Skip to main content
SpringerLink
Log in

Application of Next-Generation Maleimides (NGMs) to Site-Selective Antibody Conjugation

  • Protocol
  • First Online:
Bioconjugation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2033))

Abstract

Site-selective antibody conjugation is widely recognized as a key strategy for the optimum construction of antibody–drug conjugates (ADCs). Achieving such bioconjugation directly onto native antibodies would represent the ideal solution, as it would afford greatly improved homogeneity whilst avoiding the need for genetic engineering, and even allow the repurposing of existing antibodies “off-the shelf.” Here we describe a protocol for the use of next-generation maleimides (NGMs) for the selective modification of the four interchain disulfide bonds present in a typical IgG1 antibody format. These reagents retain the efficiency of classical maleimides whilst serving to rebridge each reduced disulfide bond, affording one attachment per disulfide. The approach is simple, uses readily available reagents, and generates robustly stable conjugates which are ideal for in vitro or in vivo applications. In addition to use in the construction of ADCs these reagents can also be used to develop antibody conjugates for imaging, bispecifics, and broadly for use across biology and medicine.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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. Wang LT, Amphlett G, Blattler WA, Lambert JM, Zhang W (2005) Structural characterization of the maytansinoid—monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci 14(9):2436–2446

    Article  CAS  Google Scholar 

  2. Junutula JR, Raab H, Clark S, Bhakta S, Leipold DD, Weir S et al (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26(8):925–932

    Article  CAS  Google Scholar 

  3. Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H et al (2010) Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res 16(19):4769–4778

    Article  CAS  Google Scholar 

  4. Hamblett KJ, Senter PD, Chace DF, Sun MMC, Lenox J, Cerveny CG et al (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10(20):7063–7070

    Article  CAS  Google Scholar 

  5. Beckley NS, Lazzareschi KP, Chih HW, Sharma VK, Flores HL (2013) Investigation into temperature-induced aggregation of an antibody drug conjugate. Bioconjug Chem 24(10):1674–1683

    Article  CAS  Google Scholar 

  6. Adem YT, Schwarz KA, Duenas E, Patapoff TW, Galush WJ, Esue O (2014) Auristatin antibody drug conjugate physical instability and the role of drug payload. Bioconjug Chem 25(4):656–664

    Article  CAS  Google Scholar 

  7. Boswell CA, Mundo EE, Zhang C, Bumbaca D, Valle NR, Kozak KR et al (2011) Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats. Bioconjug Chem 22(10):1994–2004

    Article  CAS  Google Scholar 

  8. Alley SC, Benjamin DR, Jeffrey SC, Okeley NM, Meyer DL, Sanderson RJ et al (2008) Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug Chem 19(3):759–765

    Article  CAS  Google Scholar 

  9. Shen B-Q, Xu K, Liu L, Raab H, Bhakta S, Kenrick M et al (2012) Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol 30(2):184–189

    Article  CAS  Google Scholar 

  10. Dennler P, Fischer E, Schibli R (2015) Antibody conjugates: from heterogeneous populations to defined reagents. Antibodies 4(3):197–224

    Article  CAS  Google Scholar 

  11. Jackson D, Atkinson J, Guevara CI, Zhang CY, Kery V, Moon SJ et al (2014) In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates. PLoS One 9(1):14

    Article  Google Scholar 

  12. Akkapeddi P, Azizi SA, Freedy AM, Cal P, Gois PMP, Bernardes GJL (2016) Construction of homogeneous antibody-drug conjugates using site-selective protein chemistry. Chem Sci 7(5):2954–2963

    Article  CAS  Google Scholar 

  13. Jackson DY (2016) Processes for constructing homogeneous antibody drug conjugates. Org Process Res Dev 20(5):852–866

    Article  CAS  Google Scholar 

  14. Chudasama V, Maruani A, Caddick S (2016) Recent advances in the construction of antibody-drug conjugates. Nat Chem 8(2):113–118

    Article  Google Scholar 

  15. Kuan SL, Wang T, Weil T (2016) Site-selective disulfide modification of proteins: expanding diversity beyond the proteome. Chem Eur J 22(48):17112–17129

