Influence of d-glutamine and d-glutamic acid sequences in optical peptide probes targeted against the cholecystokinin-2/gastrin-receptor on binding affinity, specificity and pharmacokinetic properties

Image-based diagnosis of tumours can be advanced and improved by targeted strategies addressing malignant molecular structures. A promising molecular target is the cholecystokinin-2-receptor (CCK2R) which can be targeted by high-affinity peptides called minigastrins. Here we present how the imaging properties of minigastrins tagged with near-infrared fluorescence (NIRF) dyes can be modulated by the introduction of different spacer sequences. We identify interactions of different probe variants with regard to target affinity, specificity and pharmacokinetic properties to optimize early detection of CCK2R-expressing tumours under clinical conditions.

The properties of optical minigastrin probes can be specifically modified by the introduction of spacer sequences. A spacer of six hydrophilic amino acids increases affinity. A mix of d-glutamic and d-glutamine acids increased target-to-background contrast. Multimerization could not increase affinity but supposedly lowered stability. The probe QE is a promising candidate for clinical evaluation in terms of diagnosis of CCK2R-expressing tumours.

Two minigastrin probes with the same near-infrared hemicyanine fluorescence dye (DY-754) for signalling and the same CCK2R-binding peptide A-Y-G-W-M/Nle-N-F-amide but different spacers were designed as follows: 'QE' with three alternating d-glutamines and d-glutamic acids and 'bivQ' with two minigastrins, each preceded by three d-glutamines. They were tested for affinity and specificity in vitro on CCK2R-expressing and CCK2R-non-expressing cells. In vivo imaging was conducted with subcutaneous tumour-bearing nude mice after i.v. probe injection (54 to 108 nmol/kg) and under competitive conditions with non-fluorescent minigastrin (n = 5/group). We also assessed probe biodistribution as well as NIRF distribution in tumour sections.

Both probes showed high affinity and specificity to CCK2R-expressing cells in vitro. In vivo tumour-to-background contrasts (tumour/background ratios (TBRs) of around 6) enabled identification of CCK2R-expressing tumours by both probes with low accumulation in CCK2R-negative tumours (TBR of around 2). Specificity of the in vivo accumulation, revealed by competition, was higher for QE. Besides renal retention, probe uptake into organs was very low.