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.

  • Letter
  • Published:

Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates

Nature Biotechnology volume 32, pages 1059–1062 (2014)Cite this article

Subjects

Abstract

Many antibody-drug conjugates (ADCs) are unstable in vivo because they are formed from maleimide-containing components conjugated to reactive thiols. These thiosuccinimide linkages undergo two competing reactions in plasma: elimination of the maleimide through a retro-Michael reaction, which results in loss of drug-linker from the ADC, and hydrolysis of the thiosuccinimide ring, which results in a derivative that is resistant to the elimination reaction. In an effort to create linker technologies with improved stability characteristics, we used diaminopropionic acid (DPR) to prepare a drug-linker incorporating a basic amino group adjacent to the maleimide, positioned to provide intramolecular catalysis of thiosuccinimide ring hydrolysis. This basic group induces the thiosuccinimide to undergo rapid hydrolysis at neutral pH and room temperature. Once hydrolyzed, the drug-linker is no longer subject to maleimide elimination reactions, preventing nonspecific deconjugation. In vivo studies demonstrate that the increased stability characteristics can lead to improved ADC antitumor activity and reduced neutropenia.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Structures and chemistry of thiol-reactive maleimides.
Figure 2: Hydrolysis rates and plasma stability of conjugates.
Figure 3: Antitumor activity, pharmacokinetics and neutropenia in rodent models.

Similar content being viewed by others

References

  1. Mullard, A. Maturing antibody-drug conjugate pipeline hits 30. Nat. Rev. Drug Discov. 12, 329–332 (2013).

    Article  CAS  Google Scholar 

  2. Sievers, E.L. & Senter, P.D. Antibody-drug conjugates in cancer therapy. Annu. Rev. Med. 64, 15–29 (2013).

    Article  CAS  Google Scholar 

  3. Okeley, N.M., Alley, S.C. & Senter, P.D. Advancing antibody drug conjugation: from the laboratory to a clinically approved anticancer drug. Hematol. Oncol. Clin. North Am. 28, 13–25 (2014).

    Article  Google Scholar 

  4. Senter, P.D. & Sievers, E.L. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol. 30, 631–637 (2012).

    Article  CAS  Google Scholar 

  5. Peddi, P.F. & Hurvitz, S.A. Trastuzumab emtansine: the first targeted chemotherapy for treatment of breast cancer. Future Oncol. 9, 319–326 (2013).

    Article  CAS  Google Scholar 

  6. Hermanson, G.T. Bioconjugate Techniques. 2nd Edn. (Academic Press, San Diego, 2008).

  7. Chari, R.V., Miller, M.L. & Widdison, W.C. Antibody-drug conjugates: an emerging concept in cancer therapy. Angew. Chem. Int. Edn Engl. 53, 3796–3827 (2014).

    Article  CAS  Google Scholar 

  8. Alley, S.C. et al. Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug. Chem. 19, 759–765 (2008).

    Article  CAS  Google Scholar 

  9. Shen, B.Q. et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30, 184–189 (2012).

    Article  CAS  Google Scholar 

  10. Baldwin, A.D. & Kiick, K.L. Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjug. Chem. 22, 1946–1953 (2011).

    Article  CAS  Google Scholar 

  11. Ryan, C.P. et al. Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis. Chem. Commun. (Camb.) 47, 5452–5454 (2011).

    Article  CAS  Google Scholar 

  12. Sassoon, I. & Blanc, V. Antibody-drug conjugate (ADC) clinical pipeline: a review. Methods Mol. Biol. 1045, 1–27 (2013).

    Article  Google Scholar 

  13. Zolot, R.S., Basu, S. & Million, R.P. Antibody-drug conjugates. Nat. Rev. Drug Discov. 12, 259–260 (2013).

    Article  CAS  Google Scholar 

  14. Chudasama, V.L. et al. Semi-mechanistic population pharmacokinetic model of multivalent trastuzumab emtansine in patients with metastatic breast cancer. Clin. Pharmacol. Ther. 92, 520–527 (2012).

    Article  CAS  Google Scholar 

  15. Strop, P. et al. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20, 161–167 (2013).

    Article  CAS  Google Scholar 

  16. Toda, N., Asano, S. & Barbas, C.F. 3rd rapid, stable, chemoselective labeling of thiols with Julia-Kocienski-like reagents: a serum-stable alternative to maleimide-based protein conjugation. Angew. Chem. Int. Edn Engl. 52, 12592–12596 (2013).