    Article  CAS  Google Scholar 

  16. Bryant P, Pabst M, Badescu G, Bird M, McDowell W, Jamieson E et al (2015) In vitro and in vivo evaluation of cysteine rebridged trastuzumab-MMAE antibody drug conjugates with defined drug-to-antibody ratios. Mol Pharm 12(6):1872–1879

    Article  CAS  Google Scholar 

  17. Badescu G, Bryant P, Bird M, Henseleit K, Swierkosz J, Parekh V et al (2014) Bridging disulfides for stable and defined antibody drug conjugates. Bioconjug Chem 25(6):1124–1136

    Article  CAS  Google Scholar 

  18. Maruani A, Smith MEB, Miranda E, Chester KA, Chudasama V, Caddick S (2015) A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nat Commun 6:6645

    Article  CAS  Google Scholar 

  19. Bahou C, Richards DA, Maruani A, Love EA, Javaid F, Caddick S et al (2018) Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones. Org Biomol Chem 16(8):1359–1366

    Article  CAS  Google Scholar 

  20. Tedaldi LM, Smith MEB, Nathani R, Baker JR (2009) Bromomaleimides; new reagents for the selective and reversible modification of cysteine. Chem Commun (43):6583–6585

    Google Scholar 

  21. Smith MEB, Schumacher FF, Ryan CP, Tedaldi LM, Papaioannou D, Waksman G et al (2010) Protein modification, bioconjugation, and disulfide bridging using bromomaleimides. J Am Chem Soc 132(6):1960–1965

    Article  CAS  Google Scholar 

  22. Ryan CP, Smith MEB, Schumacher FF, Grohmann D, Papaioannou D, Waksman G et al (2011) Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis. Chem Commun 47(19):5452–5454

    Article  CAS  Google Scholar 

  23. Castaneda L, Maruani A, Schumacher FF, Miranda E, Chudasama V, Chester KA et al (2013) Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation. Chem Commun 49(74):8187–8189

    Article  CAS  Google Scholar 

  24. Schumacher FF, Sanchania VA, Tolner B, Wright ZVF, Ryan CP, Smith MEB et al (2013) Homogeneous antibody fragment conjugation by disulfide bridging introduces ‘spinostics’. Sci Rep 3:1525

    Article  Google Scholar 

  25. Schumacher FF, Nunes JPM, Maruani A, Chudasama V, Smith MEB, Chester KA et al (2014) Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging. Org Biomol Chem 12(37):7261–7269

    Article  CAS  Google Scholar 

  26. Nunes JP, Morais M, Vassileva V, Robinson E, Rajkumar VS, Smith ME et al (2015) Functional native disulfide bridging enables delivery of a potent, stable and targeted antibody-drug conjugate (ADC). Chem Commun 51(53):10624–10627

    Article  CAS  Google Scholar 

  27. Robinson E, Nunes JP, Vassileva V, Maruani A, Nogueira J, Smith MEB et al (2017) Pyridazinediones deliver potent, stable, targeted and efficacious antibody-drug conjugates (ADCs) with a controlled loading of 4 drugs per antibody. RSC Adv

    Google Scholar 

  28. Morais M, Nunes JPM, Karu K, Forte N, Benni I, Smith MEB et al (2017) Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis. Org Biomol Chem 15(14):2947–2952

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, University College London, London, UK

    Maurício Morais, Nafsika Forte, Vijay Chudasama & James R. Baker

Authors
  1. Maurício Morais
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nafsika Forte
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vijay Chudasama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. James R. Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to James R. Baker .

Editor information

Editors and Affiliations

  1. Cellular and Molecular Immunology Laboratory, Vrije Universiteit Brussel, Brussels, Belgium

    Sam Massa

  2. In Vivo Cellular and Molecular Imaging Laboratory (ICMI), Vrije Universiteit Brussel, Brussels, Belgium

    Nick Devoogdt

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Morais, M., Forte, N., Chudasama, V., Baker, J.R. (2019). Application of Next-Generation Maleimides (NGMs) to Site-Selective Antibody Conjugation. In: Massa, S., Devoogdt, N. (eds) Bioconjugation. Methods in Molecular Biology, vol 2033. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9654-4_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9654-4_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9653-7

  • Online ISBN: 978-1-4939-9654-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation