The Experimental Study Of ⁹⁹Tc^m Labelled Anti-GRP Monoclonal Antibody D-D₃

Objective:1.To explore the 99Tcm labelling method,stability and immunocompetence of D-D3 antibody against pro-gastrin-releasing peptide?31-98??ProGRP?31-98??.2.To explore the biodistribution and pharmacokinetics of ⁹⁹Tc^m-D-D₃ antibody in healthy ICR mice and nude mice bearing human small cell lung cancer?SCLC?.3.To explore the imaging effect of ⁹⁹Tc^m-D-D₃ antibody in different tumor models.Methods:1.The GRP expression in NCI-H446,Hela,SGC-7901 and A549 cancer tissues was estimated respectively by immunohistochemistry.2.⁹⁹Tc^m-D-D₃ was prepared and labelled with 2-nercaptoethanol;⁹⁹Tc^m-D-D₃ was purified by gel column separation method;Labelling efficiency and radiochemical purity were measured by paper chromatography.⁹⁹Tc^m-D-D₃ antibody was incubated respectively with NS and healthy serum at 37°C and radiochemical purity was measured at different time points to determine the stability of labelled product.Immunocompetence of labelled product was determined with cell conjugation assay.3.Biodistribution study was performed following an i.v.injection?given via the tail vein?of ⁹⁹Tc^m-D-D₃.Healthy ICR mice were sacrificed with cervical dislocation at designated time points in group.The blood and major organs were removed,weighed and counted in a gamma scintillation counter to determine the%ID/g?percentage of injected dose related to the organ weight?for each radioactive substance.In the same way,major organs and transplanted tumor tissues of nude mice bearing SCLC,cervical cancer and gastric cancer were respectively sampled for calculating%ID/g and tumor/non-tumor?T/NT?ratios.4.After injection of ⁹⁹Tc^m-D-D₃,continuous images of the nude mice bearing SCLC,cervical cancer and gastric cancer were respectively carried out at different time points,and then tumor/base?T/B?ratios were calculated.Results:1.The results of immunohistochemistry showed that positive granules were distributed in intracytoplasm of SCLC,cervical cancer and gastric cancer,no obvious staining cells were found in lung adenocarcinoma.2.The labelling rate of ⁹⁹Tc^m-D-D₃ was 73.87±2.89%and the radiochemical purity was 94.13±4.49%.After incubation with NS at 37? for 4 h,the radiochemical purity was 82.17±1.00%and the radiochemical purity was 55.73±1.07%at 12h.Meanwhile,after incubation with healthy human serum for 4 h,the radiochemical purity was 74.13±1.67%and the radiochemical purity was 54.07±1.88%at 12h.The immunobinding rates of NCI-H446 cell and A549 cell were 81.2±2.37%and 24.3± 1.46%respectively.3.The vivo distribution of ⁹⁹Tc^m-D-D₃ in the normal ICR mice and nude mice bearing SCLC showed that the metabolism of ⁹⁹Tc^m-D-D₃ mainly depended on liver and kidney with rapid elimination in blood,the brain and muscle did not uptake much,it was consistent with general antibody.The%ID/g of tumor was higher than that of other organs at 12h and arrived at top 0.41±0.04%at 24h post-injection.The T/NT ratio increased gradually and tumor/muscle arrived at top 13.67±5.03 at 24h.4.Clear tumor image of nude mice bearing SCLC was obtained at 24h after injection with ⁹⁹Tc^m-D-D₃ antibody.The radioactivity in tumor site accumulated gradually with the time and the tumor image reached the highest clearness at 24h post-injection.There was no obvious accumulation of ⁹⁹Tc^m-D-D₃ antibody in nude mice bearing lung adenocarcinoma.The imaging results of nude mice bearing cervical cancer and gastric cancer were similar to that of nude mice bearing SCLC,but the accumulation level of ⁹⁹Tc^m-D-D₃ antibody was less then that in nude mice bearing SCLC by macroscopic observation.The T/B ratio of SCLC was higher than the ratios of cervical cancer and gastric cancer.The T/B ratios of SCLC,cervical cancer and gastric cancer were obviously higher than that of lung adenocarcinoma.Conclusion:1.⁹⁹Tc^m-D-D₃ antibody not only had high radiochemical purity but also had good stability both in vitro and in vivo,and kept good immunocompetence after being labelled with 99Tcm.2.⁹⁹Tc^m-D-D₃ antibody accumulated mailly in tumor site and took on remarkable targeting property to SCLC.⁹⁹Tc^m-D-D₃ was metabolized mainly in kidney and liver,the blood radioactivity of ⁹⁹Tc^m-D-D₃ decreased rapidly.The ratios of T/NT increased as time prolonged and arrived at top at 24h.3.⁹⁹Tc^m-D-D₃ has the potential to become radioimmunoimaging agent of SCLC.It deserves further study.