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

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Original article

Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR Yaqiong Zhou, Juan Zhang *, Haizhen Jin, Zhiguo Chen, Qinhang Wu, Weiguang Li, Ming Yue, Chen Luo, Min Wang * State Key Laboratory of Natural Medicines (China Pharmaceutical University), School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 March 2013 Accepted 24 March 2013

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 antiEGFR scFvs from a human synthetic antibody phage display library. Combined with an additional cellbased ELISA screen, a novel scFv, E10, was obtained with two-fold more potent on the binding to EGFRbearing 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. ß 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Epidermal growth factor receptor (EGFR) Prokaryotic expression Refolding Phage display Single-chain variable fragment antibody (scFv) Tumor therapy

1. 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-a 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

* Corresponding author. Tel.: +86 25 832 713 95, +86 25 832 714 83; fax: +86 25 832 713 95. E-mail addresses: [email protected] (J. Zhang), [email protected] (M. Wang).

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 TGFa 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

0753-3322/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2013.03.019

Please cite this article in press as: Zhou Y, et al. Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.03.019

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refold because of the misfolding and aggregation caused by its cysrich 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.

Table 1 List of buffers. Buffer

Buffer preparation

Lysis buffer

50 mM Tris–HCl, 5 mM EDTA, 1%TritonX-100, 1 mM PMSF, 1 mM b-ME, pH 8.0 50 mM Tris–HCl, 10 mM EDTA, 1 mg/mL DOA, 1 mM b-ME, pH 8.0 50 mM Tris–HCl, 10 mM EDTA, 5 mg/mL DOA, pH 8.0 50 mM Tris–HCl, 5 mM EDTA, 4 M urea, 500 mM NaCl, pH8.0 50 mM Tris–HCl, 6 M Gdm–HCl, 100 mM b-ME, pH 8.0 20 mM MES, 8 M urea, 500 mM NaCl, pH 5.6 20 mM MES, 8 M urea, 500 mM NaCl, 800Mm imidazole, pH 5.6 50 mM Tris, 1 mM EDTA, 3 mM GSH, 1 mM GSSG, 1 M urea, 150 mM NaCl, pH 8.9 50 mM Tris, 1 mM EDTA, 3 mM GSH, 1 mM GSSG, 8 M urea, 150 mM NaCl, pH 8.9 50 mM Tris, 1 mM EDTA, 150 mM NaCl, pH 8.9

Washing buffer A Washing buffer B Washing buffer C Denaturing buffer Purification buffer A Purification buffer B Refolding buffer A Refolding buffer B Storing buffer

2. Materials and methods 2.1. 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 8C 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 vector pHEN2 [21]. ScFvs can be expressed as soluble fragments containing c-Myc tag and His-tag, or can be displayed on the surface of bacteriophage when expressed in E. coli TG1. ScFv AK404, an anti-VEGFR2 antibody fragment screened from Griffin.1 library by colleagues, was produced at our laboratory. ScFv 11F8 derived from IMC-11F8, a high affinity antibody fragment directed against EGFR, was produced at our laboratory as previously reported by ImClone Systems [9,10]. 2.2. Cloning and expression of EGFR-tED Total mRNA was isolated from A431 cells with a FastTrack kit (Invitrogen). The gene of EGFR-tED was amplified with SuperScript One-step RT-PCR (Invitrogen) and cloned into pMD18-T (TAKARA) vector. The positive clones were selected and confirmed by DNA sequencing. The recombinant plasmid was digested with NdeI and BamHI and inserted into the pET28a(+) (Novagen) expression vector, and then, introduced into E. coli BL21 (DE3), identified by double digestion and DNA sequencing. The transformed E. coli BL21 (DE3) was cultured in LB medium with 50 mg/mL kanamycin at 37 8C with further induction by 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) for 4 h, when the optical density 600 (OD600) reached 1.0. The cells were preserved at –20 8C after centrifugation at 6000  g for 10 min at 4 8C.

