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Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

Nature protocols (2018-11-25)
Barbara Bernardim, Maria J Matos, Xhenti Ferhati, Ismael Compañón, Ana Guerreiro, Padma Akkapeddi, Antonio C B Burtoloso, Gonzalo Jiménez-Osés, Francisco Corzana, Gonçalo J L Bernardes
RÉSUMÉ

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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Millipore
Filtre membrane en PTFE, dimension de pores de 0,2 µm, Omnipore, filter diam. 90 mm, hydrophilic