Antibody–drug conjugates (ADCs) comprise antibodies covalently attached to highly potent drugs using a variety of conjugation technologies. As therapeutics, they combine the exquisite specificity of antibodies, enabling discrimination between healthy and diseased tissue, with the cell-killing ability of cytotoxic drugs. This powerful and exciting class of targeted therapy has shown considerable promise in the treatment of various cancers with two US Food and Drug Administration approved ADCs currently on the market (Adcetris and Kadcyla) and approximately 40 currently undergoing clinical evaluation. However, most of these ADCs exist as heterogeneous mixtures, which can result in a narrow therapeutic window and have major pharmacokinetic implications. In order for ADCs to deliver their full potential, sophisticated site-specific conjugation technologies to connect the drug to the antibody are vital. This Perspective discusses the strategies currently used for the site-specific construction of ADCs and appraises their merits and disadvantages.
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Ehrlich, P. Address in pathology, ON CHEMIOTHERAPY: Delivered before the seventeenth international congress of medicine. Brit. Med. J. 2, 353–359 (1913).
Mathé, G., Loc, T. B. & Bernard, J. Effet sur la leucémie 1210 de la souris d'une combinaison par diazotation d'A-méthoptérine et de γ-globulines de hamsters porteurs de cette leucémie par hétérogreffe. C.R. Hebd. Séances Acad. Sci. 246, 1626–1628 (1958).
Ghose, T. & Nigam, S. P. Antibody as carrier of chlorambucil. Cancer 29, 1398–1400 (1972).
Rowland, G. F., O'Neill, G. J. & Davies, D. A. L. Suppression of tumour growth in mice by a drug–antibody conjugate using a novel approach to linkage. Nature 255, 487–488 (1975).
Ford, C. H. J. et al. Localisation and toxicity study of a vindesine-anti-CEA conjugate in patients with advanced cancer. Brit. J. Cancer 47, 35–42 (1983).
Trail, P. A. et al. Cure of xenografted human carcinomas by Br96–doxorubicin immunoconjugates. Science 261, 212–215 (1993).
Pietersz, G. A. & Krauer, K. Antibody-targeted drugs for the therapy of cancer. J. Drug Target. 2, 183–215 (1994).
Linenberger, M. L. et al. Multidrug-resistance phenotype and clinical responses to gemtuzumab ozogamicin. Blood 98, 988–994 (2001).
Younes, A. et al. Brentuximab vedotin (SGN-35) for relapsed CD30-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. Nature Biotechnol. 30, 631–637 (2012).
Verma, S. et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N. Engl. J. Med. 367, 1783–1791 (2012).
LoRusso, P. M., Weiss, D., Guardino, E., Girish, S. & Sliwkowski, M. X. Trastuzumab emtansine: a unique antibody-drug conjugate in development for human epidermal growth factor receptor 2-positive cancer. Clin. Cancer Res. 17, 6437–6447 (2011).
Mullard, A. Maturing antibody–drug conjugate pipeline hits 30. Nature Rev. Drug Discov. 12, 329–332 (2013).
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).
Stan, A. C., Radu, D. L., Casares, S., Bona, C. A. & Brumeanu, T. D. Antineoplastic efficacy of doxorubicin enzymatically assembled on galactose residues of a monoclonal antibody specific for the carcinoembryonic antigen. Cancer Res. 59, 115–121 (1999).
Strop, P. et al. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem. Biol. 20, 161–167 (2013).
Lyons, A. et al. Site-specific attachment to recombinant antibodies via introduced surface cysteine residues. Protein Eng. 3, 703–708 (1990).
Junutula, J. R. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nature Biotechnol. 26, 925–932 (2008).
Sunbul, M. & Yin, J. Site specific protein labeling by enzymatic posttranslational modification. Org. Biomol. Chem. 7, 3361–3371 (2009).
Young, T. S., Ahmad, I., Yin, J. A. & Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395, 361–374 (2010).
Rabuka, D., Rush, J. S., deHart, G. W., Wu, P. & Bertozzi, C. R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nature Protoc. 7, 1052–1067 (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).
Junutula, J. R. et al. Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J. Immunol. Methods 332, 41–52 (2008).