at 37 8C. The supernatant was filtrated with hydrophilic polyethersulfone membrane (0.22 mm, Millipore) after centrifugation at 1,0000  g for 30 min. The harvested denatured EGFR-tED protein supernatant was stored at 4 8C for further treatment. 2.4. Purification of denatured EGFR-tED with gel filtration and IMAC A sample of 1 mL of denatured EGFR-tED protein was directly applied to a 1  100 cm Sephacryl 200HR (a 34 mm average diameter highly cross-linked agarose base matrix with an exclusion limit about 250 KD for globular proteins, GE Healthcare) column equilibrated with purification buffer A (Table 1). The peak contained EGFR-tED protein was collected for the subsequent nickel-based affinity purification with Biologic Duo flow FPLC system (Bio-Rad). The collected sample of 50 mL was applied to a 5 mL HisTrap chelating column (GE Healthcare) equilibrated with purification buffer A (Table 1). A stepwise elution chromatography process was proceeded with mixed buffers of purification buffer A and B (Table 1), containing 100 mM, 200 mM and 500 mM imidazole. The sample eluted by buffer containing 200 mM imidazole was collected, analyzed by SDS–PAGE and stored in 4 8C for refolding experiments. 2.5. Refolding of denatured EGFR-tED by urea gradient size-exclusion chromatography A Sephacryl S-200 gel column mentioned above was equilibrated with refolding buffer A (Table 1), followed by a gradual increase of refolding buffer B (Table 1) up to 100%. Consequently, a urea gradient was formed in a total volume of 25 mL (Fig. 1), with a urea concentration of 8 M at the top of the column. Afterwards, 700 mL purified EGFR-tED sample was loaded into the column and eluted with refolding buffer B. Equilibration, loading and elution were carried out at 6 8C and the collected protein solution was placed at 25 8C for 20 h [18]. After dialysis against storing buffer (Table 1), the renatured EGFR-tED protein was stored at –70 8C.

2.3. Pre-treatment of EGFR-tED inclusion bodies 2.6. Identification of EGFR-tED by western blotting E. coli cell paste (1 g wet weight) was thawed in 20 mL of lysis buffer (Table 1) containing 0.1 mg/mL lysozyme, and disrupted by sonication. Crude inclusion bodies of EGFR-tED were recovered with 20-fold volume of washing buffer A (Table 1) with agitation overnight at 25 8C, and then, washed with 20-fold volume of washing buffer B and C (Table 1) in turn. The final inclusion body pellet was solubilized in denaturing buffer (5 mL buffer per 1 g wet weight inclusion bodies) (Table 1) and incubated for 1 h in a shaker

The purified EGFR-tED was separated by 12% SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked in blocking buffer [5% skim milk in phosphate buffered saline solution–0.05% Tween20 (PBS– Tween)] (4 8C, overnight) and incubated with mouse anti-Histag monoclonal antibody (Millipore) (1 h, 37 8C), followed by peroxidase-conjugated goat anti-mouse IgG (Enogene) (1 h, 37 8C).

Please cite this article in press as: Zhou Y, et al. Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.03.019

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with 1 mM IPTG when OD600 reached 0.9 and growth was continued overnight at 30 8C. The culture supernatant containing the secreted scFvs was collected by centrifugation for subsequent monoclonal phage ELISA with EGFR-tED following a previously described procedure [22]. Then, top 15 scFvs with high affinity were selected for sequence analysis and further cell-based ELISA to select one scFv with the highest affinity. Cell-based ELISA was carried out using A431 cells. The cells were grown to confluence in an ELISA plate (Costar) and fixed by 0.25% glutaraldehyde for 10 min at RT. Thereafter, the operation was proceeded, following an optimized Cellular ELISA [23]. ScFv 11F8 was used as a positive control. ScFv AK404R, a vascular endothelial growth factor receptor 2 antibody fragment, which was previously developed in our laboratory, was used as a negative control. 2.10. Expression and purification of scFvs

Fig. 1. Experimental system for urea gradient Size-exclusion chromatography (SEC) refolding process. Prepacked Sephacryl S-200 column was pre-equilibrated as shown in the figure. Dark colors represent higher urea concentrations; light colors represent lower urea concentrations.

Protein bands were visualized using diaminobenzidine tetrahydrochloride (DAB) (Sigma–Aldrich) buffer. The concentrations of purified EGFR-tED were determined with a protein assay kit (BioRad).

