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Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates


The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.

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Figure 1: The site of conjugation modulates the therapeutic activity of antibody conjugates.
Figure 2: The plasma stability of antibody conjugates is dependent on the conjugation sites but independent of linker, drug and antibody.
Figure 3: Proposed mechanism for the influence of the conjugation site on linker stability and therapeutic activity of antibody conjugates.
Figure 4: The conjugation site influences antibody-conjugate stability in vivo.


  1. Carter, P.J. & Senter, P.D. Antibody-drug conjugates for cancer therapy. Cancer J. 14, 154–169 (2008).

    CAS  Article  Google Scholar 

  2. Polakis, P. Arming antibodies for cancer therapy. Curr. Opin. Pharmacol. 5, 382–387 (2005).

    CAS  Article  Google Scholar 

  3. Kreitman, R.J. & Pastan, I. Immunotoxins for targeted cancer therapy. Adv. Drug Deliv. Rev. 31, 53–88 (1998).

    CAS  Article  Google Scholar 

  4. Marik, J. & Junutula, J.R. Emerging role of immunoPET in receptor targeted cancer therapy. Curr. Drug Deliv. 8, 70–78 (2011).

    CAS  Article  Google Scholar 

  5. Ryan, S.M., Mantovani, G., Wang, X., Haddleton, D.M. & Brayden, D.J. Advances in PEGylation of important biotech molecules: delivery aspects. Expert Opin. Drug Deliv. 5, 371–383 (2008).

    CAS  Article  Google Scholar 

  6. McCarron, P.A. et al. Antibody conjugates and therapeutic strategies. Mol. Interv. 5, 368–380 (2005).

    CAS  Article  Google Scholar 

  7. Medintz, I.L., Uyeda, H.T., Goldman, E.R. & Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435–446 (2005).

    CAS  Article  Google Scholar 

  8. 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).

    CAS  Article  Google Scholar 

  9. Polson, A.G. et al. Antibody-drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection. Cancer Res. 69, 2358–2364 (2009).

    CAS  Article  Google Scholar 

  10. Chapman, A.P. et al. Therapeutic antibody fragments with prolonged in vivo half-lives. Nat. Biotechnol. 17, 780–783 (1999).

    CAS  Article  Google Scholar 

  11. Junutula, J.R. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925–932 (2008).

    CAS  Article  Google Scholar 

  12. Lewis Phillips, G.D. et al. Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 68, 9280–9290 (2008).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  14. Chari, R.V.J. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc. Chem. Res. 41, 98–107 (2008).

    CAS  Article  Google Scholar 

  15. Wu, A.M. & Senter, P.D. Arming antibodies: prospects and challenges for immunoconjugates. Nat. Biotechnol. 23, 1137–1146 (2005).

    CAS  Article  Google Scholar 

  16. Junutula, J.R. et al. 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, 4769–4778 (2010).

    CAS  Article  Google Scholar 

  17. Xu, K. et al. Characterization of intact antibody-drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography-mass spectrometry. Anal. Biochem. 412, 56–66 (2011).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  19. Lin, D., Saleh, S. & Liebler, D.C. Reversibility of covalent electrophile-protein adducts and chemical toxicity. Chem. Res. Toxicol. 21, 2361–2369 (2008).

    CAS  Article  Google Scholar 

  20. Knight, P. Hydrolysis of p-NN′-phenylenebismaleimide and its adducts with cysteine. Implications for cross-linking of proteins. Biochem. J. 179, 191–197 (1979).

    CAS  Article  Google Scholar 

  21. Khan, M.N. Kinetics and mechanism of the alkaline hydrolysis of maleimide. J. Pharm. Sci. 73, 1767–1771 (1984).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  23. Austin, C.D. et al. Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol. Biol. Cell 15, 5268–5282 (2004).

    CAS  Article  Google Scholar 

  24. Chen, Y. et al. Armed antibodies targeting the mucin repeats of the ovarian cancer antigen, MUC16, are highly efficacious in animal tumor models. Cancer Res. 67, 4924–4932 (2007).

    CAS  Article  Google Scholar 

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We thank our Genentech colleagues: J. Speer and E. Wu for reagent generation and inventory; D. Bumbaca for plasma stability study; J. Lau and I. Inigo for ADC efficacy studies; P. Carter and S. Panowski for critical review of the manuscript; H.B. Lowman, S. Kenkare-Mitra and I. Mellman for their insightful discussions. Anti-MMAE mouse monoclonal antibodies were a generous gift from Seattle Genetics.

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Authors and Affiliations



B.-Q.S. and K.X. designed experiments developed the methodology, performed plasma stability studies and wrote the manuscript. L.L. and M.K. performed plasma stability and LC-MS analyses. H.R. conducted ADC conjugations and S.B. generated THIOMAB constructs and performed in vitro potency studies. K.L.P.-R., J.T. and S.-F.Y. performed in vivo efficacy studies. E.M., D.L. and J.T. conducted pharmacokinetic studies. J.B. and O.M.S. quantified free MMAE levels in the plasma. S.J.S. performed internalization studies. P.J.M. and P.E.H. purified antibodies. C.E. performed antibody structural analysis. T.N., W.A.S., R.N.F. and K.M.F. designed and conducted preclinical safety studies. D.P. and S.D.S. provided project management support. L.A.K., A.E., W.L.W., R.V., S.K., M.X.S. and R.H.S. provided direction and guidance. P.P. provided direction, guidance and assisted in writing the manuscript. J.R.J. led the overall conjugation site-dependent antibody conjugate program, generated trastuzumab THIOMAB constructs, designed experiments and wrote the manuscript.

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Correspondence to Jagath R Junutula.

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All authors were employees of Genentech/Roche at the time this work was conducted.

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Shen, BQ., Xu, K., Liu, L. et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat Biotechnol 30, 184–189 (2012).

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