Antibody-drug conjugates (ADCs) are promising agents for the selective delivery of cytotoxic drugs to specific cells (for example, tumors). In this protocol, we describe two strategies for the precise modification at engineered C- or N-terminal cysteines of antibodies in IgG, diabody and small immunoprotein (SIP) formats that yield homogenous ADCs. In this protocol, cemadotin derivatives are used as model drugs, as these agents have a potent cytotoxic activity and are easy to synthesize. However, other drugs with similar functional groups could be considered. In the first approach, a cemadotin derivative containing a sulfhydryl group results in a mixed disulfide linkage. In the second approach, a cemadotin derivative containing an aldehyde group is joined via a thiazolidine linkage. The procedures outlined are robust, enabling the preparation of ADCs with a defined number of drugs per antibody in a time frame between 7 and 24 h.
Subscribe to Journal
Get full journal access for 1 year
only $41.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Aggarwal, S. What's fueling the biotech engine 2009–2010. Nat. Biotechnol. 28, 1165–1171 (2010).
Adamo, R. et al. Synthetically defined glycoprotein vaccines: current status and future directions. Chem. Sci. 4, 2995–3008 (2013).
Webb, S. Pharma interest surges in antibody drug conjugates. Nat. Biotechnol. 29, 297–298 (2011).
Webb, S. Back on target. Nat. Biotechnol. 31, 191–193 (2013).
Younes, A. et al. Brentuximab vedotin (SGN-35) for relapsed CE30-positive lymphomas. N. Engl. J. Med. 363, 1812–1821 (2010).
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).
Burris, H.A. et al. Phase II study of the antibody drug conjugate trastuzumab-dm1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J. Clin. Oncol. 29, 398–405 (2011).
Senter, P.D. Potent antibody drug conjugates for cancer therapy. Curr. Opin. Chem. Biol. 13, 235–244 (2009).
Chari, R.V.J. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc. Chem. Res. 41, 98–107 (2008).
Casi, G. & Neri, D. Antibody-drug conjugates: basic concepts, examples and future perspectives. J. Control. Release 161, 422–428 (2012).
Chalker, J.M., Bernardes, G.J.L. & Davis, B.G. A, tag-and-modify, approach to site-selective protein modification. Acc. Chem. Res. 44, 730–741 (2011).
Stephanopoulos, N. & Francis, M.B. Choosing an effective protein bioconjugation strategy. Nat. Chem. Biol. 7, 876–884 (2011).
Sletten, E.M. & Bertozzi, C.R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).
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).
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).
Axup, J.Y. et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101–16106 (2012).
Rabuka, D., Rush, J.S., deHart, G.W., Wu, P. & Bertozzi, C.R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat. Protoc. 7, 1052–1067 (2012).
Carrico, I.S., Carlson, B.L. & Bertozzi, C.R. Introducing genetically encoded aldehydes into proteins. Nat. Chem. Biol. 3, 321–322 (2007).
Katz, J., Janik, J.E. & Younes, A. Brentuximab vedotin (SGN-35). Clin. Cancer Res. 17, 6428–6436 (2011).
Voynov, V. et al. Design and application of antibody cysteine variants. Bioconjug. Chem. 21, 385–392 (2010).
Wang, L., Amphlett, G., Blättler, W.A., Lambert, J.M. & Zhang, W. Structural characterization of the maytansinoid–monoclonal antibody immunoconjugate, huN901–DM1, by mass spectrometry. Protein Sci. 14, 2436–2446 (2005).
Bernardes, G.J.L. et al. A traceless vascular-targeting antibody–drug conjugate for cancer therapy. Angew. Chem. Int. Ed. 51, 941–944 (2012).
Casi, G., Huguenin-Dezot, N., Zuberbühler, K., Scheuermann, Jr. & Neri, D. Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery. J. Am. Chem. Soc. 134, 5887–5892 (2012).
Steiner, M. et al. Spacer length shapes drug release and therapeutic efficacy of traceless disulfide-linked ADCs targeting the tumor neovasculature. Chem. Sci. 4, 297–302 (2013).
Zuberbühler, K., Casi, G., Bernardes, G.J.L. & Neri, D. Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in igg format. Chem. Commun. 48, 7100–7102 (2012).
Gerber, H.-P., Senter, P.D. & Grewal, I.S. Antibody drug-conjugates targeting the tumor vasculature: current and future developments. MAbs 1, 247–253 (2009).
Sharkey, R.M., Karacay, H., Govindan, S.V. & Goldenberg, D.M. Combination radioimmunotherapy and chemoimmunotherapy involving different or the same targets improves therapy of human pancreatic carcinoma xenograft models. Mol. Cancer Ther. 10, 1072–1081 (2011).
