Skip to main content
SpringerLink
Log in

Antibody Conjugations via Glycosyl Remodeling

  • Protocol
  • First Online:
Antibody-Drug Conjugates

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2078))

Abstract

The antibody Fc-glycans are interesting targets for conjugation of cytotoxic compounds due to their localization and their chemical composition. In striving to obtain site-specific conjugates, the antibody Fc-glycans have been explored in numerous ways. Here we present a two-step enzymatic methodology coupled to click-chemistry for conjugation at the core GlcNAc of the Fc-glycan resulting in ADCs that are homogenous with a DAR 2.0, retain antigen binding, and display cytotoxic anti-tumor effects both in vitro and in vivo.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD et al (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26(8):925–932. https://doi.org/10.1038/nbt.1480

    Article  CAS  Google Scholar 

  2. Beck A, Goetsch L, Dumontet C, Corvaia N (2017) Strategies and challenges for the next generation of antibody–drug conjugates. Nat Rev Drug Discov 16(5):315–337. https://doi.org/10.1038/nrd.2016.268

    Article  CAS  Google Scholar 

  3. Chudasama V, Maruani A, Caddick S (2016) Recent advances in the construction of antibody–drug conjugates. Nat Chem 8(2):114–119. https://doi.org/10.1038/nchem.2415

    Article  CAS  Google Scholar 

  4. Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA (2007) The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 25:21–50. https://doi.org/10.1146/annurev.immunol.25.022106.141702

    Article  CAS  PubMed  Google Scholar 

  5. Jefferis R (2009) Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action. Trends Pharmacol Sci 30(7):356–362. https://doi.org/10.1016/j.tips.2009.04.007

    Article  CAS  PubMed  Google Scholar 

  6. Nimmerjahn F, Ravetch JV (2007) Fc-receptors as regulators of immunity. Adv Immunol 96:179–204. https://doi.org/10.1016/S0065-2776(07)96005-8

    Article  CAS  Google Scholar 

  7. Zuberbühler K, Casi G, Bernandes GJ, Neri D (2012) Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format. Chem Commun 48(56):7100–7102. https://doi.org/10.1039/c2cc32412a

    Article  CAS  Google Scholar 

  8. Okeley NM, Toki BE, Zhang X, Jeffrey SC, Burke PJ, Alley SC, Senter PD (2013) Metabolic engineering of monoclonal antibody carbohydrates for antibody–drug conjugation. Bioconjug Chem 24(10):1650–1655. https://doi.org/10.1021/bc4002695

    Article  CAS  PubMed  Google Scholar 

  9. Zhou Q, Stefano JE, Manning C, Kyazike J et al (2014) Site-specific antibody–drug conjugation through glycoengineering. Bioconjug Chem 25(3):510–520. https://doi.org/10.1021/bc400505q

    Article  CAS  PubMed  Google Scholar 

  10. Wang W, Vlasak J, Li Y, Pristatsky P et al (2011) Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies. Mol Immunol 48(6–7):860–866. https://doi.org/10.1016/j.molimm.2010.12.009

    Article  CAS  PubMed  Google Scholar 

  11. Li X, Fang T, Boons GJ (2014) Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew Chem Int Ed Engl 53(28):7179–7182. https://doi.org/10.1002/anie.201402606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ramakrishnan B, Qasba PK (2002) Structure-based design of beta 1,4-galactosyltransferase I (beta 4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity: point mutation broadens beta 4Gal-T1 donor specificity. J Biol Chem 277(23):20833–20839. https://doi.org/10.1074/jbc.M111183200

    Article  CAS  PubMed  Google Scholar 

  13. Boeggeman E, Ramakrishnan B, Pasek M, Manzoni M, Puri A et al (2009) Site specific conjugation of fluoroprobes to the remodeled Fc N-glycans of monoclonal antibodies using mutant glycosyltransferases: application for cell surface antigen detection. Bioconjug Chem 20(6):1228–1236. https://doi.org/10.1021/bc900103p

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhu Z, Ramakrishnan B, Li J, Wang Y, Feng Y et al (2014) Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. MAbs 6(5):1190–1200. https://doi.org/10.4161/mabs.29889

    Article  PubMed  PubMed Central  Google Scholar 

  15. Zeglis BM, Davis CB, Aggeler R, Kang HC et al (2013) An enzyme-mediated methodology for the site-specific radiolabeling of antibodies based on catalyst-free click chemistry. Bioconjug Chem 24(6):1057–1067. https://doi.org/10.1021/bc400122c

