A comparison of the monomeric [68Ga]NODAGA-NGR and dimeric [68Ga]NOTA-(NGR)2 as aminopeptidase N ligand for positron emission tomography imaging in tumor-bearing mice.

The aminopeptidase N (APN/CD13) is a key protein specifically expressed on activated endothelial cells and by various tumors, representing a promising target for molecular imaging and therapy of malignant diseases. It is known that the tripeptide NGR is a specific ligand for CD13, therefore radiolabeled NGR peptides are auspicious radiotracers for non-invasive imaging of CD13-positive tumors. From previous studies, it is known that the target affinity could be improved by molecules with multiple ligand sequences. Therefore, the aim of this study was to compare two NGR radioligands [68Ga]NODAGA-NGR (NGR monomer) and [68Ga]NOTA-(NGR)2 (NGR dimer), the latter with two NGR ligand motifs, in vitro and in vivo. CD13 expression was determined by FACS in the human tumor cells A549, SKHep-1, and MDA-MB-231, followed by the investigation of the cell uptake of [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2. For in vivo evaluation of [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2, microPET and biodistribution were carried out in A549- and SKHep-1-bearing mice. After the final examination, tumors were cryo-conserved, cut, and stained against CD13 and CD31. A549 and SKHep-1 cells were identified as CD13 positive, whereas no CD13 expression was detected in MDA-MB-231 cells. The cell uptake study showed relatively low accumulation of both the NGR monomer and dimer in all tumor cell lines examined, with consistently higher cell uptake observed for the dimer than for the monomer. In vivo, [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2 accumulated in the tumors, with slightly higher tumor-to-muscle ratio for the NGR dimer in A549 and SKHep-1. The tumor-to-liver ratio of the NGR dimer was diminished in comparison to the NGR monomer. This finding was confirmed by biodistribution, which revealed higher accumulation in liver and spleen for the NGR dimer. Immunohistochemical staining confirmed the CD13 expression in the tumors and tumor-associated vessels. In conclusion, both the [68Ga]NODAGA-NGR and the [68Ga]NOTA-(NGR)2 were found to be suitable for PET imaging of CD13-positive tumors. Despite slight differences in tumor-to-background ratio and organ accumulation, both radiotracers can be considered comparable.