ScFvs were secreted into the periplasm in a soluble form. Flask fermentation was applied for the scFv expression in LB medium, containing 0.4 M sucrose. The cells were harvested by centrifugation at 5000  g for 10 min at 4 8C. Periplasmic proteins were isolated by osmotic shock. The bacteria pellets were resuspended with 5% of the initial volume of ice-cold 50 mM Tris–HCl, 20% sucrose, 1 mM EDTA, pH 8.0 and gently stirred for 10 min prior to centrifugation at 10,000  g for 10 min at 4 8C. The cell paste was resuspended with the same volume of ice-cold 5 mM MgSO4 and gently stirred for 15 min on ice [24]. After centrifugation, the supernatant was decanted and merged with the osmotic buffer supernatant. With further adjusting of the pooled supernatant to 0.5 M NaCl, a 1 mL HisTrap nickel-chelating column was applied for scFv purification operated according to the manufacture instructions. 2.11. Identification of scFv by western blotting

2.7. Bioactivity analysis of EGFR-tED by ELISA Bioactivity of EGFR-tED was tested by ELISA for binding to its ligand EGF. Briefly, ELISA plate was coated with EGF (5 mg/mL, Sino Biological Inc) overnight at 4 8C and blocked for 2 h. The plate was incubated with a linear concentration dilution of EGFR-tED (40, 10, 2.5, 0.625, 0.156 mg/mL) for 2 h. Then, the plate was washed three times with PBS–Tween buffer and incubated with mouse anti-Histag antibody (1:2000) for 2 h. After the plate was washed again, it was incubated with HRP-conjugated anti-mouse IgG antibody (1:5000) for 2 h, followed by TMB as the enzyme substrate. The reaction was stopped with 50 mL of 1 M H2SO4 and the absorbance was measured at 450/650 nm. 2.8. Biopanning of anti-EGFR scFvs from Griffin.1 phage display library All screening manipulations and bacteria culture and transformation were operated as described elsewhere [22]. Four rounds of biopanning were carried out with stringent conditions, i.e. the progressive reduction of EGFR-tED (50, 40, 25 and 15 mg/mL) coated on immunotubes (Nunc) and increase of washing times (10, 20, 20 and 30 times) as panning rounds increased.

Purified scFv was loaded into 15% SDS–PAGE gel for electrophoresis and then, electroblotted onto PVDF membrane. After incubation with 5% skim milk in PBS–Tween buffer for 1 h at 37 8C, the membrane was washed three times in PBS–Tween buffer and incubated for 1 h at room temperature with monoclonal antibody 9E10 for 1 h at 37 8C. The membrane was incubated with peroxidase-conjugated goat anti-mouse IgG for 1 h at 37 8C. Protein bands were visualized using DAB buffer. 2.12. Inhibition on cell proliferation by anti-EGFR scFv Inhibition on A431 cells proliferation was used to determine the relative potency of anti-EGFR scFv by an MTT assay [3]. A431 cells (1  104 cells per well) were seeded into 96-well plates and incubated for 3 days at 37 8C in 5% CO2/95% air with DMEM medium, containing 0.5% newborn calf serum and scFvs at different concentrations or without scFvs as the blank control. ScFv 11F8 was used as the positive control. Then, the plate was incubated for another 4 h with 12 mL MTT buffer. After the addition of 200 ml DMSO, the absorbance was measured at 570 nm, reference 630 nm in a microplate reader. 3. Results

2.9. Analysis of soluble scFvs by monoclonal phage ELISA and further screen by cell-based ELISA E. coli HB2151 cells infected with phages were plated onto TYE/ Amp plates and incubated overnight at 37 8C. Single colonies were inoculated in 2  TY medium, containing 100 mg/mL ampicillin and 1% glucose in 96-well plates. ScFv expression was induced

3.1. 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

Please cite this article in press as: Zhou Y, et al. Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.03.019

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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. 3.2. Expression and purification of recombinant protein

Fig. 2. Structure and the construction of the EGFR gene. (A) Schematic representation of EGFR sequence domains. The numbers represent the position of amino acids. TM: transmembrane domain, amino acids 621–644; Extracellular: extracellular domain, amino acids 1–621; Intracellular: intracellular domain, amino acids 644–1186; Target gene: EGFR-tED, amino acids 1–501. (B) Structure of EGFRtED gene in recombinant plasmid pET28a(+)-EGFR. (C) Analysis of the plasmid (pET28a(+)-EGFR) by double digestion. Lane 1, two bands generated by double digestion with NcoI and BamHI. The large one is vector (5265 bp) and the small one is target DNA EGFR-tED (1566 bp); lane 2, DNA marker.

