Enhancing Tumor Uptake for ¹⁷⁷Lu-Labeled MC1R Targeting Radioligands by Combining N-Methylations with an Albumin Binder

Objectives: Radiotracers derived from Melanotan II (MTII), a melanocortin-1 receptor (MC1R) targeting peptide, have been proposed for melanoma imaging and radionuclide therapy. We previously reported CCZ01048, an MTII derivative which showed excellent imaging properties with ⁶⁸Ga at early time points. However, when radiolabeled with ¹⁷⁷Lu, moderate tumor uptake was observed at longer time points, due to the sub-optimal in vivo stability. The aim of this study was to evaluate a panel of novel N-methylated MTIIderivatives for their binding affinity and stability, and characterize the top candidate, conjugated with an albumin binder, for tumor uptake in a preclinical model of melanoma.

Methods: The peptides were prepared by Fmoc solid phase synthesis. N-methylation was performed under Mitsunobu conditions. The DOTA-containing peptides were synthesized with one to four N-methylations on the His, Arg, Trp and Lys residues of CCZ01048. For the derivatives conjugated with an albumin binder, Fmoc-Lys(Mtt)-OH, Fmoc-Glu(OtBu)-OH and 4-(p-iodophenyl)butyric acid were used before coupling DOTA chelator. Receptor binding affinity was measured using in vitro competition binding assays on B16F10 cells. The peptides were radiolabeled with ⁶⁸Ga, and their in vivo stability was performed via radio-HPLC using urine samples at 1 h post-injection (p.i.) from C57BL/6J mice. The top candidate was conjugated with the albumin binder and radiolabeled with ¹⁷⁷Lu. SPECT/CT imaging and biodistribution studies were performed using ¹⁷⁷Lu-labeled peptides in C57BL/6J mice bearing B16F10 tumors.

Results: A panel of six novel MTII derivatives were synthesized with one to four N-methylations, which had a positive impact on their stability, with average in vivo urine stability ranging from 41.4% to ≥ 98% (n ≥ 3), compared to [⁶⁸Ga]Ga-CCZ01048 at 34%. The N-methylations had an unfavorable impact on binding affinity, with average inhibition constants ranging from 1.5 to 35.9 nM (n ≥ 3), compared to 0.31 nM for ^natGa-CCZ01048. The best candidate was CCZ01106, which had improved stability (82.5%) while maintaining excellent binding affinity (1.5 nM). An albumin binder was conjugated to CCZ01106 to afford CCZ01118. ¹⁷⁷Lu-labeled CCZ01118 was evaluated in B16F10 tumor-bearing C57BL/6J mice at 1, 4, 24 and 120 h p.i., and the tumor uptake was 24.44 ± 1.76, 29.70 ± 5.23, 18.35 ± 1.35, and 2.63 ± 0.21 percent injected dose per gram of tissue (%ID/g), respectively (n = 5). This was a significant improvement over ¹⁷⁷Lu-labeled CCZ01048 with respective tumor uptake values of 16.28 ± 3.75, 16.71 ± 3.86, 4.39 ± 1.36, and 0.41 ± 0.21%ID/g at the same time points[FB1] . SPECT imaging also showed consistent findings as much higher radioactivity accumulation was visualized in tumors. Conclusion: With the addition of N-methylations and an albumin binder, we designed CCZ01118, which showed greatly enhanced tumor uptake with ¹⁷⁷Lu in SPECT imaging and biodistribution studies. CCZ01118 is therefore a promising compound for radionuclide therapy of melanoma.