LOCALIZATION OF IODINE-125-MIP-DES-MET(14) BOMBESIN (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, [I-125]-Tyr(4)- bombesin was compared with [I-125]-mIP-bombesin (a seven amino acid bo mbesin analog) for in vitro binding and internalization into tumor cel ls 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 localizat ion with these radiolabeled peptides, Methods: [I-125]-mIP-bombesin wa s synthesized and compared with [I-125]-Tyr(4)-bombesin in internaliza tion assays using BNR-11 cells (mouse fibroblast cells stably transfec ted with GRPr) over a 24-hr period. In vitro binding assays used BNR-1 1, and A427, HeLa and SKOV3.ip1 human cancer cells, which were either uninfected or infected with AdCMVGRPr. Biodistribution studies were pe rformed in normal BALB/c mice and in athymic nude mice bearing orthoto pic SKOV3.ip1 ovarian cancer tumors. The SKOV3.ip1 tumors were induced to express GRPr with the AdCMVGRPr adenoviral vector. Results: Intern alization assays showed that [I-125]-Tyr(4)-bombesin was rapidly inter nalized and catabolized at 37 degrees C with approximate to 10% of the radioactivity remaining intracellularly at 4 hr, compared with approx imate to 30% with [I-125]-mIP-bombesin. HeLa, A427 and SKOV3.ip1 cells were all induced to express levels of GRPr that were higher than thos e seen with the positive control BNR-11 cells. Normal mice showed a lo wer level of radioactivity in both the blood and thyroid for [I-125]-m IP-bombesin [0.26% +/- 0.10% injected dose per gram (ID/g) and 0.24% /- 0.05% ID] than for [I-125]-Tyr(4)-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 ce ll inoculation followed by [I-125]-mIP-bombesin i.p. at day 7 showed 1 6.5% +/- 4.8% ID/g in tumor compared with 5.9% +/- 3.0% ID/g with [I-1 25]-Tyr(4)-bombesin at 4 hr postinjection. Tumor bearing mice given sa line or a control adenovirus expressing the beta-galactosidase (LacZ) gene showed significantly lower tumor uptake values of both bombesin p eptides. Conclusion: Internalization assays showed that [I-125]-mIP-bo mbesin has favorable characteristics compared with [I-125]-Tyr(4)-bomb esin with regards to cellular internalization and retention. The resul ts demonstrate successful in vitro and in vivo transduction of human t umor cells with a recombinant adenoviral vector-expressing GRPr. Addit ionally, tumors transduced in vivo to express GRPr demonstrated signif icantly greater localization of [I-125]-mIP-bombesin when compared wit h [I-125]-Tyr(4)-bombesin.