The recombinant expression plasmid (pET28a(+)-EGFR) was transformed into E. coli BL21 (DE3) and the target protein (57.9 KD) was then highly expressed after the induction by IPTG (Fig. 2A), which accumulated in insoluble inclusion bodies. We developed a purification protocol that involved two-step purification under denatured conditions followed by a urea gradient gel filtration refolding step. Cell lysates were recovered from bacteria, collected after 4 h induction and the inclusion bodies were isolated and extensively washed to remove the soluble proteins (Fig. 3A). Approximately 25 mg of proteins were recovered from the denatured buffer derived from 500 mL bacteria culture. First, the denatured protein was subjected to gel filtration on FPLC system. Column flowthrough was monitored by UV absorption at 280 nm, and was separated into fractions by an auto-fraction collector. Three distinct peaks can be seen (Fig. 3B), analyzed by SDS–PAGE (Fig. 3C). Peak 2 contained monomeric EGFR-tED (> 90% purity)

Fig. 3. Expression, purification and characterization of EGFR-tED. (A) SDS–PAGE analysis of EGFR-tED expression. Lane 1: induction of BL21 (DE3)-pET28a; lane 2: induction of BL21 (DE3)-pET28a- EGFR; lane3: the washed inclusion bodies of EGFR- tED. (B) and (C) Purification of the denatured EGFR-tED by gel filtration. (B) Chromatography trace by FPLC. (C) SDS–PAGE analysis. Lane 1: P1; lane 2: P2; lane 3: P3. (D) SDS–PAGE analysis. Lane1: EGFR-tED purified by IMAC; lane 2: EGFR-tED refolded by SEC. (E) Western blotting analysis of purified EGFR-tED by a mouse anti-His antibody. All lanes M in the SDS–PAGE represent protein marker.

Please cite this article in press as: Zhou Y, et al. Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.03.019

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Table 2 Enrichment factors during panning. Round

Input (tu)

Output (tu)

Recovery rate (out/in)

1 2 3 4

1.8  1014 1.4  1013 4.0  1013 3.8  1012

4.5  106 3.2  106 8.2  106 2.5  107

2.5  10 2.3  10 2.1  10 6.6  10

8 7 7 6

Enrichment (ratio n/ratio n – 1) – 9.2 0.9 31

Transducing units (tu) before and after panning rounds were determined using titration of transduced colonies. Recovery rate, i.e. output vs input in the same round, and enrichment, i.e. the factor by which the ratio of rescued phages differed from round n – 1 to round n was calculated.

3.4. Enrichment of high affinity scFvs after four rounds of biopanning

Fig. 4. On-column refolding of EGFR-tED by urea gradient gel filtration. Elution flow rate: 0.11 mL/min. The urea concentration change is shown by the change of conductivity curve. P1 represents the peak of refolded EGFR protein. P2 represents the peak of b-ME. * Represents the peak of saline compounds in the loading protein.

while the other two peaks probably contained aggregated or truncated forms and the other impurities. The recombinant protein was further purified and concentrated by Ni-NTA chromatography eluted with 200 mM imidazole (Fig. 3D). The final yield of purified EGFR-tED was 2.6 mg. 3.3. EGFR-tED refolding by urea gradient size-exclusion chromatography (SEC) The urea gradient refolding process was demonstrated by the change of the conductivity curve (Fig. 4). The urea concentration at the top of the gel column was the same as that in the sample of denatured protein. When denatured protein moved downward from the top of the gel column, it underwent a slow change and gentle drop of urea concentration, and move exactly into the refolding buffer containing lower urea concentration at the end of the column by selecting a suitable gradient length (data not shown). The refolded protein was analyzed by SDS–PAGE (Fig. 3D) and western blotting (Fig. 3E). The final yield of refolded protein was 1.3 mg per 500 mL culture. The concentration-dependent manner between the renatured EGFR-tED protein and EGF was detected by a quantitative ELISA assay. The binding of serial diluted EGFR-tED to immobilized EGF increased gradually and achieved a platform at high concentration (Fig. 5), indicating that the renatured EGFR-tED could bind to EGF in a concentration-dependent manner.

