title: Development of bispecific molecules for the in situ detection of protein-protein interactions and protein phosphorylation.
abstract: Investigation of protein-protein interactions (PPIs) and protein phosphorylation in clinical tissue samples can offer valuable information about the activation 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 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 "dual binders" (DBs), which are bispecific detection agents consisting of two Fab fragment molecules joined by a 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 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.
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Jan
authors: J
authors: van Dieck
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Volker
authors: V
authors: Schmid
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Dieter
authors: D
authors: Heindl
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Sebastian
authors: S
authors: Dziadek
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Michael
authors: M
authors: Schraeml
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Michael
authors: M
authors: Gerg
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Petra
authors: P
authors: Massoner
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Alfred M
authors: AM
authors: Engel
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Georg
authors: G
authors: Tiefenthaler
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Serhat
authors: S
authors: Vural
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Simon
authors: S
authors: Stritt
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Fabian
authors: F
authors: Tetzlaff
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Monika
authors: M
authors: Soukupova
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Erhard
authors: E
authors: Kopetzki
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Birgit
authors: B
authors: Bossenmaier
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Marlene
authors: M
authors: Thomas
authors: Pharma Research and Early Development, Roche Glycart AG, 8952 Schlieren, Switzerland.
authors: Christian
authors: C
authors: Klein
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Alfred
authors: A
authors: Mertens
authors: Pharma Research and Early Development, Roche Diagnostics GmbH, 82377 Penzberg, Germany.
authors: Astrid
authors: A
authors: Heller
authors: Roche Professional Diagnostics, Roche Diagnostics GmbH, 82377 Penzberg, Germany. Electronic address: michael.tacke@roche.com.
authors: Michael
authors: M
authors: Tacke
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D045744:Cell Line, Tumor; D019281:Dimerization; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D061986:MCF-7 Cells; D051379:Mice; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D009841:Oligonucleotides; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D011994:Recombinant Proteins; C581292:ERBB3 protein, human