Transglutaminase-catalyzed covalent multimerization of Camelidae anti-human TNF single domain antibodies improves neutralizing activity

Plagmann, I.; Chalaris, A.; Kruglov, A.; Nedospasov, S.; Rosenstiel, P.; Rose-John, S.; Scheller, J.

Авторы: Plagmann I., Chalaris A., Kruglov AA, Nedospasov S., Rosenstiel P., Rose-John S., Scheller J.
Журнал: Journal of Biotechnology
Том: 142
Номер: 2
Год издания: 2009
Издательство: Elsevier BV
Местоположение издательства: Netherlands
Первая страница: 170
Последняя страница: 178
DOI: 10.1016/j.jbiotec.2009.04.002
Аннотация: Tumor necrosis factor (TNF) plays an important role in chronic inflammatory disorders, such as Rheumatoid Arthritis and Crohn’s disease. Recently, monoclonal Camelidae variable heavy-chain domain-only antibodies (VHH) were developed to antagonize the action of human TNF (hTNF). Here, we show that hTNF–VH H does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF–VHH molecules in vitro catalyzed by microbial transglutaminases (MTG). The ribonuclease S-tag-peptide was shown to act as a peptidyl substrate in covalent protein cross-linking reactions catalyzed by MTG from Streptomyces mobaraensis. The S-tag sequence was C-terminally fused to the hTNF–VHH and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF–VHH-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF–VH H was not susceptible to protein crosslinking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF–VHH proteins were much more active than the monomeric hTNF–VHH. We hypoth- esize that improved inhibition by dimeric and multimeric single chain hTNF–VHH proteins is caused by avidity effects.
Добавил в систему: Недоспасов Сергей Артурович

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Transglutaminase-catalyzed covalent multimerization of Camelidae anti-human TNF single domain antibodies improves neutralizing activityстатья

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