteome Res. 17, 1844–1851 (2018).

    Article  CAS  PubMed  Google Scholar 

  53. Brademan, D. R., Riley, N. M., Kwiecien, N. W. & Coon, J. J. Interactive peptide spectral annotator: a versatile web-based tool for proteomic applications. Mol. Cell. Proteom. 18, S193–S201 (2019).

    Article  CAS  Google Scholar 

Download references

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.).

Author information

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

Authors
  1. Green Ahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Steven M. Banik
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Caitlyn L. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nicholas M. Riley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jennifer R. Cochran
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carolyn R. Bertozzi
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Carolyn R. Bertozzi.

Ethics declarations

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.

Additional information

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

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Source data

Supplementary information

Supplementary Information

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

Reporting Summary

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 1

Unprocessed gel.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed blots.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 3

Unprocessed blots.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 5

Unprocessed blots.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 6

Unprocessed blots.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Unprocessed gel.

Source Data Extended Data Fig. 7

Unprocessed gel.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Statistical source data.

Source Data Extended Data Fig. 9

Unprocessed blot.

Source Data Extended Data Fig. 10

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00770-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research