| Full text | |
| 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: | 15/07509-1 - Site-specific modification of proteins via aqueous Horner-Wadsworth-Emmons reaction followed by Wolff rearrangement |
| Grantee: | Barbara Bernardim de Souza |
| Support Opportunities: | Scholarships abroad - Research Internship - Doctorate |
| 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 Opportunities: | Regular Research Grants |
| FAPESP's process: | 17/13168-8 - Construction of chemically defined antibody-drug conjugates using carbonylacrylic reagents |
| Grantee: | Barbara Bernardim de Souza |
| Support Opportunities: | Scholarships abroad - Research Internship - Post-doctor |