Comparison of the binding and internalization properties of 12 DOTA-coupled and 111In-labelled CCK2/Gastrin receptor binding peptides: a collaborative project under COST Action BM0607

Specific overexpression of cholecystokinin 2 (CCK2)/Gastrin receptors has been demonstrated in several tumors of neuroendocrine origin. In some of these cancer types, such s medullary thyroid cancer (MTC), there are no effective systemic treatments for metastatic disease. Peptide Receptor Radionuclide Therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CK2R targeted radiopharmaceuticals have been described in recent years. As part of the EU COST action BM0607 we studied the in vitro and in vivo characteristics of 12 DOTA‐conjugated CK2R binding peptides. In the present study, we analyzed binding and internalization characteristics. Stability, biodistribution, and imaging studies have been performed in parallel. methods. Determination of IC50 was performed using autoradiography, with peptides complexed to Indium‐115 displacing 125I‐CCK on tissue sections from human tumors. Saturation binding and internalization experiments were performed using 111In‐labelled peptides (specific activity 2‐10 MBq/nmol). The rat AR42J cell line and the human A431‐CCK2R transfected cell line were utilized for in vitro experiments. Dissociation constants (Kd) and apparent number of binding sites (Bmax) were determined with the aid of graphical analysis software. Internalization was determined in receptor expressing cells by incubating with tracer amounts of peptide at 37 and 4°C for different times up to 120 min. Surface bound peptide was then stripped either by acid wash or incubation with 1μM cold peptide at 4°C. Results. All peptides showed high and similar receptor affinity with IC50 values ranging from 0.2 to 3.4 nM. Saturation experiments also showed high affinity with Kd values in the 10‐9 ‐10‐8 M range. Bmax values estimated in A431‐CCK2R cells ranged from 1 .0‐ 4.6 E6 per cell. Slight differences in the levels of non‐specific binding to cells were observed for the different peptides. All peptides showed high levels of internalization when incubated at 37°C. Conclusion. All DOTA‐conjugated peptides showed similar receptor binding and internalization properties. Issues pertaining to in vivo stability and biodistribution will be important in determining the most promising candidates for further development and clinical application. This study is part of COST Action BM0607‐WG5