Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents

Autor(es)

Bernardim, Barbara ^{[1, 2]} ; Matos, Maria J. ^[2] ; Ferhati, Xhenti ^[3] ; Companon, Ismael ^[3] ; Guerreiro, Ana ^[4] ; Akkapeddi, Padma ^[4] ; Burtoloso, Antonio C. B. ^[5] ; Jimenez-Oses, Gonzalo ^[3] ; Corzana, Francisco ^[3] ; Bernardes, Goncalo J. L. ^{[4, 2]}

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Carlos, Dept Quim, Sao Carlos, SP - Brazil

^[2] Univ Cambridge, Dept Chem, Cambridge - England

^[3] Univ La Rioja, Dept Quim, Ctr Invest Sintesis Quim, Logrono - Spain

^[4] Univ Lisbon, Inst Med Mol, Fac Med, Lisbon - Portugal

^[5] Univ Sao Paulo, Inst Quim Sao Carlos, Sao Carlos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Nature Protocols; v. 14, n. 1, p. 86-99, JAN 2019.
• Citações Web of Science: 2

Resumo

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. (AU)

• Processo FAPESP: 15/07509-1 - Modificação específica de proteínas via reação de Horner-Wadsworth-Emmons seguida de rearranjo de Wolff
• Beneficiário: Barbara Bernardim de Souza
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Doutorado
• Processo FAPESP: 13/25504-1 - Desenvolvimento de novas metodologias para a-funcionalização assimétrica de compostos carbonílicos utilizando catalisadores quirais
• Beneficiário: Antonio Carlos Bender Burtoloso
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/13168-8 - Construção de conjugados anticorpo-droga quimicamente definidos utilizando reagentes carbonilacrílicos
• Beneficiário: Barbara Bernardim de Souza
• Modalidade de apoio: Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado