Efficient and irreversible antibody–cysteine bioconjugation using carbonylacrylic reagents

Abstract

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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Fig. 1: A general approach for cysteine selective and irreversible bioconjugation of antibodies.
Fig. 2: Antibody–cysteine bioconjugation with carbonylacrylic reagent 1.
Fig. 3: Synthesis of carbonylacrylic reagents bearing the desired payload for site-selective antibody–cysteine bioconjugation.
Fig. 4: Synthesis of a functional ADC through site-selective conjugation with caa-ValCit-PAB-MMAE 2.
Fig. 5: Construction of homogeneous ADCs through cysteine-selective conjugation with caa-ValCit-PAB-MMAE (2) and caa-crizotinib (3).

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Acknowledgements

We thank FAPESP (BEPE 2015/07509-1 and 2017/13168-8 to B.B., and 2013/25504-1 to A.C.B.B.), Xunta de Galicia (M.J.M.), FCT Portugal (FCT Investigator to G.J.L.B., IF/00624/2015), the EU (Marie Sklodowska-Curie ITN Protein Conjugates, GA 675007, including a PhD Studentship to X.F.), the Ministerio de Economía y Competitividad (projects CTQ2015-67727-R and UNLR13-4E-1931 to F.C. and CTQ2015-70524-R and RYC-2013-14706 to G.J.O.) and the Universidad de La Rioja (FPI Studentship to I.C.). We also thank S. Massa and N. Devoogdt (Vrije Universiteit Brussel (VUM), Brussels) for the generous gift of the Her2-targeting nanobody 2Rb17c, Genentech for providing Thiomab LC-V205C and trastuzumab antibodies, and D. Neri’s laboratory (Swiss Federal Institute of Technology (ETH Zürich), Zurich) for the generous gift of the F16 antibody. G.J.L.B. is a Royal Society University Research Fellow (UF110046 and URF/R/180019) and the recipient of a European Research Council Starting Grant (TagIt, GA 676832).

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Contributions

G.J.L.B. designed and supervised the research. B.B. and M.J.M. performed reactions on antibodies and MS analysis. F.C., X.F. and I.C. designed and performed the synthesis of caa-ValCit-PAB-MMAE and caa-crizotinib. A.G. and P.A. studied antibody binding and performed flow cytometry analysis. A.C.B.B. and G.J.-O. contributed to the design of the carbonylacrylic reagent. B.B. and G.J.L.B. wrote the paper with contributions from all authors.

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Correspondence to Gonçalo J. L. Bernardes.

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Key references using this protocol

Stenton, B. J., Oliveira, B. L., Matos, M. J., Sinatra, L. & Bernardes, G. J. L. Chem. Sci. 9, 4185–4189 (2018): http://pubs.rsc.org/en/Content/ArticleLanding/2018/SC/C8SC00256H#!divAbstract

Bernardim, B. et al. Nat. Commun. 7, 13128 (2016): https://www.nature.com/articles/ncomms13128

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Supplementary Figures 1–25 and Supplementary Methods

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Bernardim, B., Matos, M.J., Ferhati, X. et al. Efficient and irreversible antibody–cysteine bioconjugation using carbonylacrylic reagents. Nat Protoc 14, 86–99 (2019). https://doi.org/10.1038/s41596-018-0083-9

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