DOTA-MGS5, a New Cholecystokinin-2 Receptor-Targeting Peptide Analog with an Optimized Targeting Profile for Theranostic Use

Abstract

Molecular imaging and targeted radiotherapy with radiolabeled cholecystokinin-2 receptor (CCK2R) targeting peptide probes holds high promise to improve the clinical management of patients with metastatic medullary thyroid carcinoma and other CCK2Rexpressing malignancies. Low stability and suboptimal targeting of currently available radiolabeled peptide analogs has prompted us to seek new stabilization strategies. In this study, we present a new minigastrin analog with site-specific C-terminal modifications showing a highly optimized targeting profile. Methods: DOTA-D-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH 2 (DOTA-MGS5) radiolabeled with 111 In, 68 Ga, and 177 Lu was evaluated in extensive in vitro stability studies. For 177 Lu-DOTA-MGS5, additional metabolic studies were performed on BALB/c mice. Receptor affinity and cell uptake were studied in A431 human epidermoid carcinoma cells transfected with human CCK2R (A431-CCK2R), as well as the same cell line transfected with the empty vector (A431-mock). A431-CCK2R/A431-mock xenografted athymic BALB/c nude mice were used for biodistribution studies and small-animal SPECT/CT. Results: DOTA-MGS5 radiolabeled with 111 In and 177 Lu showed a highly increased stability against enzymatic degradation in different media up to 24 h of incubation. Similar results were observed for 68 Ga-DOTA-MGS5 incubated up to 4 h. In the blood of mice injected with 177 Lu-DOTA-MGS5, at least 70% intact radiopeptide was detected up to 1 h after injection. The unlabeled peptide and the complexes with the natural isotopes showed retained receptor affinity, and the radiopeptides showed unexpectedly high cell uptake in A431-CCK2R cells (.60% at 4 h). Regardless of the radiometal used for labeling, impressively high uptake in A431-CCK2R xenografts was found (∼20% injected activity/g 1-4 h after injection), whereas the uptake in A431-mock xenografts was negligible. Low background activity and favorable tumor-to-kidney ratios (4-6) allowed for high image contrast in small-animal SPECT/CT. Conclusion: The excellent targeting properties of DOTA-MGS5 support future clinical studies evaluating the diagnostic and therapeutic potential in patients with progressive or metastatic medullary thyroid carcinoma, as well as other advanced-stage CCK2R-expressing malignancies.

Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177

“…The successful clinical use of radiolabeled somatostatin analogs for targeting somatostatin receptors in patients with neuroendocrine tumors could not yet be translated to radiolabeled minigastrin analogs targeting CCK2R. In our recent studies, we could develop a new stabilization strategy leading to increased stability of the linear peptide sequence against enzymatic degradation in vivo and improving the targeting properties [ 28 , 29 ]. In this study, we have further explored our stabilization strategy by introducing an additional modification in the N -terminal region of the peptide backbone in our lead compound DOTA-MGS5.…”

“…Only at the later time point of 2 h after incubation, an almost complete breakdown occurred in kidney homogenate, whereas in liver homogenate >50% intact radiopeptide was still present. The resistance against enzymatic degradation in rat liver homogenate was also improved when compared to DOTA-MGS5 labeled with different radiometals for which less than 30% intact radiopeptide was found for the same time point, suggesting a potential additive stabilizing effect of the insertion of Pro in position 2 [ 28 ].…”

“…In our recent studies we could introduce new modifications within the C-terminal sequence Trp-Met-Asp-Phe-NH 2 , known to be essential for CCK2R binding [ 27 , 28 , 29 ]. Most favorable properties were found for the new MG analog with the sequence DOTA-DGlu-Ala-Tyr-Gly-Trp-( N -Me)Nle-Asp-1Nal-NH 2 (DOTA-MGS5), in which methionine is replaced by N -methylated norleucine (( N -Me)Nle) and phenylalanine by 1-naphtylalanine (1Nal) [ 28 ]. Besides leading to highly improved stability in vivo, the introduced modifications also led to an enhanced receptor-specific cell uptake, as well as to a highly increased tumor uptake, when radiolabeled with different radiometals.…”

“…In nude BALB/c mice bearing CCK2R-expressing tumor xenografts, a tumor uptake of more than 20% of the injected activity per gram (IA/g) was observed. This corresponds to a three-fold improvement compared to PP-F11 and PP-F11N (~6.7% and 6.9% IA/g) [ 14 , 28 ]. Nevertheless, some degradation products were still detected by radio-HPLC analysis of blood obtained from mice injected with 177 Lu-labeled DOTA-MGS5 [ 28 ].…”

Cholecystokinin 2 Receptor Agonist 177Lu-PP-F11N for Radionuclide Therapy of Medullary Thyroid Carcinoma: Results of the Lumed Phase 0a Study

“…Another approach to reduce nephrotoxicity is to modify the compound itself: Amino acid chains with more than 5 glutamic acids in their sequence play an important role in the kidney reuptake mechanism and may reduce kidney uptake (10,11). Such an approach was implemented by several groups and resulted in the development of a library of improved radiolabeled minigastrin analogues (12)(13)(14)(15)(16)(17). Some of these new compounds show higher tumor uptake, higher tumor-to-kidney uptake ratios and higher serum stability, compared to the previously developed minigastrin analogues.…”