Comparison of the binding and internalization propertiesof 12 DOTA-coupled and 111In-labelled CCK2/gastrinreceptor binding peptides: a collaborative projectunder COST Action BM0607

Purpose Specific overexpression of cholecystokinin 2 (CCK2)/gastrin receptors has been demonstrated in several tumours of neuroendocrine origin. In some of these cancer types, such as medullary thyroid cancer (MTC), a sensitive diagnostic modality is still unavailable and therapeutic options for inoperable lesions are needed. Peptide receptor radionuclide therapy (PRRT) may be a viable therapeutic strategy in the management of these patients. Several CCK2R-targeted radiopharmaceuticals have been described in recent years. As part of the European Union COSTAction BM0607 we studied the in vitro and in vivo characteristics of 12 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R binding peptides. In the presentstudy, we analysed binding and internalization characteristics. Stability, biodistribution and imaging studies have been performed in parallel by other centres involved in the project. Methods Determination of IC50 values was performed using autoradiography, with DOTA-peptides displacing 125I-CCK from receptors on tissue sections from human tumours. Saturation binding and internalization experiments were performed using 111In-labelled peptides. 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. 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 subsequent incubation with 1 μM unlabelled peptide at 4°C. Results All peptides showed high 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 0.6 to 2.2×106 per cell. All peptides showed high levels of internalization when incubated at 37°C. Conclusion All DOTA-conjugated peptides showed high receptor binding and internalization properties and appear suitable for further characterization, as described in other articles of this issue.