Bacterial Strain and plasmid
Escherichia coli (E. coli) BL21 was used as host strain with an IPTG-inducible T7 RNA polymerase. And pGEX-4T-1 plasmid (Amersham, Italy) was used for protein construction and maintenance. Recombinant proteins are expressed as fusion protein with Glutathione S transfer (GST). The vector allows high level Isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible expression for the gene of interest in E. coli.
The DNA sequence for the wild type CR2-R domain and its mutant CR2-K domain of human EGFR was obtained from the National Center for Biotechnology Information (NCBI) at https://www.ncbi.nlm.nih.gov/ [Gene ID: 1956]. The wild type of DNA sequences for CR2 domain and its mutated form were flanked with restriction site EcoRI and XhoI. After that, the wild type of CR2 domain and its mutant form were synthesized and cloned in pGEX4T-1 plasmid (GeneCust, Luxembourg). The resulting construct, named [CR2-R-pGEX4T-1 and CR2-K-pGEX4T-1] were analyzed by DNA sequencing to ascertain its correctness.
Transformation of recombinant plasmids to E. coli BL21
40 ng of the recombinant plasmids (CR2-R-pGEX4T-1 and CR2-K-pGEX4T-1) were transformed into the competent cells of E. coli BL21 strain by electroporation using a Gene Pulser Xcell system (Bio-Rad, USA) under the following setting: 200 Ω, 2500 V, 20 µF. Then, 100 µl of the electro-transformed cells were plated onto LB agar medium supplemented with ampicillin at 100 µg/ml as a selective antibiotic. After incubation over night at 37°C; the positive colonies which has recombinant plasmids were picked up and confirmed by colony PCR technique using specific primers targeting CR2 domain (Forward: ʹ5-GAATTCGTGATAATTTCAGGAAAC-ʹ3) & (Reverse: ʹ5-CTCGAGCCCATTCGTTGGACAGCC -ʹ3). After that, the recombinant plasmids were purified using QIAprep Spin Miniprep Kit (cat# 27106), and were checked by using 1% of agarose gel electrophoresis and were sequenced using a common forward primer for pGEX4T-1 (ʹ5-GGGCTGGCAAGCCACGTTTGGTG- ʹ3) and analyzed with the standard nucleotide blast program from NCBI databank.
Recombinant expression of CR2-R and CR2-K proteins in E. coli BL21
The expression of soluble fusion protein for CR2 domain of human EGFR, BL21-λDE3 cells carrying pGEX4T1-CR2-R plasmids, pGEX4T1-CR2-K plasmids and pGEX4T1 empty plasmids were grown at 37˚C at 250 rpm in 3 ml LB media with 100µg/ml Amp to get the OD 600=0.8. Secondary culture was inoculated with 1% inoculums in 20 ml LB culture media having 100µg/ml Amp. Culture was grown till OD 600=0.8 and induced with 0.25 mM IPTG, 37˚C at 250 rpm for 4 hrs. As a control uninduced BL21-λDE3 cells were taken. After cell lysis, the bacterial cells lysate was harvested by centrifugation at 4,000 rpm for 20 min at 4˚C. The pellets were resuspended with 5 ml of lysis buffer in (1/5th of the original culture volume) at 4˚C on ice. The cells were lysed by sonication 6 X 30 seconds (3 minute) with break of 20 seconds (Amplitude 10) on ice. Afterwards, Triton X-100 was added to 1% of lysis volume and mixed on a rocker for 1 hour at 4˚C. finally, the samples were centrifuged at 13200 rpm for 30 minutes at 4˚C and the supernatant was saved at -80˚C considering to be our specific total soluble protein for CR2 domain of human EGFR. The expression was assessed by 15% SDS-PAGE and the protein bands were visualized by staining with Brilliant Blue R-250, followed by Western blotting with anti-GST antibody. Also, the protein concentration was determined using the Bradford method (34).
Western blot analysis
The equivalent amounts of protein samples were resolved on a 15% SDS-PAGE and the separated bands were transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 0.1% of 0% fat milk, in Tris-buffered saline, pH 7.4 (TBS) containing 0.05% Tween 20 for 1 hour at room temperature. Western blotting analysis was performed with horseradish peroxidase (HRP)-conjugated anti-GST antibody (Invitrogen) (diluted 1:10000). Target proteins were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham, GE Healthcare).
Purification and endotoxin removal of recombinant protein of CR2 domain
The recombinant proteins CR2-R and CR2-K were applied in GSTrap FF affinity column (GE health, United Kingdom) for purification using the standard procedure described according to manufacture ̓s instruction. The purified proteins (CR2-R and CR2-K) in 1X PBS were passed through Ion Exchange column at PH 7.4 and found in flow through. The flow through were dialysed with 1X PBS under endotoxin free condition at 4˚C. Purified proteins were analyzed on 12% SDS PAGE.
Production of monoclonal antibodies against CR2 domain
The purified proteins (CR2R and CR2-K) were sent to ProMab Biotechnologies, Inc. (California, U.S.A) for custom monoclonal antibodies production. All immunizations and subsequent steps in production of hybridoma cells/purified antibodies were completed by ProMab Biotechnologies, Inc. Briefly, for immunization of Balb/c mice, ProMab Biotechnologies used the recombinant protein as well as the following conjugated free peptides (CWGPEPRD and CWGPEDKD) corresponding to each variant. The antibody response titer was evaluated using ELISA before proceeding to fusion. Approximately 3-4 mg protein immunogen or 2 mg conjugated peptide immunogen and 0.5 mg free were required. Hybridoma fusion was performed using splenocytes from mouse with the best titer and Sp2/0 myeloma cells. After that, supernatants from the growing hybridoma wells were screened by ELISA. 20 clones were positive and sent to life science department in the Arabian Gulf University for evaluation. Two ELISAs assay were performed one using the peptides and the second the recombinant proteins. Six double positive clones were selected (2 clones specific to CR2R, 2 clones specific to CR2K and two recognizing both CR2R & CR2K,. The monoclonal antibodies isotyping was carried out using Mouse MAb ID kit (HRP): ZYMED Cat#: 90-6550; Lot.60907259.