Woo, H. J., Lotz, M. M., Jung, J. U. & Mercurio, A. M. Carbohydrate-binding protein 35 (Mac-2), a laminin-binding lectin, forms functional dimers using cysteine 186. J. Biol. Chem. 266, 18419–18422 (1991).
Wootton, S. K. & Yoo, D. Homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages. J. Virol. 77, 4546–4557 (2003).
Greenberg, C. S., Birckbichler, P. J. & Rice, R. H. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J. 5, 3071–3077 (1991).
Kanaji, T. et al. Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112. J. Biol. Chem. 268, 11565–11572 (1993).
Kashiwagi, T. et al. Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J. Biol. Chem. 277, 44252–44260 (2002).
Jeger, S. et al. Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew. Chem. Int. Ed. 49, 9995–9997 (2010).
Wu, P. et al. Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag. Proc. Natl Acad. Sci. USA 106, 3000–3005 (2009).
Drake, P. M. et al. Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes. Bioconjugate Chem. 25, 1331–1341 (2014).
Zimmerman, E. S. et al. Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjugate Chem. 25, 351–361 (2014).
Sapra, P. et al. Long-term tumor regression induced by an antibody-drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells. Mol. Cancer Ther. 12, 38–47 (2013).
Tian, F. et al. A general approach to site-specific antibody drug conjugates. Proc. Natl Acad. Sci. USA 111, 1766–1771 (2014).
Sun, M. M. et al. Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides. Bioconjugate Chem. 16, 1282–1290 (2005).
Doronina, S. O. et al. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nature Biotechnol. 21, 778–784 (2003).
Beckley, N. S., Lazzareschi, K. P., Chih, H.-W., Sharma, V. K. & Flores, H. L. Investigation into temperature-induced aggregation of an antibody drug conjugate. Bioconjugate Chem. 24, 1674–1683 (2013).
Shen, B. Q. et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nature Biotechnol. 30, 184–189 (2012).
Lyon, R. P. et al. Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nature Biotechnol. 32, 1059–1062 (2014).
Badescu, G. et al. Bridging disulfides for stable and defined antibody drug conjugates. Bioconjugate Chem. 25, 1124–1136 (2014).
Nunes, J. P. M. et al. Functional native disulfide bridging enables delivery of a potent, stable and targeted antibody-drug conjugate (ADC). Chem. Commun. 51, 10624–10627 (2015).
Maruani, A. et al. A plug-and-play approach to antibody-based therapeutics via a chemoselective dual click strategy. Nature Commun. 6, 6645 (2015).
Maruani, A. et al. Site-selective multi-porphyrin attachment enables the formation of a next-generation antibody-based photodynamic therapeutic. Chem. Commun. 51, 15304–15307 (2015).
Lee, M. T. W., Maruani, A., Baker, J., Caddick, S. & Chudasama, V. Next-generation disulfide stapling: Reduction and functional re-bridging all in one. Chem. Sci. 7, 799–802 (2016).
Hinman, L. M. et al. Preparation and characterization of monoclonal antibody conjugates of the calicheamicins: a novel and potent family of antitumor antibiotics. Cancer Res. 53, 3336–3342 (1993).
Hamann, P. R. et al. An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors. Choice of linker and overcoming drug resistance. Bioconjugate Chem. 16, 346–353 (2005).
Wang, W. et al. Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies. Mol. Immunol. 48, 860–866 (2011).
Zhou, Q. et al. Site-specific antibody-drug conjugation through glycoengineering. Bioconjugate Chem. 25, 510–520 (2014).
Li, X., Fang, T. & Boons, G. J. Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions. Angew. Chem. Int. Ed. 53, 7179–7182 (2014).
Jefferis, R. Glycosylation of recombinant antibody therapeutics. Biotechnol. Prog. 21, 11–16 (2005).
We are grateful to UCL, UCLB, NIHR BRC, MRC, BBSRC and EPSRC for support of our work in this area.
V.C. and S.C. are co-founders and directors of the company ThioLogics.
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Chudasama, V., Maruani, A. & Caddick, S. Recent advances in the construction of antibody–drug conjugates. Nature Chem 8, 114–119 (2016). https://doi.org/10.1038/nchem.2415
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