Journal
NATURE PROTOCOLS
Volume 14, Issue 1, Pages 86-99Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-018-0083-9
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Funding
- FAPESP [BEPE 2015/07509-1, 2017/13168-8, 2013/25504-1]
- Xunta de Galicia
- FCT Portugal (FCT Investigator) [IF/00624/2015]
- EU (Marie Sklodowska-Curie ITN Protein Conjugates) [GA 675007]
- Ministerio de Economia y Competitividad [CTQ2015-67727-R, UNLR13-4E-1931, CTQ2015-70524-R, RYC-2013-14706]
- Universidad de La Rioja
- Royal Society University Research Fellow [UF110046, URF/R/180019]
- European Research Council Starting Grant (TagIt) [GA 676832]
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There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.
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