    Article  CAS  Google Scholar 

  17. Jeffrey, S.C. et al. A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. Bioconjug. Chem. 24, 1256–1263 (2013).

    Article  CAS  Google Scholar 

  18. Knight, P. Hydrolysis of p-N,N′-phenylenebismaleimide and its adducts with cysteine. Biochem. J. 179, 191–197 (1979).

    Article  CAS  Google Scholar 

  19. Kalia, J. & Raines, R.T. Catalysis of imido group hydrolysis in a maleimide conjugate. Bioorg. Med. Chem. Lett. 17, 6286–6289 (2007).

    Article  CAS  Google Scholar 

  20. Badescu, G. et al. A new reagent for stable thiol-specific conjugation. Bioconjug. Chem. 25, 460–469 (2014).

    Article  CAS  Google Scholar 

  21. Agarwal, P., van der Weijden, J., Sletten, E.M., Rabuka, D. & Bertozzi, C.R.A. Pictet-Spengler ligation for protein chemical modification. Proc. Natl. Acad. Sci. USA 110, 46–51 (2013).

    Article  Google Scholar 

  22. Tian, F. et al. A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. USA 111, 1766–1771 (2014).

    Article  CAS  Google Scholar 

  23. Doronina, S.O. et al. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. 21, 778–784 (2003).

    Article  CAS  Google Scholar 

  24. Francisco, J.A. et al. cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood 102, 1458–1465 (2003).

    Article  CAS  Google Scholar 

  25. Lyon, R.P., Meyer, D.L., Setter, J.R. & Senter, P.D. Conjugation of anticancer drugs through endogenous monoclonal antibody cysteine residues. Methods Enzymol. 502, 123–138 (2012).

    Article  CAS  Google Scholar 

  26. Hamblett, K.J. et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10, 7063–7070 (2004).

    Article  CAS  Google Scholar 

  27. Sanderson, R.J. et al. In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin. Cancer Res. 11, 843–852 (2005).

    CAS  PubMed  Google Scholar 

  28. Wahl, A.F. et al. The anti-CD30 monoclonal antibody SGN-30 promotes growth arrest and DNA fragmentation in vitro and affects antitumor activity in models of Hodgkin's disease. Cancer Res. 62, 3736–3742 (2002).

    CAS  PubMed  Google Scholar 

  29. Dubowchik, G.M. et al. Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity. Bioconjug. Chem. 13, 855–869 (2002).

    Article  CAS  Google Scholar 

  30. Sun, M.M. et al. Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjug. Chem. 16, 1282–1290 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank our Seattle Genetics colleagues H. Kostner for ELISA analysis of rat pharmacokinetics samples and S. Alley for assistance in pharmacokinetic modeling.

Author information

Authors and Affiliations

  1. Seattle Genetics, Bothell, Washington, USA

    Robert P Lyon, Jocelyn R Setter, Tim D Bovee, Svetlana O Doronina, Joshua H Hunter, Martha E Anderson, Cindy L Balasubramanian, Steven M Duniho, Chris I Leiske, Fu Li & Peter D Senter

Authors
  1. Robert P Lyon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jocelyn R Setter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tim D Bovee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Svetlana O Doronina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joshua H Hunter
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Martha E Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cindy L Balasubramanian
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Steven M Duniho
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Chris I Leiske
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Fu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Peter D Senter
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.P.L. and P.D.S. conceived the project rationale and wrote the manuscript; R.P.L. performed hydrolysis rate studies with aminoethyl maleimide; J.R.S. performed hydrolysis rate and stability studies with ADCs; T.D.B. and S.O.D. synthesized the drug-linkers; J.H.H. performed radiolabeling and quantitation for pharmacokinetic studies; M.E.A. performed the xenograft studies and rat pharmacokinetic studies; C.L.B. and S.M.D. performed the rat toxicology studies; C.I.L. prepared ADCs for all in vivo studies; F.L. discovered differential antibody pharmacokinetics in SCID and NSG mice.

Corresponding author

Correspondence to Robert P Lyon.

Ethics declarations

Competing interests

All authors are employees of Seattle Genetics, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 469 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lyon, R., Setter, J., Bovee, T. et al. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nat Biotechnol 32, 1059–1062 (2014). https://doi.org/10.1038/nbt.2968

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.2968

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