Abstract
As a critical feature of the next generation of antibody–drug conjugates (ADCs), site-specific bioconjugation approaches can help to optimize stability, pharmacokinetics, efficacy, and safety as well as improve manufacturing consistency. The SMARTag® technology platform offers a practical and efficient chemoenzymatic solution for site-specific protein modifications. A bioorthogonal aldehyde handle is introduced through the oxidation of a cysteine residue, embedded in a specific peptide sequence (CxPxR), to the aldehyde-bearing formylglycine (fGly). This enzymatic modification is carried out by the formylglycine-generating enzyme (FGE). The broad recognition of this short sequence by FGE within the context of heterologous proteins allows for the introduction of fGly residues at chosen sites in proteins expressed in prokaryotic and eukaryotic systems. The protocol presented here describes the methods for expressing fGly-containing antibodies in eukaryotic cells and subsequent site-specific conjugation with a payload-linker using aldehyde-specific Hydrazino-Iso-Pictet–Spengler (HIPS) chemistry.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wu AM, Senter PD (2005) Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 23(9):1137–1146
Beck A, Goetsch L, Dumontet C et al (2017) Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov 16(5):315–337
Wang L, Amphlett G, Blättler WA et al (2005) Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry. Protein Sci 14(9):2436–2446
Kim MT, Chen Y, Marhoul J et al (2014) Statistical modeling of the drug load distribution on trastuzumab emtansine (Kadcyla), a lysine-linked antibody drug conjugate. Bioconjug Chem 25(7):1223–1232
Bross PF, Beitz J, Chen G et al (2001) Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res 7(6):1490–1496
Hamblett KJ, Senter PD, Chace DF et al (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin Cancer Res 10(20):7063–7070
Wakankar A, Chen Y, Gokarn Y et al (2014) Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs 3(2):161–172
Junutula JR, Raab H, Clark S et al (2008) Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol 26(8):925–932
Axup JY, Bajjuri KM, Ritland M et al (2012) Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc Natl Acad Sci U S A 109(40):16101–16106
Zimmerman ES, Heibeck TH, Gill A et al (2014) Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem 25(2):351–361
VanBrunt MP, Shanebeck K, Caldwell Z et al (2015) Genetically encoded azide containing amino acid in mammalian cells enables site-specific antibody-drug conjugates using click cycloaddition chemistry. Bioconjug Chem 26(11):2249–2260
Hofer T, Skeffington LR, Chapman CM et al (2009) Molecularly defined antibody conjugation through a selenocysteine interface. Biochemistry 48(50):12047–12057
Li X, Fang T, Boons G-J (2014) The preparation of well-defined antibody–drug conjugates through glycan remodeling and strain promoted azide-alkyne cycloadditions. Angew Chem Int Ed Engl 53(28):7179–7182
Okeley NM, Toki BE, Zhang X et al (2013) Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation. Bioconjug Chem 24(10):1650–1655
Zhu Z, Ramakrishnan B, Li J et al (2014) Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar. MAbs 6(5):1190–1200
Tang F, Wang LX, Huang W (2017) Chemoenzymatic synthesis of glycoengineered IgG antibodies and glycosite-specific antibody-drug conjugates. Nat Protoc 12(8):1702–1721
Zhou Q, Stefano JE, Manning C et al (2014) Site-specific antibody-drug conjugation through glycoengineering. Bioconjug Chem 25(3):510–520
Strop P, Liu SH, Dorywalska M et al (2013) Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol 20(2):161–167
Dennler P, Chiotellis A, Fischer E et al (2014) Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjug Chem 25(3):569–578
Stefan N, Gébleux R, Waldmeier L et al (2017) Highly potent, anthracycline-based antibody-drug conjugates generated by enzymatic, site-specific conjugation. Mol Cancer Ther 16(5):879–892
York D, Baker J, Holder PG et al (2016) Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II). BMC Biotechnol 16:23. https://doi.org/10.1186/s12896-016-0254-0
Rabuka D, Rush JS, de Hart GW et al (2012) Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Nat Protoc 7(6):1052–1067
Kalia J, Raines RT (2008) Hydrolytic stability of hydrazones and oximes. Angew Chem Int Ed Engl 47(39):7523–7526
Agarwal P, Kudirka R, Albers AE et al (2013) Hydrazino-Pictet-Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjug Chem 24(6):846–851
Drake PM, Albers AE, Baker J et al (2014) 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. Bioconjug Chem 25(7):1331–1341
Drake PM, Carlson A, McFarland JM et al (2018) CAT-02-106, a site-specifically conjugated anti-CD22 antibody bearing an MDR1-resistant maytansine payload yields excellent efficacy and safety in preclinical models. Mol Cancer Ther 17(1):161–168
Widdison WC, Wilhelm SD, Cavanagh EE et al (2006) Semisynthetic maytansine analogues for the targeted treatment of cancer. J Med Chem 49(14):4392–4408
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Liu, J., Barfield, R.M., Rabuka, D. (2019). Site-Specific Bioconjugation Using SMARTag® Technology: A Practical and Effective Chemoenzymatic Approach to Generate Antibody–Drug Conjugates. 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_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9654-4_10
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