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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents

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Author(s):
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]
Total Authors: 10
Affiliation:
[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
Total Affiliations: 5
Document type: Journal article
Source: Nature Protocols; v. 14, n. 1, p. 86-99, JAN 2019.
Web of Science Citations: 2
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. (AU)

FAPESP's process: 13/25504-1 - Development of new methodologies for the asymmetric a-functionalization of carbonyl compounds using chiral catalysts
Grantee:Antonio Carlos Bender Burtoloso
Support type: Regular Research Grants
FAPESP's process: 15/07509-1 - Site-specific modification of proteins via aqueous Horner-Wadsworth-Emmons reaction followed by Wolff rearrangement
Grantee:Barbara Bernardim de Souza
Support type: Scholarships abroad - Research Internship - Doctorate
FAPESP's process: 17/13168-8 - Construction of chemically defined antibody-drug conjugates using carbonylacrylic reagents
Grantee:Barbara Bernardim de Souza
Support type: Scholarships abroad - Research Internship - Post-doctor