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

Dieck, Jan van; Schmid, Volker; Heindl, Dieter; Dziadek, Sebastian; Schraeml, Michael; Gerg, Michael; Massoner, Petra; Engel, Alfred M.; Tiefenthaler, Georg; Vural, Serhat; Stritt, Simon; Tetzlaff, Fabian; Soukupova, Monika; Kopetzki, Erhard; Bossenmaier, Birgit; Thomas, Marlène; Klein, Christian; Mertens, Alfred; Heller, Astrid; Tacke, Michael

doi:10.1016/j.chembiol.2013.12.018

Cited by 8 publications

(7 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, HER dimerization pattern is context-dependent and correlates with the downstream effective networks. HER dimers have been investigated with co-immunoprecipitation and immunohistochemical methods [ 41 ]. In the present study we employed in situ PLA to identify membrane-bound protein–protein interactions of HER receptors at various conditions [ 42 ].…”

Section: Discussionmentioning

confidence: 99%

HER-3 targeting alters the dimerization pattern of ErbB protein family members in breast carcinomas

et al. 2015

View full text Add to dashboard Cite

Breast carcinogenesis is a multi-step process in which membrane receptor tyrosine kinases are crucial participants. Lots of research has been done on epidermal growth factor receptor (EGFR) and HER-2 with important clinical results. However, breast cancer patients present intrinsic or acquired resistance to available HER-2-directed therapies, mainly due to HER-3. Using new techniques, such as proximity ligation assay, herein we evaluate the dimerization pattern of HER-3 and the importance of context-dependent dimer formation between HER-3 and other HER protein family members. Additionally, we show that the efficacy of novel HER-3 targeting agents can be better predicted in certain breast cancer patient sub-groups based on the dimerization pattern of HER protein family members. Moreover, this model was also evaluated and reproduced in human paraffin-embedded breast cancer tissues.

show abstract

Section: Discussionmentioning

confidence: 99%

HER-3 targeting alters the dimerization pattern of ErbB protein family members in breast carcinomas

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Recently, new applications for bispecific antibodies have been demonstrated, such as gene-mediated therapy and immunodiagnostic applications [64][65][66]. Given the encouraging clinical data, improvements in the design and the efficacy of therapeutic bispecific antibodies are likely to continue rapidly.…”

Section: Future Perspectives Limitations and Therapeutic Benefitsmentioning

confidence: 97%

Advances in Bispecific Antibodies Engineering: Novel Concepts for Immunotherapies

Spasevska¹,

Duong²,

Klein³

et al. 2015

J Blood Disord Transfus

View full text Add to dashboard Cite

Bispecific antibodies are emerging as novel approach for immunotherapies by combining two antigen-recognizing elements into a single construct that is able to simultaneously bind to two distinct targets. Bispecific antibodies can be applied to recruit immunological effector cells for killing of tumor cells or to simultaneously block two signaling pathways or cytokines. Subsequently, they have prompted significant interest for a number of therapeutic applications, both in cancer and in other indications. The growing interest in therapeutic antibodies along with the rapid progress in antibody engineering have yield a multitude of bispecific antibodies formats and derived molecules that differ in size, shape and function. Presently, two major classes of bispecific antibodies are most widely studied: immunoglobulin-like and small single chain Fv (scFv)-based bispecific antibodies. This review summarizes selected key methods used to generate bispecific antibodies, reports new concepts developed for immunotherapy and discusses their potential development and therapeutic benefits.

show abstract

“…The "Dual Binders" approach requires significant efforts for the development of different bispecific molecules for every PPC target. 17 Herein, we describe a new fully automated, broadly applicable, solid-phase IHC assay that detects PPCs through protein proximity using caged haptens.…”

Section: ■ Introductionmentioning

confidence: 99%

Fully Automated Protein Proximity Assay in Formalin-Fixed, Paraffin-Embedded Tissue Using Caged Haptens

et al. 2020

View full text Add to dashboard Cite

The ability to interrogate for the presence and distribution of protein−protein complexes (PPCs) is of high importance for the advancement of diagnostic capabilities such as determining therapeutic effects in the context of pharmaceutical development. Herein, we report a novel assay for detecting and visualizing PPCs on formalin-fixed, paraffin-embedded material using a caged hapten. To this end, we synthetically modified a nitropyrazole hapten with an alkaline phosphatase (AP)-responsive self-immolative caging group. The AP-labile caging group abrogates antibody binding; however, upon exposure to AP, the native hapten is regenerated. These caged haptens were applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the population of PPCs, was then visualized by an anti-hapten antibody conjugated to horseradish peroxidase, followed by diaminobenzidine detection. We provide proof of concept for the detection of protein proximity pairs (β-catenin−E-cadherin and EGFR−GRB2), and confirm assay specificity through technical controls involving reagent omission experiments, and biologically by treatment with small-molecule kinase inhibitors that interrupt kinase− adaptor complexes.

show abstract

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

Cited by 8 publications

References 38 publications

HER-3 targeting alters the dimerization pattern of ErbB protein family members in breast carcinomas

HER-3 targeting alters the dimerization pattern of ErbB protein family members in breast carcinomas

Advances in Bispecific Antibodies Engineering: Novel Concepts for Immunotherapies

Fully Automated Protein Proximity Assay in Formalin-Fixed, Paraffin-Embedded Tissue Using Caged Haptens

Contact Info

Product

Resources

About