Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents

• Tipo: Artigo em Revista Científica Internacional
• Data: 2019

Autores

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

Revista

Título: Nature Protocols

Vol. 14

Páginas: 86-99

ISSN: 1754-2189

Editora: Springer Nature

Indexação

ISI Web of Knowledge - 12 Citações

Scopus - 13 Citações

Outras Informações

ID Authenticus: 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.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 14