- Cell Culture
Cell lines used in this study were as follows VERO, MDA-MB-468, VERO expressing EGFR (VERO/EGFR), VERO expressing HER2 (VERO/HER2), and VERO expressing both EGFR and HER2 (VERO/EGFR/HER2). VERO and MDA-MB-468 were obtained from the Pasteur Institute of Iran (Tehran, Iran) and maintained in the appropriate media recommended by the provider. VERO/HER2 cell line has been established previously (15-17).
VERO/EGFR cells were generated from transfected VERO cells using the linearized plasmid pCMV-encoding full-length EGFR. VERO/EGFR/HER2 was also generated through transfection of VERO/EGFR cells with pCVN-encoding full-length HER2. The selection of the cells was carried out via culture in the presence of 500 µg/ml hygromycin for VERO/EGFR, and 500 µg/ml hygromycin and 500 µg/ml G418 for VERO/EGFR/HER2. Both cell lines were cultured in an appropriate medium in 250 µg/ml maintenance dose of hygromycin and G418.
- Library preparation
The Tomlinson J and I phage display antibody libraries (Source BioScience, UK) were used to select antibody fragments against the extracellular subdomain II (dimerization arm) of EGFR expressed on the surface of VERO/EGFR cells. These are semisynthetic phage libraries based on a single human framework with side-chain diversity incorporated at positions in the antigen-binding site. Stocks of recombinant M13 phages displaying pIII-scFv fusion on their surface were prepared, according to the recommended protocol (16). Briefly, aliquots of each library were cultured in 200 ml of 2xTY medium supplemented with 1% glucose and 100 μg/ml ampicillin at OD600 = 0.4. Approximately 2 ´ 1011 of M13K07 helper phages were added to 50 ml of the culture medium and incubated at 37°C in a water bath for 30 min. Bacterial pellets were collected by centrifugation at 3300 ´ g for 30 min and the pellets were then resuspended in 100 ml of 2X TY medium supplemented with 0.1% glucose, 100 μg/ml ampicillin, and 50 μg/ml kanamycin. Recombinant phages were precipitated from the overnight culture with PEG/NaCl solution (20% Polyethylene glycol 6000, 2.5 M NaCl).
- Subtractive panning on the cells
The first round of panning was performed on MDA-MB-468, a cancer cell line overexpressing EGFR, to reduce non-specific bindings. The process of antibody selection is schematically shown in Figure 1.
Each library (~1012 pfu/ml) was incubated with the MDA-MB-468 cell line for 2 hours at room temperature with gently rotating. The cells were then pelleted by centrifugation at 140 ´ g for 20 min, and the supernatant was moved to a new tube. The next round was performed on EGF-stimulated (60 ng/ml) and then –non-stimulated VERO/EGFR cells (19). The cells were pelleted and washed twice with HEPES washing buffer. The bound phages were then eluted with 1 ml glycine buffer (pH=2.2) and then used for reinoculation of exponentially growing TG1 bacteria. Next, 20 ml of 2xTY-Amp-Glu medium was inoculated with 10 to 20 μl bacterial suspension to yield an OD600 = 0.1, and grown at 37°C and 140 ´ g at OD600 = 0.5. The bacteria were then inoculated with M13K07 helper phage in a multiplicity of infection (MOI: phage/bacteria) of 10 to 20 for 30 min at 37°C in a water bath. Bacteria were pelleted and resuspended in 100 ml 2XTY containing 100 mg/ml ampicillin and 50 mg/ml kanamycin culture medium and incubated overnight at 30°C on a shaker. The same procedure was performed for the VERO/EGFR cell line for two more rounds of panning.
- PCR and fingerprinting
To evaluate the results of the selection process of single-chain antibodies, variety in selected clones was examined by PCR and fingerprinting at the end of the last round of panning. PCR was carried out on several clones using two primers, LMB3: CAGGAAACAGCTATGAC and pHEN: CTATGCGGCCCCATTCA. Positive samples were digested by BstNI restriction enzyme overnight at 37°C and loaded on a 2% agarose gel.
- Polyclonal phage cell ELISA
To confirm binding of the pool of isolated phages to their ligands on the cells, a phage ELISA was performed with EGF-stimulated and –non-stimulated VERO/EGFR cells, using phages from all three rounds of panning. Approximately 7 ´ 103 VERO/EGFR cells were seeded in each well of 96-well culture plates (SPL, South Korea) one day before the experiment. The experiment was performed in triplicate. Cells were washed twice with cold phosphate-buffered saline (PBS) and blocked with 200 μl 2% bovine serum albumin (BSA) for 1 hour at room temperature. Then, 100 μl of the polyclonal phage particles from each round of selection was added to each well and incubated for 2 hours at room temperature. The wells were washed three times with 200 μl tris-buffered saline, 0.1% tween 20 (TBST), then incubated with 100 μl of 1:5000 dilutions of HRP-conjugated anti-M13 antibody (Amersham Pharmacia, US) in 2% blocking buffer for 1 hour at room temperature. Finally, the wells were washed three times with TTBS and incubated with tetramethylbenzidine (TMB) solution (Sigma-Aldrich) for 15 min at room temperature. The reactions were stopped by 3N HCl, and the absorbance was read at 450 nm on an ELISA plate reader (Organon-Teknika, The Netherlands).
