Original article
Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR

https://doi.org/10.1016/j.biopha.2013.03.019Get rights and content

Abstract

The epidermal growth factor receptor (EGFR), overexpressed in many epithelial tumors, is emerging as an attractive target for cancer therapy. Antibodies to the extracellular region of EGFR play a key role in the development of a mechanistic understanding and cancer therapy. In the present study, we demonstrated for the first time that EGFR-truncated extracellular domain (EGFR-tED), which was expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies, could be purified and renatured. The EGFR-tED protein was purified by gel filtration and Ni-NTA affinity chromatography with high purity (> 90%) and refolded by a urea gradient size-exclusion chromatography, which could bind its ligand EGF in a concentration-dependent manner. The renatured EGFR was used for biopanning anti-EGFR scFvs from a human synthetic antibody phage display library. Combined with an additional cell-based ELISA screen, a novel scFv, E10, was obtained with two-fold more potent on the binding to EGFR-bearing tumor cells (the epidermoid carcinoma cell line A431) and the inhibition of A431 cells proliferation than scFv 11F8, suggesting that the E10 has the potential to be developed as therapeutic agents to solid tumors associated with EGFR overexpression.

Introduction

Epidermal growth factor receptor (EGFR or HER) family comprises of four distinct transmembrane receptors: EGFR or HER1/erbB1, HER2/erbB2, HER3/erbB3, and HER4/erbB4 [1]. The first molecularly cloned EGFR is a large (1186 residues), modular glycoprotein with an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular tyrosine kinase domain [2]. EGFR is overexpressed in a large number of human tumors, including carcinomas of the head and neck, breast, colon, prostate, lung, ovaries and sinonasal squamous cell [3], [4]. Overexpression of EGFR is correlated with an unfavorable prognosis, altered response to chemotherapy, and decreased survival [2], [5], It is also frequently accompanied by the production of its natural ligands, mainly EGF or TGF-α by tumor cells, suggesting that an autocrine loop participates in malignant transformation [3], which makes it a significant therapeutic target for cancer [6], [7].

Two main classes of compounds that have been currently developed targeting EGFR are the small-molecule inhibitors of the intracellular tyrosine kinase domain and monoclonal antibodies (mAbs) directed against the extracellular region of the receptor. Even though a few anti-EGFR mAbs are currently in clinic or clinical trials, they exhibit varying properties [8], For example, cetuximab and panitumumab were recently marketed for colon, head and neck, and/or lung cancers, covering limited ranges of solid tumors. Phase II study of IMC-11F8 [9], [10] in patients with colorectal cancer has been completed (NCT00835185). Considering the diversity of the EGFR-associated solid tumors, it is necessary to develop more anti-EGFR antibodies to be applied to other solid tumors [6], [8], [11].

EGFR have been expressed or extracted from different eukaryotic hosts for the structure-based drug design and screen or preparation of its antibodies, such as HEK293 cell line, rat astrocytes, breast cancer cell line SKBR3 and Chinese hamster ovary cells [12]. The extracellular portion of EGFR is divided into four domains (I–IV). Domain I and III of the receptor have been shown to play minor and major roles, respectively, in the ligand binding, while domain II mediates receptor dimerization [13]. A truncated EGFR extracellular domain (1–501 residues, including domain I to III and the first module of domain IV) expressed in HEK293 cell line, binds human EGF or TGFα with 13-fold higher affinity than the full-length EGFR ectodomain [14], [15].

However, prokaryotic hosts, e.g. Escherichia coli (E. coli), has not been reported before to obtain this kind of recombinant molecules mainly for the formation of inclusion bodies, which are hard to refold because of the misfolding and aggregation caused by its cys-rich domains. Nevertheless, due to the limitations of eukaryotic expression systems, such as low yield of recombinant protein, high cost, complex to construct and time-consuming for expression, strategies of solubilizing and refolding recombinant proteins for a high-level and rapid production of foreign proteins in bacteria were developed [16], [17]. Size-exclusion chromatography (SEC) for the removal of denaturants and the separation of folding intermediates has been extensively used on refolding proteins that is difficult to refold by conventional methods since 1990s [18], [19].

In this study, we seek to clone and express the truncated EGFR extracellular domain (EGFR-tED, 1–501 residues) in E. coli as inclusion bodies and screen a novel scFv against EGFR from a human synthetic antibody phage display library. This provides the potential for the development of human antibody-targeting tumor therapy.

Section snippets

Cells and reagents

A431 cells, an EGFR-overexpressed epidermoid carcinoma cell line, were purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences, and maintained in DMEM containing 10% newborn calf serum (Gibco). Cell culture was maintained at 37 °C in a 5% CO2 humidified incubator.

Griffin.1 library is a large naive human scFv phage library, derived by recloning synthetic heavy and light chain variable genes (VH and VL) from human synthetic Fab lox library vectors [20] into the phagemid

Construction of the recombinant expression plasmid pET28a(+)-EGFR

The truncated extracellular domain of EGFR gene was initially amplified from the mRNA of A431 cell by RT-PCR, cloned into pMD18-T simple vector (TaKaRa) (Fig. 2A), and finally inserted into the prokaryotic expression vector (pET28a(+)) with NdeI and BamHI (Fig. 2B). After pET28a(+)-EGFR plasmid was digested with NcoI and BamHI, the constructed target gene (1 kb) was visible in the gel (Fig. 2C). The inserted DNA was confirmed by sequencing.

Expression and purification of recombinant protein

The recombinant expression plasmid (pET28a(+)-EGFR) was

Discussion

The block of EGFR signaling by anti-EGFR antibodies has proven its anti-tumor effect in clinic or clinical trials [8]. In this current study, we have successfully carried out prokaryotic expression and renaturation of a truncated extracellular domain of EGFR, which can bind to its ligand EGF in a concentration-dependent manner. With the renatured EGFR-tED and further cell-based ELISA, scFv E10 was screened, which shows two-fold more effect on A431 cells inhibition than scFv 11F8.

EGFR-tED

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgments

This work has been supported by National Natural Science Foundation of China (81102364, 81072561 and 81273425), the Project Program of State Key Laboratory of Natural Medicines (JKGP201101, China Pharmaceutical University); Provincial Science and technology supporting program (BE2009675) and Colleges and universities in Jiangsu Province plans for graduate research and innovation projects (CX10B-376Z); Jiangsu Province Qinglan Project (2010).

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