| Objective: To produce and screen the ABCG2 specific single-chain antibody of human lung adenocarcinoma, further detect the efficiency and label the scFv antibody with radionuclide for radioimmunoimaging study in tumor-bearing mice. This study will lay a foundation for lung adenocarcinoma resistance reversal and radioimmunotherapy, and provide promising approach for molecular targeting therapy of lung adenocarcinoma patients.Methods and results:Part 1 Construction of scFv antibody library1. Synthesis of scFv gene: The total RNA was extracted from the positive lymph nodes near the lung adenocarcinoma. The c DNA was amplified by reverse transcription. Take the c DNA as a template, the VH and VL genes were amplified by PCR. Then, the VH and VL genes were amplified to add a pair of linker sequence in their upstream anddownstream respectively. Equal amount of VH-Linker- and Linker+-VL fragments were used to assemble the scFv gene by SOE-PCR. The scFv fragments were detected and purified by 1% agaros gel electrophoresis.Our results showed that the ratio of A260/A280 of the total RNA is 1.98 which is good purity and unconspicuous degradation. Specific bands of 360 bp, 340 bp, 410 bp, 390 bp and 750 bp were observed on the gel,representing the VH,VL, VH-Linker-, Linker+-VL and scFv gene fragment respectively.2. Preparation of primary antibody library: After Sfi I/Not I digestion,the purified scFv fragments were cloned into phage display vector p CANTAB5 E. Ligation products were used to transforma competent E.coli TG1 by electroporation. The clones were then identified by plasmid PCR and Sfi I/Not I enzyme digestion analysis, the positive insert rate was 100%(12/12). The positive plasmids were then analyzed by sequence analysis.The consistency of sequence alignment with the coding sequence of scFv was reached to 99%.3. Expressing of phage antibody: The positive plasmids were collected by 2×YT culture medium. The scFv fragments were induced for expression with helper phage M13KO7.Part 2 Selection and immunoreactivity detection of scFv antibody1. Screening on A549 cells and ABCG2 antigen: The primary phage library was screened on A549 lung adenocarcinoma cells for three roundsof “adsorption-elution-amplification”. Then three other rounds were performed against the ABCG2 antigen. The harvest rate of first round was1.54×10-6, and after six rounds of panning, the number was 1.7×10-4. The biopanning process resulted in a 110-fold enrichment of affinity.2. Expression, purification and identification of soluble scFv antibody:The colonies giving high signals against ABCG2 antigen in ELISA were chosen for soluble expression. E. coli HB215 l was infected with these chosen colonies with 1 mmol/L IPTG. The scFv fragments were purified over a Hitrap TM Anti-E Tag column. The soluble antibodies were identified by SDS-PAGE, western-blot was also used to testify the soluble expression with anti E-tag primary antibody(1: 3000). A clear 34 k Da band was observed, confirming the scFv fragments had been soluble expressed in E.coli HB215 l. The specificity of scFv was analyzed by ELISA and immunocytochemistry(ICC) and non-targeting scFv antibody(primary scFv before biopanning) was used as a control: the scFv was highly specific that could bind to A549 cells, but not to CNE2 and HCT116 cells.Antibody affinity analysis by competitive ELISA: results showed that at low concentrations of cell extract(1: 500 diluted), the purified scFv showed a feeble inhibition effect to competition antibody Ig G(inhibition rate, 4.56 % ± 1.96 %), whereas the inhibition rate of the high concentration group(1: 1 diluted) ascended to 68.09 % ± 4.27 %,indicating that the scFv has high affinity to lung adenocarcinoma cells.Part 3 Proliferation inhibition and chemosensitization of the scFv antibody1. CCK-8, colony formation, migration and flow cytometry analysis:A549 cells were incubated with scFv for 48 h, the CCK-8 analysis showed a time and dose dependent growth inhibition of the scFv to A549 cells. In both the colony formation and migration assays, scFv significantly reduced the number of cells which could establish a clone or migrate through the insert membranes. Apoptosis and cell cycle analyses were further performed. Compared with control group, an increasing apoptosis rate(47.95 % ± 5.43 % vs. 9.21 % ± 2.38 %, P < 0.01) was observed in scFv-treated group accompanied by G1 cell cycle arrest(70.70 % ± 1.81 %vs.52.44 % ± 2.72 %, P < 0.01).2. Cisplatin sensitization of the scFv: After treated with scFv, A549 cells showed significantly enhanced sensitivity to cisplatin with decreased IC50 values(5.31 ± 0.49 μg/m L vs. 0.06 ± 0.03 μg/m L, P < 0.01).3. ScFv regulates ABCG2 expression: Lysates of A549 cells incubated with different concentrations of scFv were examined by Western blot.Compared with control group, scFv caused a dose-dependently decrease in ABCG2 and MDR1 expressions.Part 4 Radioimmunoimaging study of scFv antibodyThe soluble scFv was radiolabeled with 131 I using the chloramine T method. The nude mice bearing A549 cells were injected via the tail veinwith purified 131I-scFv. At different times, the tumor and major organs were removed, weighed and counted on a γ-counter to determine the %ID/g for biodistribution study. Mice were scanned at designated times for SPECT/CT imaging. The results showed that the tumor/blood,tumor/muscle and tumor/brain signal ratios were gradually increased after injection, and reached the peak of 3.45 ± 1.06, 4.51 ± 0.89, and 23.65 ±3.00 at 24 h. The radioactivity was aggregated in tumor locations and the tumor imaging was clearly observed at 24 h by use of SPECT/CT fused images.Part 5 In vivo antitumor activity of scFv antibodyTo generate xenograft models, freshly dissociated A549 cells(2×106/100 μL PBS) were subcutaneously injected into the back of each mouse. When the average tumor size reached approximately 500 mm3,mice were then divided randomly into four groups: normal saline(NS),scFv alone, DDP alone and pre-treatment with scFv followed by DDP(scFv+DDP) with four in each group, and subjected to the corresponding treatment. The therapy used in this study was scFv 10 mg/kg, i.p., d1-3 and DDP 2.5 mg/kg, i.p., d 4 and 7. Tumor growth and tumor volumes were monitored every other day. Tumor tissues were harvested and fixed in 4%formaldehyde solution and subsequently embedded in paraffin. Tumor specimens were stained using Ki-67 and cleaved caspase 3 antibody for cell proliferation and apoptosis. Smaller tumor volumes as well as significantlydecreased proliferation and increased apoptosis were observed in scFv+DDP group.Conclusion: We constructed the scFv antibody of lung adenocarcinoma against ABCG2 by phage display technology. The immunoreactivity, chemosensitization and anti-proliferation ability in vivo or in vitro were analyzed. The scFv antibody was further labeled with radionuclide. Biodistribution study and SPECT/CT imaging were performed. The results showed that the ABCG2 specific scFv antibody of lung adenocarcinoma has been prepared successfully. The scFv showed high affinity and specificity to A549 cells, strong anti-proliferation and pro-apoptosis effects on them as well as significantly increased chemosensitivity to cisplatin. The satisfactory radioimmunoimaging effect,specific affinity to target tumor and good dynamic characteristics implicate its potential application in lung adenocarcinoma imaging diagnosis and target therapy. |
PDF Full Text Request