Peptabody‐EGF: A novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells

Abstract

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy. ' 2006 Wiley-Liss, Inc.

Multivalency: the hallmark of antibodies used for optimization of tumor targeting by design

“…(76) Peptabody directed against EGFR exhibited significantly higher binding activity for this receptor than a corresponding therapeutic mAb and had a strong antiproliferative effect on cancer cells, overexpressing EGFR. (77) Ankirin repeats High-affinity binders were selected from designed ankyrin repeat protein libraries. (78) The advantages of designed ankirin repeats are: high expression level, thermodynamic stability, absence of cysteines and fast enrichment of binders.…”

Heptameric Targeting Ligands against EGFR and HER2 with High Stability and Avidity

“…In the previous self-association approaches for the generation of trimeric and pentameric complexes, additional cysteine (Cys) residues were introduced to stabilize the oligomeric structure through the formation of inter-molecular disulfide bonds [10], [11], [14], [17]. However, the formation of undesired disulfide bonds is common, resulting in mis-folding, aggregation, and loss of target-binding functions [1].…”

“…Targeting ligands with di-, tri-, tetra-, and pentavalency have been developed, however, those with a valency higher than five have not been reported and it is the focus of this study [4]–[14]. One protein class featuring heptameric structures is the Sm or Sm-like (Lsm) RNA-binding protein that has been implicated in a variety of RNA processing events in all eukaryotic organisms [15].…”

Tribody: Robust Self-Assembled Trimeric Targeting Ligands with High Stability and Significantly Improved Target-Binding Strength

“…Different scaffolds that allow for enhanced avidity have been reported. 1,2,5–7 These scaffolds include the bacteriophage T4 foldon domain, collagen like peptide (Gly-Pro-Pro) 10 , NC1 domain of collagen XV and XVIII domain, and GCN leucine zipper domain for trimers, 1,2,8,9,10 streptavidin and transcription factor p53 for tetramers, 11 the B-subunit of bacterial verotoxin and cartilage oligomeric matrix protein (COMP) for pentamers, 12,13 and recently the hyperthermophilic Sm protein for heptamers. 6 However, most of these scaffolds are derived from non-human proteins and have limited clinical application due to immunogenicity.…”