Fig. 5. Binding of EGFR-tED to EGF measured by ELISA. EGF (5 mg/mL) was coated on the ELISA plate at first. The concentration of EGFR-tED varied from 40 mg/mL to 0.156 mg/mL by quadrupling dilution.

The titer of the eluted phages of each round of panning was determined to monitor the recovery. As shown in Table 2, the recovery increased gradually as expected in spite of the enhanced intensity in elution. A 31-fold increase in the ratio of output vs input phage titer, expressed as the enrichment factor, was observed from round 3 to round 4 (Table 2). The recovery of round 4 was increased by approximately 260-fold than that of round 1, which indicated a significant enrichment through the rounds of panning. 3.5. Selection of scFv E10 with the highest affinity by monoclonal phage ELISA and cell-based ELISA assay In the monoclonal phage ELISA assay of 192 randomly picked colonies, the soluble scFvs expressed by 70% colonies reacted against EGFR-tED (data not shown), from which, top 15 clones with higher affinity were sequenced and six different scFvs were obtained for A431 cell-based ELISA assay (Fig. 6). It was possible to rank the scFvs with respect to their binding to EGFR and the ranking was reproducible. Among them, scFv E10 (GenBank accession no. JQ306330) bound most strongly to EGFR, approximately two-fold more potent than scFv 11F8 and therefore, was selected as the target scFv for further study. 3.6. Purification and characterization of scFv E10 Periplasmic proteins isolated by osmotic shock were passed through a nickel-chelating Histrap column. After the column was washed with buffer containing 50 mM imidazole, the bound scFv

Fig. 6. Binding of scFvs to EGFR-bearing tumor cells A431 measured by cell-based ELISA. ScFvs (30 mg/mL) were added to an ELISA plate coated with A431 cells and skim milk. A431 blank control is for cell-based ELISA without the addition of scFvs. 2G6, C11, 2E2, E10, C5, and 2B12 are scFvs screened from Griffin.1 Library by EGFRtED. ScFv 11F8 was used as a positive control. ScFv AK404 was used as a negative control. Bound scFvs were detected by mouse anti-His-tag antibody.

Please cite this article in press as: Zhou Y, et al. Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.03.019

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Fig. 7. Purification and characterization of E10. (A) Purification of E10 by Ni-NTA column on 15% coomassie brilliant blue stained SDS–PAGE. Lane 1, flowthrough of E10 protein from the column; lane 2, elution from the Ni-NTA column with 50 mM imidazole; lane 3, elution with 100 mM imidazole. (B) Western blotting analysis for purified E10 shown by arrow. All lanes M in the SDS–PAGE represent protein marker.

was eluted with 100 mM imidazole as a single peak with purity above 90% (Fig. 7A). We routinely obtained 650 mg purified scFv from 1L culture. Western blotting was carried out by using monoclonal antibody 9E10, which could specifically recognize the c-Myc tag at the end of scFv, as confirmed by the band of right size (Fig. 7B). 3.7. Inhibition of scFv E10 on A431 cells proliferation The potency of E10 to inhibit A431 cells proliferation was determined in vitro by an MTT assay. A431 cells were cultured in the presence of various concentrations of scFv E10 or 11F8. The measurement of the vital cell quantities after culture for 48 h demonstrated that proliferation was inhibited in a dose-dependent manner (Fig. 8), with a maximum inhibition of 75%. The IC50 values of E10 for inhibition on cells proliferation was 54 nM compared with 94 nm for that of scFv 11F8, which showed that a novel scFv with two-fold more potent was obtained. 4. 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

Fig. 8. Inhibition on A431 cells proliferation by scFvs. The concentrations of scFv E10 were 200, 100, 50, 25, 12.5, 2.5 nM. The concentrations of scFv 11F8 were 120, 60, 30, 15, 7.5 nM by doubling dilution.