Neri, D. & Bicknell, R. Tumour vascular targeting. Nat. Rev. Cancer 5, 436–446 (2005).
Villa, A. et al. A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo. Int. J. Cancer 122, 2405–2413 (2008).
Tarli, L. et al. A high-affinity human antibody that targets tumoral blood vessels. Blood 94, 192–198 (1999).
Holliger, P. & Hudson, P.J. Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–1136 (2005).
Borsi, L. et al. Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. Int. J. Cancer 102, 75–85 (2002).
Kim, K.M. et al. Anti-CD30 diabody-drug conjugates with potent antitumor activity. Mol. Cancer Ther. 7, 2486–2497 (2008).
Rybak, J.-N., Roesli, C., Kaspar, M., Villa, A. & Neri, D. The extra-domain a of fibronectin is a vascular marker of solid tumors and metastases. Cancer Res. 67, 10948–10957 (2007).
Pettit, G.R. et al. The isolation and structure of a remarkable marine animal antineoplastic constituent: dolastatin 10. J. Am. Chem. Soc. 109, 6883–6885 (1987).
Pettit, G.R. et al. Isolation and structure of the cytostatic linear depsipeptide dolastatin 15. J. Org. Chem. 54, 6005–6006 (1989).
Simmons, T.L., Andrianasolo, E., McPhail, K., Flatt, P. & Gerwick, W.H. Marine natural products as anticancer drugs. Mol. Cancer Ther. 4, 333–342 (2005).
Sun, X. et al. Design of antibodymaytansinoid conjugates allows for efficient detoxification via liver metabolism. Bioconjug. Chem. 22, 728–735 (2011).
Ellman, G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77 (1959).
G.J.L.B. thanks the European Molecular Biology Organization (EMBO) and the Novartis Foundation for generous funding. We thank N. Huguenin-Dezot and S. Trüssel for their contributions during these projects. We also thank S. Wulhfard, A. Villa and K. Schwager for their help in protein engineering and expression. Financial contributions from ETH Zürich, the Swiss National Science Foundation, SwissBridge/Stammbach Stiftung, Kommission für Technologie und Innovation (KTI), Philochem AG and Philogen SpA are gratefully acknowledged.
G.C. is employed at Philochem AG. D.N. is a co-founder and shareholder of Philogen SpA.
Integrated supplementary information
The total ion chromatogram, combined ion series and deconvoluted spectra are shown for the starting material and product of the reaction of SIP(F8)-Ellman's with CemCH2-SH. Identical analyses were carried out for all the conjugation reactions performed in this work.
a) ESI-MS spectrum; b) SDS-PAGE; M, molecular marker; 1, SIP(F8) non-reducing conditions; 2, SIP(F8) reducing conditions; 3, purified SIP(F8)-SS-Ellman's; 4, purified SIP(F8)-SS-CH2Cem c) size-exclusion chromatography; arrows indicate standard proteins (11 mL: ferritin 440 kDa; 14.1 mL: BSA 67 kDa; 15.4 mL: lactoglobulin 35 kDa); and d) Biacore analysis towards recombinant 11A12 fibronectin. Data previously described in [Bernardes, G.J.L. et al., A traceless vascular-targeting antibody–drug conjugate for cancer therapy. Angew. Chem. Int. Ed. 51, 941-944 (2012)]. A single peak after deconvolution of the multiply-charged ions spectrum should be detected by LCMS analysis of SIP(F8)-SS-CH2Cem at 39415 Da; SDS-PAGE analysis should give a single major band at ~ 39 kDa; in some cases, SDS-PAGE shows a very minor band at ∼ 75 kDa (see TROUBLESHOOTING); a peak eluting at a retention volume of 15.3 mL corresponding to the noncovalent homodimeric form of SIP(F8)-SS-CH2Cem should be detected by gel filtration; and finally, SIP(F8)-SS-CH2Cem should remain immunoreactive towards recombinant 11A12 fibronectin as observed by direct comparison of the sensorgram profiles of modified and nonmodified antibody fragments.
About this article
Cite this article
Bernardes, G., Steiner, M., Hartmann, I. et al. Site-specific chemical modification of antibody fragments using traceless cleavable linkers. Nat Protoc 8, 2079–2089 (2013). https://doi.org/10.1038/nprot.2013.121
European Journal of Organic Chemistry (2019)
Chemical Communications (2019)
Journal of Molecular Liquids (2019)
Biotechnology Progress (2019)
High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains
Biotechnology Journal (2019)