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Agard NJ, Prescher JA, Bertozzi CR (2004) A strain promoted [3 +2] azide – alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 126:15046–15047. https://doi.org/10.1021/ja044996f

    Article  CAS  PubMed  Google Scholar 

  17. Jewett JC, Bertozzi CR (2010) Cu-free click cycloaddition reactions in chemical biology. Chem Soc Rev 39(4):1272–1279

    Article  CAS  Google Scholar 

  18. Cook BE, Adumeau P, Membreno R, Carnazza KE, Brand C, Reiner T, Agnew BJ, Lewis JS, Zeglis BM (2016) Pretargeted PET imaging using a site-specifically labeled immunoconjugate. Bioconjug Chem 27(8):1789–1795. https://doi.org/10.1021/acs.bioconjchem.6b00235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zeglis BM, Davis CB, Abdel-Atti D, Carlin SD et al (2014) Chemoenzymatic strategy for the synthesis of site-specifically labeled Immunoconjugates for multimodal PET and optical imaging. Bioconjug Chem 25(12):2123–2128. https://doi.org/10.1021/bc500499h

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Adumeau P, Vivier D, Sharma SK, Wang W, Zhang T, Chen A, Agnew BJ, Zeglis BM (2018) Site-specifically labeled antibody–drug conjugate for simultaneous therapy and immunoPET. Mol Pharm 15(3):892–898. https://doi.org/10.1021/acs.molpharmaceut.7b00802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Collin M, Olsén A (2001) EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. EMBO J 20(12):3046–3055. https://doi.org/10.1093/emboj/20.12.3046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sjögren J, Struwe WB, Cosgrave EF, Rudd PM, Stervander M, Allhorn M, Hollands A, Nizet V, Collin M (2013) EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein. Biochem J 455(1):107–118. https://doi.org/10.1042/BJ20130126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sjögren J, Cosgrave EF, Allhorn M, Nordgren M, Björk S, Olsson F, Fredriksson S, Collin M (2015) EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans. Glycobiology 25(10):1053–1063. https://doi.org/10.1093/glycob/cwv047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. van Geel R, Wijdeven MA, Heesbeen R, Verkade JM, Wasiel AA, van Berkel SS, van Delft FL (2015) Chemoenzymatic conjugation of toxic payloads to the globally conserved N-glycan of native mAbs provides homogeneous and highly efficacious antibody–drug conjugates. Bioconjug Chem 26(11):2233–2242. https://doi.org/10.1021/acs.bioconjchem.5b00224

    Article  CAS  PubMed  Google Scholar 

  25. Tang F, Yang Y, Tang Y, Tang S et al (2016) One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody–drug conjugates. Org Biomol Chem 14(40):9501–9518. https://doi.org/10.1039/c6ob01751g

    Article  CAS  PubMed  Google Scholar 

  26. Gao P, Pinkston KL, Wilganowski N, Robinson H, Azhdarinia A, Zhu B, Sevick-Muraca EM, Harvey BR (2015) Deglycosylation of mAb by EndoS for improved molecular imaging. Mol Imaging Biol 17(2):195–203. https://doi.org/10.1007/s11307-014-0781-9

    Article  CAS  PubMed  Google Scholar 

  27. Hald A, Nielsen C, MacCann D, Doran N, Morgan H, Buick R, Behrendt N, Engelholm L (2018) Generation of an ADC against sarcoma and glioblastoma. Paper presented at 8th Annual World ADC Europe, Berlin, 26–28 Mar 2018

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Genovis AB, Lund, Sweden

    Hanna Toftevall, Helén Nyhlén, Fredrik Olsson & Jonathan Sjögren

Authors
  1. Hanna Toftevall
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Helén Nyhlén
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fredrik Olsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jonathan Sjögren
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jonathan Sjögren .

Editor information

Editors and Affiliations

  1. Department of Pharmacy and Pharmaceutical Sciences, Binghamton University, Binghamton, NY, USA

    L. Nathan Tumey

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Toftevall, H., Nyhlén, H., Olsson, F., Sjögren, J. (2020). Antibody Conjugations via Glycosyl Remodeling. In: Tumey, L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 2078. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9929-3_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9929-3_9

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9928-6

  • Online ISBN: 978-1-4939-9929-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation