| Objective:To evaluate the biodistribution of (131)I labeled anti-ProGRP(31-98) scFv in healthy Kunming mice, tumor-bearing mice and primarily perform the imaging in nude mice bearing human small cell lung cancer so as to investigate the feasibility of (131)I- anti- ProGRP(31-98) scFv as a new tumor tracer for diagnosing of certain tumors.Methods:1. The ProGRP expression in NCI-H446, Hela, H460 and A549 cell lines was detected respectively by flow cytometry and its expression in these tumor tissues was estimated by immunohistochemistry.2. Chloramine-T method was used for (131)I labeling anti-ProGRP(31-98) scFv; (131)I-anti-ProGRP(31-98) scFv was purified by gel column separation method; Labeling efficiency and radiochemical purity were measured by paper chromatography; (131)I-anti-ProGRP(31-98) scFv was incubated with healthy serum at 37℃, and radiochemical purity was measured at different time to determine the stability of labeled product; Immunocompetence of labeled product was determined with cell conjugation assay. 3. Biodistribution study was performed following an i.v.injection (given via the tailvein) of (131)I-anti-ProGRP(31-98) scFv. Healthy Kunming mice were sacrificed by cutting off carotid artery at designated time points in group, and 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 human small cell lung cancer were sampled for calculating %ID/g and tumor/non-tumor (T/NT) ratios.4. After injection of (131)I-anti-ProGRP(31-98) scFv, continuous images of the nude mice bearing SCLC were initially carried out at different time, and then tumor/base(T/B)ratios were calculated.Results:1. The expression rates of ProGRP in NCI-H446, Hela, H460, A549 cell lines were 95%, 77%, 53% and 4% respectively. The results of immunohistochemistry showed that positive granules (DAB staining) were distributed in intracytoplasm of small cell lung cancer, cervical cancer and large cell lung cancer, no obvious staining cells were found in lung adenocarcinoma.2. The labeling rate of (131)I-anti-ProGRP(31-98) scFv was 93.35±0.67%, and the radiochemical purity was 98.49±1.21%. After incubation at 37οC for 24 h, the radiochemical purity was 94.30±0.41%, and still maintained above 90% at 48 h. Meanwhile, after incubation with healthy human serum for 24 h, the radiochemical purity was 83.61±2.19%, and still maintained above 80% at 48 h. The immunobinding rates of NCI-H446 cell and A549 cell were 85.36% and 21.02%.3. The vivo distribution of (131)I-anti-ProGRP(31-98) scFv in the normal Kunming mice and nude mice bearing human small cell lung cancer showed that the metabolism of (131)I-anti-ProGRP(31-98) scFv mainly depended on liver and kidney with rapid elimination in blood and other main organs, the brain and muscle did not uptake much, and it was consistent with general scFv. The %ID/g of tumor was obviously higher than that of other organs at 12 h and arrived at top 5.38±0.92% at 24 h post-injection. The T/NT ratio increased gradually and arrived at top at 24 h.4. The tumor of nude mice bearing SCLC was visualized at 1 h after injection of (131)I-anti-ProGRP(31-98) scFv, The radioactivity in tumor site accumulated gradually and the tumor image became the clearest at 24 h post-injection.Conclusions:1. Labeling of (131)I-anti-ProGRP(31-98) scFv is easy, it not only has high labeling efficiency and radiochemical purity, but also has good stability in vitro and in vivo. (131)I-anti-ProGRP(31-98) scFv was metabolized mainly in kidney and liver, the blood and other major organs radioactivity of (131)I-anti-ProGRP(31-98) scFv decreased rapidly. 2. The biodistribution in SCLC tumor-bearing mice showed that (131)I-anti-ProGRP(31-98) scFv can be accumulated in tumor, the ratios of T/NT increased as time prolongs and arrived at top at 24 h.3. (131)I-anti-ProGRP(31-98) scFv has the potential to become radioimmunoimaging agent of SCLC. It deserves further study. |
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