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 protein expressed by E. coli existed in the form of inclusion bodies mainly due to its cys-rich domains. At first, the different kinds of ion-exchange chromatography, such as Q, S, DEAE and CM Sepharose ion-exchangers (GE Healthcare) were tried to purify the EGFR-tED protein. However, the protein could barely bind to cation-exchange column, while anion-exchange chromatography had a low resolution despite the consideration of pH, the ionic strength and the flow rate (data not shown). The possible reason could be that the denatured recombinant protein is difficult to be positively charged and binds to anion-exchange column similar to impurity when negatively charged. The exclusion of ion-exchange chromatography, Sephacryl S-200 gel filtration and Ni-NTA affinity chromatography were applied for the purification with a satisfactory result (Fig. 3). Dilution and dialysis were employed in the refolding of the recombinant protein. However, most protein was precipitated during the refolding process probably due to the formation of the aggregates caused by the strong hydrophobic property or the mismatch of the disulfide bonds (data not shown). A new strategy of gradually decreasing urea concentration in SEC had been developed [25] to remove the denaturant more slowly than in general SEC in order to decrease the aggregation. Meanwhile, urea at low concentration (< 2 M [19]) in SEC can also improve the yield of correctly folded protein [26]. Therefore, the gradual removal of the denaturant by Sephacryl S-200 gel filtration with urea gradient could refold the EGFR-tED protein in a much gentle environment by stabilizing the correctly folded intermediates of different refolding stages, thus, minimizing the likelihood of aggregation. A relatively high refolding yield was therefore achieved in comparison with the usual dilution refolding (Fig. 1). The renatured protein binding EGF in a concentrationdependent manner indicated that EGFR-tED was obtained with specific bioactivity (Fig. 5). Moreover, refolding using a chromatographic process is attractive for its automation and simultaneous partial purification [27]. The successful preparation of EGFR-tED by prokaryotic expression and renaturation in this study is much more convenient and comes with lower cost

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method than eukaryotic expression, which can be used for the screening of its antibody. Furthermore, antibody fragments were screened with the renatured EGFR-tED from Griffin.1 library derived from human B-cell lymphocyte, which is capable of avoiding HAMA response and possess a longer half-life in vivo [28,29]. Among the six scFvs screened and tested, five were bound to both purified EGFR and EGFR-bearing tumor cells A431 (Fig. 6). One of the possible explanations for the result that one scFv did not bind to A431 cells may be that the renatured EGFR-tED and the EGFR on the tumor cells surface have differences in conformation or glycosylation. Besides, the recombinant protein has an additional 21 amino acids at the N-terminus, which needs further study to prove whether the scFv could bind the 21 amino acids peptide or not. After EGFR-tED protein biopanning and further cell-based ELISA screening, the scFv E10 with the strongest binding to EGFR-bearing tumor cell A431 was selected, which inhibited the proliferation of A431 with approximately two-fold potent than scFv 11F8 (Fig. 8). E10 was almost 10-fold more potent on binding to A431 cells than C11 even if only four amino acids differences on the heavy chain complementarity-determining region 3 (CDR3) and two amino acids differences on the light chain CDR3. The significant impact of CDR3 on the antibody affinity indicates a promising modification site for antibody affinity maturation in vitro [30]. In the future study, the scFv E10 can be cloned into mammalian cell expression vectors containing DNA coding for the constant regions of human inmmunoglobulins to form a whole antibody molecule which can be administered to human patients for cancer therapy.

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Disclosure of interest

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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). References [1] Kumar A, Petri ET, Halmos B, Boggon TJ. Structure and clinical relevance of the epidermal growth factor receptor in human cancer. J Clin Oncol 2008;26: 1742–51. [2] Boersma YL, Chao G, Steiner D, Wittrup KD, Plueckthun A. Bispecific designed ankyrin repeat proteins (DARPins) targeting the epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling. J Biol Chem 2011;286(48):41273–85. [3] Bleeker WK, Lammerts van Bueren JJ, van Ojik HH, Gerritsen AF, Pluyter M, Houtkamp M, et al. Dual mode of action of a human anti-epidermal growth factor receptor monoclonal antibody for cancer therapy. J Immunol 2004;173:4699–707. [4] Lopez F, Llorente JL, Oviedo CM, Vivanco B, Alvarez Marcos C, Garcia-Inclan C, et al. Gene amplification and protein overexpression of EGFR and ERBB2 in sinonasal squamous cell carcinoma. Cancer 2012;118(7):1818–26.

Please cite this article in press as: Zhou Y, et al. Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomed Pharmacother (2013), http://dx.doi.org/10.1016/j.biopha.2013.03.019