In vitro and in vivo evaluation of melanin-binding decapeptide 4B4 radiolabeled with 177Lu, 166Ho, and 153Sm radiolanthanides for the purpose of targeted radionuclide therapy of melanoma

Abstract

Melanoma is a malignancy with increasing incidence. Although primary tumors that are localized to the skin can be successfully treated by surgical removal, there is no satisfactory treatment for metastatic melanoma, a condition that has currently an estimated 5-year survival of just 6%. During the last decade, β- or α-emitter-radiolabeled peptides that bind to different receptors on a variety of tumors have been investigated as potential therapeutic agents in both the preclinical and clinical settings with encouraging results. A recent study demonstrated that 188-Rhenium ((188)Re)-labeled, via HYNIC ligand, fungal melanin-binding decapeptide 4B4 was effective against experimental MNT1 human melanoma and was safe to normal melanized tissues. The availability of radiolanthanides with diverse nuclear emission schemes and half-lives provides an opportunity to expand the repertoire of peptides for radionuclide therapy of melanoma. The melanin-binding decapeptide 4B4 was radiolabeled with (177)Lu, (166)Ho, and (153)Sm via a DO3A chelate. The stability studies of Ln*-DO3A-4B4 in phosphate-buffered saline, serum, and a hydroxyapatite assay demonstrated that (177)Lu-labeled peptide was more stable than (166)Ho- and (153)Sm-labeled peptides, most likely because of the smallest ionic radius of the former allowing for better complexation with DO3A. Binding of Ln*-DO3A-4B4 to the lysed highly melanized MNT1 melanoma cells demonstrated the specificity of peptides binding to melanin. In vivo biodistribution data for (177)Lu-DO3A-4B4 given by intraperitoneal administration to lightly pigmented human metastatic A2058 melanoma-bearing mice demonstrated very high uptake in the kidneys and low tumor uptake. Intravenous administration did not improve the tumor uptake. The plausible explanation of low tumor uptake of (177)Lu-DO3A-4B4 could be its decreased ability to bind to melanin during in vitro binding studies in comparison with (188)Re-HYNIC-4B4, exacerbated by the very fast clearance from the blood and the kidneys "sink" effect.

FIG. 1.

The peptide constructs employed in this study. DO3A was coupled to the melanin-binding 4B4 peptide and the irrelevant PA1 control using standard solid-phase peptide synthesis techniques. Both peptide constructs were radiolabeled with ^natLu, ¹⁷⁷Lu, ¹⁵³Sm, and ¹⁶⁶Ho. PEG, 8-amino-3,6-dioxaoctanoic acid.

FIG. 2.

HPLC trace of ^natLnDO3A-4B4 coinjected with ¹⁷⁷Lu-DO3A-4B4. The coelution of the ¹⁷⁷Lu and ^natLn peptide complexes demonstrates that the ¹⁷⁷Lu complex possesses the same chemical structure of the characterized ^natLnDO3A-4B4.

FIG. 3.

Optimization of the radiochemical yield for labeling reactions of DO3A-4B4 with ¹⁷⁷Lu. The radiochemical yield was determined by counts under the radiopeak of ¹⁷⁷Lu-DO3A-4B4 divided by the counts in the total spectra.

FIG. 4.

Cell binding assay of *Ln-DO3A-4B4 in highly pigmented MNT1 cells. Cells were osmotically lysed prior to addition of the radiolabeled complex. For the blocking experiment, 50 μg/mL DO3A-4B4 peptide was used before addition of radiolabeled peptides. Data are given in Supplementary Table S3.