Panoply™ Human CCKBR Over-expressing Stable Cell Line

Targeting tumors that express the cholecystokinin-2 receptor (CCK2R, also known as CCKBR) using radiolabeled minigastrin (MG) analogs is often hindered by the rapid enzymatic degradation of these linear peptides in vivo. Here, researchers synthesized a novel MG analog-DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2-featuring site-specific substitutions at amino acid positions 2, 6, and 8; concurrently, they also synthesized various potential metabolites of this molecule. Subsequently, they evaluated the interactions between this peptide and selected metabolites with the receptor in CCK2R-expressing cell lines. The researchers assessed the enzymatic stability of the 177Lu-labeled peptide analog not only in various in vitro media but also in vivo in BALB/c mice (within one hour post-injection), identifying the resulting metabolites via radio-HPLC analysis. The results demonstrated a significantly enhanced in vivo stability for this novel radiolabeled peptide: one hour after injection into BALB/c mice, over 56% of the radioactive peptide in the bloodstream remained in its intact form. The study confirmed that only the intact peptide molecule possesses high affinity and efficient cellular uptake capabilities; enzymatic cleavage occurring within the receptor-specific C-terminal amino acid sequence results in a complete loss of both affinity and cellular uptake capacity. Favorable biodistribution characteristics were observed in BALB/c mice, characterized by low background radioactivity, predominant renal excretion, and sustained uptake within CCK2R-expressing tissues. This novel, stability-optimized MG analog demonstrates immense potential for applications in both diagnostic and therapeutic fields.

For [¹⁷⁷Lu]Lu-1 co-incubated with CCK2R-overexpressing A431 cells, significant internalization was observed, with uptake levels reaching 44.4 ± 2.7% after 1 hour of incubation (Figure 1). As expected, the ¹⁷⁷Lu-labeled metabolites exhibited no internalization into the CCK2R-overexpressing A431 cells. Furthermore, after 2 hours of incubation, the exceptionally high receptor-specific uptake capability of [¹⁷⁷Lu]Lu-1 was further substantiated, as its uptake levels increased to 66.6 ± 0.3%; in contrast, the ¹⁷⁷Lu-labeled metabolites continued to show no internalization. The specificity of this cellular uptake was confirmed through parallel incubation experiments using A431-mock cells, wherein the cellular uptake of [¹⁷⁷Lu]Lu-1 remained below 0.2% and 0.4% at the 1-hour and 2-hour time points, respectively.

Figure 1. Cell uptake of (a) [¹⁷⁷Lu]Lu-1 in A431-CCK2R and A431-mock cells and (b) ¹⁷⁷Lu-labeled M1-M4 in A431-CCK2R cells, after 1 h and 2 h incubation. (Hörmann A A, et al., 2020)