Reactivity Assessment of the mAbs against hybridomas by ELISA
The reactivity and speciଁcity of the recombinant monoclonal antibodies examined by ELISA, using hybridoma clones. The free peptide antigens (CWGPEPRD) were coated on ELISA plates overnight at 4°C. After being washed with PBS and blocked by 1% BSA, the puriଁed clone supernatant from 24-well plate (primary antibody) was added and incubated for 1 hour at room temperature. The wells were then washed and detected by 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) substrate after incubation with HRP labeled anti IgG (secondary antibody). The reaction was stopped by addition of 2M H2SO4 and the absorbance was measured at 450 nm by a microtiter ELISA plate reader. The same protocol used with the free peptide antigen (CWGPEPKD).
Molecular and Cellular Characterization of mAb EGFR/CR2-RK
We selected 3 purified monoclonal antibodies named mAb-CR2-R (specific for wild type form of CR2 domain), mAb-CR2-K (specific for mutated form of CR2 domain) and mAb-CR2-RK. We carried out molecular and cellular characterization on mAb-CR2-RK it targets both the wild type and mutated form of the CR2 domain.
A. Analysis by western blot
Recombinant CR2 proteins were labelled using the purified anti-CR2RK antibodies in a concentration of 1:10000 in TBST overnight at 4°C with shaking. The membrane was washed as previous and a secondary antibody (anti mouse IgG HRP) diluted in TBS-T (1:4000) was added. The blocking by 0.1% of 0% fat milk for 1 hr. The detection by Amersham ECL Plus western blotting detection kit and LI‑COR C‑DiGit Chemiluminescence Western Blot Scanner.
B. Analysis by ELISA
Same previous ELISA procedure was used to evaluate the specificity and sensitivity between mAb-EGFR/CR2-RK (50µg/ml) with different concentrations (0.25 mg, 0.5 mg & 1 mg) of both recombinant purified CR2R and CR2K antigens and mAb-EGFR/CR2-RK antibody was tested against each antigen (CR2R & CR2K) in triplicate.
C. Analysis by IHC
The IHC study on formalin-fixed paraffin embedded tissues sections on a total of 10 tissue samples from lung adenocarcinoma of cancer patients from the Arabian patients was carried out at Johns Hopkins Aramco healthcare center. The biopsies were taken before the patients undergo any therapeutic course. The Ventana iView TM DAB detection kit (Cat #760-091) was used to assess the binding of anti-EGFR mAb-CR2-RK with a HRP conjugated anti-mouse IgG as secondary antibody. A normal skin tissue was used as positive tissue control, whereas the same tumor tissue lacking mAb were used as negative control. All the steps of IHC procedure including interpretation of the results, the patient's morphologic findings and pertinent clinical data were confirmed by a qualified pathologist according to the American Society of Clinical Oncology/College of American Pathologists reporting guidelines (ASCO/CAP) using the same standardized reading recommendations of the CAP for Her2neu (35). The intensity of staining was scored using the following scale: no staining, 0; weak staining, 1+; moderate membranous staining in 10-30% of the tumor cells, 2+; and strong staining, 3+ in >30% of tumor cells. We classified scores of 0 and +1 as negative and scores of 2+ and 3+ as positive.
D. Analysis by Surface Plasmon Resonance (Biacore)
The binding affinities between the mAbEGFR/CR2-RK & CR2 recombinant protein was performed using Biacore. The experiment was done at the Biacore Molecular Interactions Shared Resource of Lombardi Comprehensive Cancer Center at Georgetown University in Washington DC, USA. BiacoreT200 (GE Healthcare) was used to determine the kinetic parameters for the binding of Anti-CR2RK-mAb used as a ligand (~150 kDa, 1.5 mg/ml, stock in PBS) with Peptide 1 (labelled as “P1”, CWGPEPRD, 959.04kDa, 10 mg/ml stock in PBS) and Peptide 2 (labelled as “P2”, CWGPEPKD, 1931.03kDa, 10 mg/ml stock in PBS) were used as analytes. Recombinant GST (~26 kDa, 0.2 mg/ml stock) was also used as a control analyte. The experiments were performed using carboxymethyl-dextran (CM5) sensor chips. Briefly, Flow cell FC1 was used as reference for FC2. Anti-EGFR/CR2-Ab was diluted (1:50 dilution, 30 µg/ml diluted concentration) in HBS-P and captured onto FC2 to a level of ~8000 RU. Right after antibodies were captured, 20s pulse of NHS-EDC followed by 20s pulse of Ethanolamine were injected in order to crosslink the antibodies onto the surface. Capture/crosslinking was carried out in the presence of HBS-P. Overnight kinetics were performed for the analytes in the presence of the HBS-P. The flow rate of all the solutions was maintained at 50 µL/min. Analyte’s concentrations were 0 µM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1000 nM. One 30s pulse of 1M NaCl was utilized to regenerate sensor surface. Sensorgrams from overnight kinetics were evaluated by 1:1 kinetics model fitting.