Synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates Journal Article


Authors: Davydova, M.; Dewaele Le Roi, G.; Adumeau, P.; Zeglis, B. M.
Article Title: Synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates
Abstract: Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent - dubbed 'PODS' - as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.
Journal Title: Journal of Visualized Experiments
Volume: 2019
Issue: 145
ISSN: 1940-087X
Publisher: MYJoVE Corporation  
Date Published: 2019-03-01
Start Page: e59063
Language: English
DOI: 10.3791/59063
PUBMED: 30907883
PROVIDER: scopus
PMCID: PMC7169956
DOI/URL:
Notes: Source: Scopus
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  1. Brian Zeglis
    106 Zeglis