Development of Bispecific Molecules for the In Situ Detection of Protein-Protein Interactions and Protein Phosphorylation

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• 作者：Jan van Dieck¹ ; ⁴ ; Volker Schmid¹ ; ⁴ ; Dieter Heindl² ; Sebastian Dziadek² ; Michael Schraeml² ; Michael Gerg² ; Petra Massoner² ; Alfred M. Engel² ; Georg Tiefenthaler¹ ; Serhat Vural² ; Simon Stritt¹ ; Fabian Tetzlaff¹ ; Monika Soukupova² ; Erhard Kopetzki¹ ; Birgit Bossenmaier¹ ; Marlene Thomas¹ ; Christian Klein³ ; Alfred Mertens¹ ; Astrid Heller¹ ; Michael Tacke² ; ^{michael.tacke@roche.com" class="auth_mail}
• 刊名：Chemistry & Biology
• 出版年：20 March, 2014
• 年：2014
• 卷：21
• 期：3
• 页码：357-368
• 全文大小：2411 K

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Summary

Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation聽status and function of proteins involved in disease progression. However, existing antibody-based methods for phosphorylation detection have been found to lack specificity, and methods developed for examining PPIs in聽vitro cannot be easily adapted for tissues samples. In this study, we eliminated some of these limitations by developing a specific immunohistochemical staining method that uses 鈥渄ual binders鈥?(DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a聽flexible linker, to detect PPIs and protein phosphorylation. We engineered DBs by selecting Fab fragments with fast off-rate kinetics, which allowed us to demonstrate that stable target binding was achieved only upon simultaneous, cooperative binding to both聽epitopes. We show that DBs specifically detect the activated HER2/HER3 complex in formalin-fixed, paraffin-embedded cancer cells and exhibit superior detection specificity for phospho-HER3 compared to the corresponding monoclonal antibody. Overall, the performance of DBs makes them attractive tools for future development for clinical applications.