Production And Radioimmunoimaging Of Novel Fully Human Phage Display Recombinant Antibodies And Growth Inhibition Of Lung Adenocarcinoma Cell Line Overexpressing Prx I

Currently, lung cancer is one of the malignant tumors with the highest mortality around the world. Nowadays, the lung cancer has become one of the greatest enemies to human health and life comparing that at the early 20th century. Surgery, chemotherapy and radiation therapy are the main methods of treatments in clinics. However, there is no significant symptom of the inchoate lung cancer, and most lung cancer patients are in advanced stage when diagnosed, leading to an unsatisfactory curative effect. The 5 year–survival rate of lung cancer in developing country is about 15%, and the number in our country is less than 10%. Finding new therapeutic tool is the breakthrough to enhance the curative effect of lung cancer. The targeted therapy of cancers has the good prospects for the development. Specificity therapy is the salient feature of targeted therapy, and the normal cells can be protected when the malignant tumor cells have been killed. Based on the principle of radioimmunotherapy, to couple the radionuclide with the specificity targeted therapy antibodies, we can combine the antibody–mediated cell killing effect and the therapeutic action of radionuclide together, in order to enhance the curative effect.The key to a successful targeted therapy depends on the choice and construction of antibodies. The monoclonal antibody (McAb) is the most potential therapy antibody. There are three phases of the McAb development: humanization reform of the murine McAb phase, micromolecule antibody phase, and phage display library phase. The phage display antibody is the deputy of the 3rd phase antibody, representing a new development of gene engineering antibody technology.The oxidoreduction status has close relation with the tumour generation. The oxidoreduction protein can be used for tumor study. Peroxiredoxins are newly discovered peroxidase proteins, playing an important role in active oxygen clearance. Peroxiredoxin I(Prx I)is a member of Peroxiredoxin family, overexpressing in lung adenocarcinoma. Prxs can depress the generation of active oxyradicals in cells, and protect cells from apoptosis. Thus, Prx I is a candidate therapy target of lung adenocarcinoma.In order to overcome the malpractice of the heterology and low penetrating power of the murine McAb in clinic use, we attempted to prepare the human single–chain variable fragment (scFv) antibody by phage display technology with an target of lung adenocarcinoma cells overexpressing Prx I. The proliferation inhibition ability will be studied, and the antibodies will be used for radioimmunoimaging in bearing lung adenocarcinoma nude mice.Objective: To prepare the anti–Prx I lung adenocarcinoma human single–chain antibodies, and detect the antibody efficiency. Label the scFv antibody with radionuclide 131I and make radioimmunoimaging study in bearing lung adenocarcinoma nude mice. This study will lay the foundation for lung cancer radioimmunotherapy, and provide useful adjunct treatment to lung cancer patients.Methods and results:Part 1 Construction of phage antibody library1. Preparation of human scFv antibody: The B cells were obtained from the lymph nodes near the lung adenocarcinoma, and the total RNA was extracted. The cDNA gene were prepared and amplified by RT–PCR. The primer pairs for VH and VL gene amplified were determined by graticule bolting. Take the cDNA as a template, the VH and VL gene were amplified by PCR. Then, the VH–linker and VL–linker were amplified from the VH and VL, fragments with their respective primers. The amplified VH–linker and VL–linker fragments were then connected and amplified by SOE–PCR to form scFv, and the Sfi I and Not I restriction sites were inlet in scFv. The scFv fragments were purified by gel electrophoresis. The gel electrophoresis results showed that the total RNA of B cells has 2 bands (28S and 18S). A 370 bp band and 350 bp band were observed on the gel, representing the VH and VL gene respectively. The scFv fragments were detected on gel as a 750 bp band.2. Ligation of scFv fragments into phage vector: After Sfi I/Not I digestion, the purified scFv fragments were cloned into phage display vector pCANTAB–5E. Ligation mixtures were used to transform Escherichia coli TG1 through electroporation, and 2.8×107cfu/μg clones were obtained. The clones were then identified by Sfi I/Not I digestion analysis, the positive insert rate was 88% (44/50). The positive clones were then analyzed by sequence analysis.3. Expressing of phage antibody: The positive plasmids were collected by 2×YT culture medium. The scFv fragments were induced for expression with help phage M13KO7 in a sugar–free environment. Part 2 Phage antibody library selection1. Screening on lung adenocarcinoma cells: Before selection, normal human bronchial epithelial cells (HBE16) were used to deplete the phage library of nonspecific binders. Then, the phage library were screened on lung adenocarcinoma cells A549 3 rounds. The number of eluted phages increased after 3 rounds panning.2. Screening