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Antibody Conjugations via Glycosyl Remodeling

  • Hanna Toftevall
  • Helén Nyhlén
  • Fredrik Olsson
  • Jonathan SjögrenEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

ADC conjugation Antibody glycosylation Glycosyl remodeling Site-specific conjugation Click-chemistry 

References

  1. 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.1480CrossRefGoogle Scholar
  2. 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.2016.268CrossRefGoogle Scholar
  3. 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.2415CrossRefGoogle Scholar
  4. 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.25.022106.141702CrossRefPubMedGoogle Scholar
  5. 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.2009.04.007CrossRefPubMedGoogle Scholar
  6. 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)96005-8CrossRefGoogle Scholar
  7. 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/c2cc32412aCrossRefGoogle Scholar
  8. 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/bc4002695CrossRefPubMedGoogle Scholar
  9. 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/bc400505qCrossRefPubMedGoogle Scholar
  10. 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.2010.12.009CrossRefPubMedGoogle Scholar
  11. 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.201402606CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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.M111183200CrossRefPubMedGoogle Scholar
  13. 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/bc900103pCrossRefPubMedPubMedCentralGoogle Scholar
  14. 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.29889CrossRefPubMedPubMedCentralGoogle Scholar
  15. 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/bc400122cCrossRefPubMedPubMedCentralGoogle Scholar
  16. 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/ja044996fCrossRefPubMedGoogle Scholar
  17. 17.
    Jewett JC, Bertozzi CR (2010) Cu-free click cycloaddition reactions in chemical biology. Chem Soc Rev 39(4):1272–1279CrossRefGoogle Scholar
  18. 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.6b00235CrossRefPubMedPubMedCentralGoogle Scholar
  19. 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/bc500499hCrossRefPubMedPubMedCentralGoogle Scholar
  20. 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.7b00802CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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/20.12.3046CrossRefPubMedPubMedCentralGoogle Scholar
  22. 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/BJ20130126CrossRefPubMedPubMedCentralGoogle Scholar
  23. 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/cwv047CrossRefPubMedPubMedCentralGoogle Scholar
  24. 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.5b00224CrossRefPubMedGoogle Scholar
  25. 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/c6ob01751gCrossRefPubMedGoogle Scholar
  26. 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/s11307-014-0781-9CrossRefPubMedGoogle Scholar
  27. 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 2018Google Scholar

Copyright information

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

Authors and Affiliations

  • Hanna Toftevall
    • 1
  • Helén Nyhlén
    • 1
  • Fredrik Olsson
    • 1
  • Jonathan Sjögren
    • 1
    Email author
  1. 1.Genovis ABLundSweden

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