Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor

Abstract

Intracellular expression of single-chain antibodies (scFvs) represents a promising approach for selective interference with cellular proto-oncogenes such as the epidermal growth factor receptor (EGFR). Previously, we have shown that intrabodies targeted to the lumen of the endoplasmic reticulum prevent the transit of EGFR or the related ErbB2 molecule to the cell surface, thereby inactivating their transforming potential. While intramolecular disulfide bridges important for antibody stability are correctly formed during expression in the secretory pathway, scFvs expressed in the reducing environment of the cytosol are often inactive. To overcome this problem and to generate antibody fragments that interact with the intracellular domain of human EGFR in the cytoplasm, here we have chosen a two-step approach combining classical selection of scFvs by phage display with subsequent expression in yeast. After enrichment of EGFR-specific antibody fragments from a combinatorial library by biopanning, a yeast two-hybrid screen was performed using the intracellular domain of EGFR as bait. Screening of 1.5 Â 10 5 preselected scFv plasmids under highly stringent conditions yielded 223 colonies that represented at least five independent scFv clones functional in the intracellular milieu of eukaryotic cells. Interaction of selected antibody fragments with the intracellular domain of EGFR was confirmed in GST pull-down and coimmunoprecipitation experiments. Upon cytoplasmic expression in human tumor cells, scFvs colocalized with EGFR at the plasma membrane demonstrating their functionality in vivo.

Novel antibodies as anticancer agents

“…Intrabodies have been used to modulate the expression of overexpressed tumor proteins, such as Erb2 in breast and ovary tumors (Deshane et al, 1995a, b;Alvarez et al, 2000), cyclin E in breast cancer (Strube and Chen, 2002) and EGFR in epithelial cancers (Hyland et al, 2003).…”

Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation

“…The intrabodies have been proven very effective in inhibiting the function of culprit proteins both in vitro and in animal models of a wide spectrum of diseases, for example, in HIV infection (6,47,48), in neurodegenerative diseases, such as Alzheimer's (46), in diseases caused by mutated proteins, such as Huntington's (5, 46 -48), and others. In the field of oncology, intrabodies have been used to modulate the expression of proteins up-regulated in tumors, such as ErbB2 and Cyclin E in breast and ovarian cancer (1, 2, 4, 49), interleukin-2 receptor in some sorts of leukemia (3), and epidermal growth factor receptor in glioblastoma and epithelial cancers (50). In most of these cases, intrabodies were used as means to alter the intracellular trafficking and localization of their targets, for example, by trapping oncogene products (e.g., epidermal growth factor receptor and ErbB2) that were destined for cell surface expression in the endoplasmic reticulum or isolating transcription factors (e.g., Cyclin E) in the cytoplasm.…”

A bispecific transmembrane antibody simultaneously targeting intra‐ and extracellular epitopes of the epidermal growth factor receptor inhibits receptor activation and tumor cell growth

“…13 For comparison, similar 225.TM and TM.30 sequences were generated, which encode membrane-anchored extracellular scFv(225) or membrane-anchored intracellular scFv (30). For inducible expression, antibody sequences were inserted downstream of a tetracycline-inducible promoter into retroviral transfer plasmid pES.1-Ptet-T6, 14 modified to include an enhanced green fluorescent protein (EGFP) marker gene driven by the spleen focus forming virus (SFFV) promoter.…”

“…Using a yeast two-hybrid screen we previously isolated a panel of scFv antibodies from a preselected library that retained binding to the intracellular domain of EGFR upon cytoplasmic expression. 13 Here, we identified the epitope within EGFR recognized by scFv (30), the most active of these molecules, and used scFv(225) and scFv (30) to generate a novel type of bispecific transmembrane antibody simultaneously targeting intraand extracellular EGFR epitopes. This bispecific molecule termed 225.TM.30 carries scFv(225) as an extracellular domain at its N-terminus, linked via a flexible hinge region and a transmembrane domain (TM) to an intracellular scFv (30) domain.…”