on Prx I: The phage library after 3 rounds panning on A549 cells were then selected on Prx I for 3 rounds. So, the panning were performed 6 rounds altogether. The harvest rate of first round was 1.3×10-6, and after 6 rounds panning, the number was 2.3×10-4, which increased 180 times.3. Expressing of soluble scFv antibody: The colonies giving high signals against Prx I were chosen for soluble expression. E. coli HB215l was infected with these chosen colonies with 1 mmol/L IPTG. The scFv fragments were purified over a HitrapTM Anti–E Tag column.4. The identification of soluble antibodies: SDS–PAGE identification: The soluble antibodies were identified by SDS–PAGE. The protein of antibodies was separated on a polyacrylamide gel and stained with Coomassie Blue R–250. A clear 30 ku band was observed, confirming the scFv fragments had been soluble expressed in E. coli HB215l. The reactivity of soluble scFv analyzed by ELISA: The ELISA result showed the A405 nm value of scFv on A549 cells was 0.63±0.08, and the value on MDA–MB–435 cells and HBE16 cells were 0.36±0.05 and 0.37±0.06, which confirming the specificity of the scFv to lung adenocarcinoma cells. Antibody affinity analysis by competitive ELISA: The results showed that, at high concentrations of cell extract (1:1 diluted), the purified scFv showed a significant inhibition effect to competition antibody IgG (inhibition rate, 66.1%), whereas the inhibition rate of the low concentration group (1:250 diluted) descended to 4.5%. immunocytochemistry analysis: The result showed that the Prx I scFv stained the A549 cells much more significant than that on MDA–MB–435 cells and HBE16 cells, indicating the Prx I–specific purified scFv with high affinity to A549 cells.Part 3 Proliferation inhibition study on lung adenocarcinoma cells1. Internalization study: The scFv were labeled with radionuclide 125I using the chloramine T method. The A549 cells were incubated with purified antibodies at 37°C for 30 min and 120 min. As the controls, unselected scFv and irrelevant antibody anti–iASPP scFv (prepared in our laboratory) were labeled with 125I as well. The temperature control for 125I–scFv was held at 4°C for 120 min. The cells were washed, for the removal of surface bound 125I-scFv. Then, the cells were lysed with 2% SDS and cell lysate radioactivity was measured on aγ–counter. The total amount of 125I–scFv bound to cells was estimated from the sum of the acid washes (surface bound 125I–scFv) and cell lysate (internalized 125I–scFv) radioactivity. The result showed that, compared with the control group, the Prx I specificity scFv can combine the A549 cells and be internalized effectly.2. MTT and flow cytometry analysis: After 72 h incubation with A549 cells, The MTT analysis was operated. The scFv showed a dose-dependent growth inhibition to A549 cells. The apoptosis of A549 cells were analyzed by flow cytometry (AnnexinV–FITC/PI). The scFv caused a significant apoptosis comparing with the control group.3. Expression of Prx I protein analysis: After 72 h incubation with scFv, the expression of Prx I protein in A549 cells was examined by Western blot. Compared with the control group, scFv caused a decrease in Prx I expression in A549 cells, and the Prx I protein expressed in A549 cells was about 60% of that in control group.Part 4 Radiolabeling of scFv and identificationThe soluble scFv was radiolabeled with 131I using the chloramine T method and purified by gel–filtration on a Sephadex G200 column. The labeling yield, specific activity, radiochemical purity and the stability of 131I-scFv in serum were tested. The results showed that the labeling yield was 83.2±2.8%,specific activity was 2.8±0.2MBq/μg, and radiochemical purity was 95.6±3.7%. After 48 h incubation with fresh human serum, the radiochemical purity of the 131I–scFv showed no significant depression. Part 5 Biodistribution study and SPECT imaging:The nude mice bearing lung adenocarcinoma were injected via the tail vein with purified 131I–scFv. At various times, the tumor and major organs were removed from groups, and were weighed and counted on aγ-counter to determine the %ID/g. The lung adenocarcinoma bearing nude mice were injected with 131I–scFv and fixed on boards for radioimmunoimaging analysis at designated times. The results showed that the tumor/blood and tumor/muscle values were increased after injection, and reached the peak of 4.19±0.13 and 4.89±0.56 at 48 h. The radioactivity was aggregated in tumor locations and the tumor imaging was clearly observed. Conclusion: We prepared the anti–Prx I lung adenocarcinoma human scFv antibodies by phage display technology. The specificity and proliferation ability were analyzed. The scFv antibodies were labeled with radionuclide. Biodistribution study and SPECT imaging were performed. The results showed that the lung adenocarcinoma specificity human scFv antibodies have been prepared successfully. The scFv showed strong antiproliferative effects on lung adenocarcinoma cells. The satisfactory radioimmunoimaging effect and specific affinity to target tumor implicates its potential application in lung adenocarcinoma imaging diagnosis and immunotherapy.