The Experimental Study Of Anti-GRP_31-98 Monoclonal Antibody

Objective1. To explore the expression rate and location of gastrin-releasing peptide (GRP) antigen in cell lines and tissues of small cell lung cancer (SCLC).2. To explore the ¹³¹I labeling method, stability and immunocompetence of E-B5 antibody against pro-gastrin-releasing peptide _31-98 (ProGRP_31-98).3. To explore the biodistribution and pharmacokinetics of ¹³¹I-E-B₅ antibody in healthy Kunming mice and nude mice bearing SCLC.4. To explore the imaging effect and the best imaging time of ¹³¹I-E-B₅ antibody in different tumor models.5. To explore the radioimmunotherapy feasibility and curative effect of ¹³¹I-E-B₅ antibody in treating SCLC.Method1. The expression of GRP antigen in small cell lung cancer cell line NCI-H446 and lung adenocarcinoma cell line A549 cell was detected by flow cytometer and the expression of GRP antigen in tissues of human SCLC was observed by immunohistochemistry.2. Chloramine-T method was used for ¹³¹I labeling E-B₅ antibody. Labeling efficiency and radiochemical purity were estimated by using paper chromatography method. ¹³¹I-E-B₅ antibody was incubated with healthy serum at 37℃, and radiochemical purity at different time points was demonstrated to know the stability of ¹³¹I-E-B₅ antibody. Immunocompetence of ¹³¹I- E-B₅ was detected with cell conjugation assay.3. After healthy Kunming mice were injected with ¹³¹I-E-B₅ antibody through tail veil, mice were sacrificed by cutting off carotid artery at different time points. The blood and organs were harvested for calculating dose per gram of tissue (%ID/g) and pharmacokinetic parameters. Nude mice bearing human small cell lung cancer were sacrificed at different time points after infusion with ¹³¹I-E-B₅ antibody through tail vein, and their organs were harvested for calculating %ID/g and tumor/non-tumor (T/NT) ratio.4. Continuous images of the nude mice bearing SCLC, lung adenocarcinoma and colon cancer were carried out respectively at different time points after injection of ¹³¹I-E-B₅ antibody, and then transplantation tumor images were observed and tumor/base(T/B) ratios were calculated.5. The nude mice bearing SCLC were divided into 3 groups, including ¹³¹I-E-B₅ antibody, Na¹³¹I and control group. From the first to the fourth week after intratumoral injections, the tumor sizes were measured and tumor growth curves were drew.Results1. The positive ratios of GRP expression were 89% and 12% on NCI-H446 cells and A549 cells respectively. The results of immunohistochemistry showed that there were conspicuous distribution of positive cells in tissues of human small cell lung cancer and brown particles were found in cytoplasm of tumor cells.2. The labeling efficiency of ¹³¹I-E-B₅ antibody was 90.8%, the radiochemical purity was 99.28% and the specific activity was 2.69MBq/μg. The radiochemical purity still maintained above 90% and 70% after incubation in water bath at 37℃for 6 h and 24 h respectively. After incubation with healthy serum for 24 h, the radiochemical purity still reached 68.1%. The immunobinding ratios were 71.6% and 33.2% for NCI-H446 cells and A549 cells respectively.3. The vivo distribution and elimination of ¹³¹1-E-B₅ antibody were consistent with a two-compartment model .The metabolism of ¹³¹I-E-B₅ antibody mainly depended on liver and kidney and with rapid elimination in blood. The %ID/g of transplantation tumor was obviously higher than that of other organs and arrived at top at 72 h post-injection. The T/NT ratio increased gradually with the time.4. Clear tumor image of nude mice bearing SCLC was obtained at 24 h after injection with ¹³¹I-E-B5 antibody. The radioactivity in tumor site accumulated gradually with the time and the tumor image reached the highest clearness at 72 h and 96 h post-injection. There was no obvious accumulation of ¹³¹I-E-B5 antibody in nude mice bearing lung adenocarcinoma. The imaging results of nude mice bearing colon cancer were similar to that of nude mice bearing SCLC, but the accumulation level of ¹³¹I-E-B₅ antibody was less then that in nude mice bearing SCLC by macroscopic observation. The T/B ratio of SCLC obviously higher than the ratio of colon cancer. The T/B ratio of SCLC and colon cancer were higher than lung adenocarcinoma, then T/B ratio of lung adenocarcinoma haven't obviously change.5. The inhibition ratio of the ¹³¹I-E-B₅ group was conspicuous higher than that of Na¹³¹I group. The tumor size decreased obviously after two weeks' therapy of ¹³¹I-E-B₅ antibody. There were not only obvious punctiform anabrosis but also gradually enlarged necrosis on the surface of tumor. Aecidioid anabrosis was found in lOOuCi and 200 uCi group after four weeks' therapy. However, almost disappearance of tumor was seen in 400μCi and 600μCi group. The tumor growth in Na¹³¹I group was slowly and with no significant decrease in tumor size. There were punctiform anabrosis on the surface of tumor in 400μCi group and 600μCi.Conclusion1. ¹³¹I-E-B₅ antibody not only has high labeling efficiency and radiochemical purity ,but also has good stability both in vitro and in vivo, and keep good immunocompetence after being labeled with ¹³¹I.2. ¹³¹I-E-B₅ antibody accumulates mainly in tumor site and takes on remarkable targeting property to SCLC. The local injection of ¹³¹I-E-B₅ not only inhibits the growth of tumor, but also destroys tumor.3.¹³¹I-E-B₅ antibody has potential clinical value and can be used in radioimmumoimaging and radioimmunotherapy of SCLC. Further investigation should be done.