Heterobivalent dual-target probe for targeting GRP and Y1 receptors on tumor cells.

Receptor targeting ligands for imaging and/or therapy of cancer are limited by heterogeneity of receptor expression by tumor cells, both inter-patient and intra-patient. It is often more important for imaging agents to identify local and distant spread of disease than it is to identify a specific receptor presence. Two natural hormone peptide receptors, GRPR and Y1, are specifically interesting because expression of GRPR, Y1 or both is up-regulated in most breast cancers. We describe here the design and development of a new heterobivalent peptide ligand, truncated bombesin (t-BBN)/BVD15-DO3A, for dual-targeting of GRPR and Y1, and validation of its dual binding capability. Such a probe should be useful in imaging cells, tissues and tumors that are GRPR and/or Y1 positive and should target radioisotopes, for example, (68)Ga and/or (177)Lu, to more tumors cells than single GRPR or Y1 targeted probes. A GRP targeting ligand, J-G-Abz4-QWAVGHLM-NH(2) (J-G-Abz4-t-BBN), and an Y1 targeting ligand, INP-K[ε-J-(α-DO3A-ε-DGa)-K]-YRLRY-NH(2)([ε-J-(α-DO3A-ε-DGa)-K]-BVD-15), were synthesized and coupled to produce the heterobivalent ligand, t-BBN/BVD15-DO3A. Competitive displacement binding assays using t-BBN/BVD15-DO3A against (125)I-Tyr(4)-BBN yielded an IC(50) value of 18 ± 0.7 nM for GRPR in T-47D cells, a human breast cancer cell line. A similar assay using t-BBN/BVD15-DO3A against porcine (125)I-NPY showed IC(50) values of 80 ± 11 nM for Y1 receptor in MCF7 cells, another human breast cancer cell line. In conclusion, it is possible to construct a single DO3A chelate containing probe that can target both GRPR and Y1 on human tumor cells.

Identification of ideal peptides for heterovalent ligands.

Recent use of hetero-dimerization to improve the affinity of peptide ligands has made peptides an attractive alternative to small molecules and proteins. Using this strategy, we have developed peptides with affinities comparable to antibodies and specificities often better than small molecules or antibodies. These peptides can be used as a delivery vehicle for drugs or diagnostics, especially in the case of tumor targeting cytotoxic drugs or targeted diagnostics. We describe here an assay to identify an ideal pair of peptides suitable for heterovalent ligands.

A high-affinity near-infrared fluorescent probe to target bombesin receptors.

PURPOSE: This study aimed to create new optical surgical navigation NIRF probes for prostate and breast cancers. PROCEDURES: IR800-linker-QWAVGHLM-NH2 with linker = GSG, GGG, and G-Abz4 were synthesized and characterized. IC50 for bombesin receptors (BBN-R) in PC-3 prostate and T47D breast cancer cells, fluorescence microscopy in PC-3 cells, and NIRF imaging in mice PC-3 tumor xenografts were studied. RESULTS: GGG, GSG, and G-Abz4 derivatives had IC50 (nM) for BBN-R+ PC-3 cells = 187 ± 31, 56 ± 5, and 2.6 ± 0.2 and T47D cells = 383 ± 1, 57.4 ± 1.2, and 3.1 ± 1.1, respectively. By microscopy the Abz4 derivative showed the highest uptake, was competed with by BBN, and had little to no binding to BBN-R- cells. In NIRF imaging the G-Abz4 probe was brighter than GGG probe in BBN-R+ tissues in vivo and tissues, tumors, and tumor slices ex vivo. Uptake could be partially blocked in BBN-R+ pancreas but not visibly in tumor. CONCLUSIONS: Linker choice can dominate peptidic BBN-R binding. The G-Abz4 linker yields a higher affinity and specific BBN-R binder in this series of molecules.