- Dimerization inhibition test
Dimerization inhibition test was performed with phages obtained from each round of selection. The VERO/EGFR/HER2 cell line was serum-starved for 24 hours, and then cells were detached by a cell scraper and washed with PBS. The cells were stimulated with 60 ng/ml of EGF for 30 min on ice, following by incubation with selected phages from each round of panning for 30 min. Crosslinking of HER2 and EGFR was performed with a cell membrane-impermeable protein crosslinking agent, sulfosuccinimidyl (bis) substrate, (Thermo Fisher Scientific, Inc., MA, USA), according to the manufacturer’s instructions. Briefly, the cells were washed twice with ice-cold PBS and incubated with freshly prepared bis substrate at room temperature for 30 min. Cells were lysed using a radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) containing 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 mL of protease inhibitor cocktail, and 1 mM sodium orthovanadate.
The total cell lysate was immunoprecipitated, using mouse anti-EGFR (Clone D8, Santa Cruz Biotechnology) antibody. Briefly, 500 µg of total cell lysate was incubated with protein A/G plus agarose in the presence of 5 µg of anti-EGFR antibody overnight at 4 °C with gentle shaking. The antigen-antibody complexes were then pelleted and washed 4 times. The complex was detached using loading buffer (62.5 mM Tris·Cl, pH 6.8, 2% w/v SDS, 2% v/v glycerol 0.1% w/v bromphenol blue, and 300 mM 2-mercaptoethanol) and heated at 75°C for 10 min.
To show the ability of selected scFv to inhibit EGFR homodimerization and EGFR/HER2 heterodimerization, the anti-EGFR immunoprecipitated proteins were separated under a denaturing and reducing SDS-PAGE and then transferred to Polyvinylidene difluoride (PVDF) membranes (Santa Cruz Biotechnology) in a tank transfer system (Bio-Rad, CA, USA) at 100 V for 60 min. Membranes were then separately probed with anti-EGFR and anti-HER2 as the primary antibodies, and the goat anti-mouse conjugated HRP as the secondary antibody. The blots were developed with the ECL kit (Parstous Biotec, Mashhad, Iran) for 5 min and imaged by the G:BOX imaging system (Syngene, Cambridge, UK).
- Production and purification of phage free scFv
Soluble scFv was produced to evaluate the function of the selected antibody pools. To do this, 100 µl of the eluted phages from each round of selection were incubated with 2 ml of exponentially growing HB2151 bacteria (OD600 = 0.4) for 30 min at 37°C in a water bath. Then, the bacteria were pelleted and plated on the TYE-Amp-Glu medium. Bacterial colonies were then scraped by a spatula. Then, 50 µl of each phage pool was inoculated in 50 ml 2XTY-Amp and incubated with shaking at 37°C for approximately 6 hours until OD600 reached 0.8 to 1. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and incubated with shaking at 30°C overnight. Bacterial were then pelleted and dissolved in 2 ml of hyper-osmotic lysis solution containing 50 mM Tris, 20% saccharose, and 1 mM EDTA at pH 8. Cell debris was pelleted by centrifugation at 20,000 ´ g for 30 min at 4°C, and the supernatant containing the desired antibodies were collected and stored at -20 °C for subsequent use.
Soluble scFvs were separated from the protein mixture using HisTrap HP affinity chromatography with an ÄKTA purifier. Briefly, the HisTrapTM HP column was equilibrated with the binding buffer, containing 50 mM Tris, 1M NaCl, and 40 mM Imidazole, and then the protein mixture was injected into the column. The His-tagged recombinant protein was eluted with the elution buffer, containing 50 mM Tris, 500 mM NaCl, and 500 mM imidazole at pH 8, fractions containing eluted proteins were collected and analyzed using SDS-PAGE. The concentration of the collected antibodies was measured using the Bradford assay (19). To confirm the presence of soluble scFvs, the final products of purification were subjected to immunoblotting. The purified proteins were immunoblotted as described earlier using HRP-conjugated anti-His tag antibody (Clone H-3, Santa Cruz Biotechnology).