Localization of idine-125-mIP-Des-Met14bmbesin (7-13)NH₂ in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer

The gastrin releasing peptide receptor (GRPr) has a high affinity for the 14 amino acid bombesin peptide. For this analysis, f-4-Tyr4bombesin was compared with [125l]-mlP-bombesin (a seven amino acid bombesin analog) for in vitro binding and internalization into tumor cells and for tumor localization in vivo. Also, a recombinant adenoviral vector (AdCMVGRPr) was used for gene transfer to induce the expression of GRPr in human ovarian cancer cells for binding and tumor localization with these radiolabeled peptides. Methods: [125l]-mlP-bombesin was synthesized and compared with [125l]-Tyr4-bombesin in internalization assays using BNR-11 cells (mouse fibroblast cells stably transfected with GRPr) over a 24-hr period. In vitro binding assays used BNR-11, and A427, HeLa and SKOV3.ip1 human cancer cells, which were either uninfected or infected with AdCMVGRPr. Biodistribution studies were performed in normal BALB/c mice and in athymic nude mice bearing orthotopic SKOVS.ipl ovarian cancer tumors. The SKOV3.ip1 tumors were induced to express GRPr with the AdCMVGRPr adenoviral vector. Results: Internalization assays showed that [125l]-Tyr4bombesin was rapidly internalized and catabolized at 37°C with =10% of the radioactivity remaining intracellularly at 4 hr, compared with =30% with f 25l]-mlP-bombesin. HeLa, A427 and SKOVS.ipl cells were all induced to express levels of GRPr that were higher than those seen with the positive control BNR-11 cells. Normal mice showed a lower level of radioactivity in both the blood and thyroid for [125l]-mlP-bombesin [0.26% ±0.10% injected dose per gram (ID/g) and0.24% ±0.05% ID]thanfor[125l]-Tyr4-bombesin(3.5% ±1.6% ID/g and 5.2% ±4.4% ID) at 4 hr postinjection. Mice bearing intraperitoneal (i.p.) SKOV3.ip1 tumors and given AdCMVGRPr i.p. 5 days after tumor cell inoculation followed by [125l]-mlP-bombesin i.p. at day 7 showed 16.5% ±4.8% ID/g in tumor compared with 5.9% ± 3.0% ID/g with f 25l]-Tyr4-bombesin at 4 hr postinjection. Tumor bearing mice given saline or a control adenovirus expressing the β-galactosidase (LacZ) gene showed significantly lower tumor uptake values of both bombesin peptides. Conclusion: Internalization assays showed that [125l]-mlP-bombesin has favorable characteristics compared with [125l]-Tyr4-bombesin with regards to cellular internalization and retention. The results demonstrate successful in vitro and in vivo transduction of human tumor cells with a recombinant adenoviral vector-expressing GRPr. Additionally, tumors transduced in vivo to express GRPr demonstrated significantly greater localization of [125l]-mlP-bombesin when compared with f-4ll-Tyr4bombesin.