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Proteins adopt unique folded secondary and tertiary structures that are responsible for their remarkable biological properties. This structural complexity is key in designing efficacious peptides that can mimic the three-dimensional structure needed for biological function. In this study, we employ different chemical strategies to induce and stabilize a β-hairpin fold of peptides targeting cholecystokinin-2 receptors for theranostic application (combination of a targeted therapeutic and a diagnostic companion). The newly developed peptides exhibited enhanced folding capacity as demonstrated by circular dichroism (CD) spectroscopy, ion-mobility spectrometry-mass spectrometry, and two-dimensional (2D) NMR experiments. Enhanced folding characteristics of the peptides led to increased biological potency, affording four optimal Ga-68 labeled radiotracers ([ 68 Ga]Ga- 4b , [ 68 Ga]Ga- 11b-13b ) targeting CCK-2R. In particular, [ 68 Ga]Ga- 12b and [ 68 Ga]Ga- 13b presented improved metabolic stability, enhanced cell internalization, and up to 6 fold increase in tumor uptake. These peptides hold great promise as next-generation theranostic radiopharmaceuticals.

Minigastrin (MG) analogues, known for their high potential to target cholecystokinin-2 receptor (CCK2R) expressing tumors, have limited clinical applicability due to low enzymatic stability. By introducing site-specific substitutions within the C-terminal receptor-binding sequence, reduced metabolization and improved tumor targeting can be achieved. In this work, the influence of additional modification within the N-terminal sequence has been explored. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R ligands with proline substitution at different positions were synthesized. Substitution did not affect CCK2R affinity, and the conjugates labeled with indium-111 and lutetium-177 showed a high enzymatic stability in different incubation media as well as in vivo (57-79% intact radiopeptide in blood of BALB/c mice at 1 h p.i.) combined with enhanced tumor uptake (29-46% IA/g at 4 h in xenografted BALB/c nude mice). The inclusion of Pro contributes significantly to the development of CCK2R ligands with optimal targeting properties for application in targeted radiotherapy.

Two cyclized minigastrin analogues for gastrin receptor scintigraphy were synthesized and derivatized with HYNIC at the N-terminus for labeling with 99mTc. Radiolabeling efficiency, stability, cell internalization, and receptor binding on CCK-2 receptor expressing AR42J cells were studied and the biodistribution evaluated in tumor bearing nude mice, including NanoSPECT/CT imaging. Metabolites in urine, liver, and kidneys were analyzed by radio-HPLC. Radiolabeled cyclic MG showed high stability in vitro and receptor mediated uptake in AR42J cells. In the animal tumor model, fast renal clearance and low nonspecific uptake in most organs were observed. A tumor uptake >3% was calculated ex vivo 1 h p.i. for both 99mTc-EDDA-HYNIC-cyclo-MG1 and 99mTc-EDDA-HYNIC-cyclo-MG2. In an imaging study with 99mTc-EDDA-HYNIC-cyclo-MG1, the tumor was clearly visualized. The metabolite analysis indicated rapid enzymatic degradation in vivo.