Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents

• Type: Article in International Scientific Journal
• Date: 2019

Authors

Bernardim, B (Author) Other; Matos, MJ (Author) FCUP; Ferhati, X (Author) Other; Companon, I (Author) Other; Guerreiro, A (Author) Other; Akkapeddi, P (Author) Other; Burtoloso, ACB (Author) Other; Jimenez Oses, G (Author) Other; Corzana, F (Author) Other; Bernardes, GJL (Author) Other

Journal

Title: Nature Protocols

Vol. 14

Pages: 86-99

ISSN: 1754-2189

Publisher: Springer Nature

Indexing

ISI Web of Knowledge - 12 Citations

Scopus - 13 Citations

Other information

Authenticus ID: P-00P-YQA

DOI: 10.1038/s41596-018-0083-9

Abstract (EN): 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 14