Dataset Information

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

ABSTRACT:

Introduction

The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR.

Methods

Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488).

Results

These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α).

Discussion

EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.

SUBMITTER: Comez D

PROVIDER: S-EPMC9726709 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

Comez Dehan D Glenn Jacqueline J Anbuhl Stephanie M SM Heukers Raimond R Smit Martine J MJ Hill Stephen J SJ Kilpatrick Laura E LE

Frontiers in immunology 20221123

<h4>Introduction</h4>The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR ...[more]

PMID: 36505413

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Project description:Introduction: Surgical resection is one of the main treatment options for several types of cancer, the desired outcome being complete removal of the primary tumor and its local metastases. Any malignant tissue that remains after surgery may lead to relapsing disease, negatively impacting the patient's quality of life and overall survival. Fluorescence imaging in surgical oncology aims to facilitate full resection of solid tumors through the visualization of malignant tissue during surgery, following the administration of a fluorescent contrast agent. An important class of targeting molecules are Nanobodies® (Nbs), small antigen-binding fragments derived from camelid heavy chain only antibodies. When coupled with a fluorophore, Nbs can bind to a specific receptor and demarcate tumor margins through a fluorescence camera, improving the accuracy of surgical intervention. A widely investigated target for fluorescence-guided surgery is the epidermal growth factor receptor (EGFR), which is overexpressed in several types of tumors. Promising results with the fluorescently labeled anti-EGFR Nb 7D12-s775z in murine models motivated a project employing the compound in a pioneering study in dogs with spontaneous cancer. Methods: To determine the safety profile of the study drug, three healthy purpose-bred dogs received an intravenous injection of the tracer at 5.83, 11.66, and 19.47 mg/m2, separated by a 14-day wash-out period. Physical examination and fluorescence imaging were performed at established time points, and the animals were closely monitored between doses. Blood and urine values were analyzed pre- and 24 h post administration. Results: No adverse effects were observed, and blood and urine values stayed within the reference range. Images of the oral mucosa, acquired with a fluorescence imaging device (Fluobeam®), suggest rapid clearance, which was in accordance with previous in vivo studies. Discussion: These are the first results to indicate that 7D12-s775z is well tolerated in dogs and paves the way to conduct clinical trials in canine patients with EGFR-overexpressing spontaneous tumors.

Dataset Information

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

Introduction

Methods

Results

Discussion

Publications

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

Similar Datasets

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