Chelator Conjugation Chemistry Matters: Squaramide Linker Enhances Tumor Contrast in ⁶⁸ Ga-labeled Anti-CEA Nanobody Tracer

Complexation of radiometals by chelators allows for convenient radiolabeling of molecules of interest for the preparation of radiopharmaceuticals. In the chelator family, triazacyclononane (TACN)-based macrocycles have been used ubiquitously over the last 40 years, and many bifunctional derivatives have been developed. Despite this diversity, researchers commonly make their chelator selection based on practical factors like (commercial) availability and compatibility with the desired radionuclide, acknowledging that these considerations often outweigh achieving ideal in vivo pharmacokinetics. In this study, we generated and preclinically evaluated four gallium-68-labeled anti-CEA Nanobody-based tracers carrying different TACN-derivatives: p-NCS-Bn-NOTA, p-NCS-Bn-NODAGA, NODAGA-Sq, and NODAGA-NHS. Since the macrocyclic chelator is highly similar in these derivatives─they all present a NOTA ((1,4,7-triazacyclononane-1,4,7-triacetic acid) scaffold─the effect of the bioconjugation handle on the pharmacokinetic properties could be examined in a side-by-side comparison. The four PET tracers were prepared and could easily be labeled with gallium-68. Then, their stability, target affinity, and hydrophilic character were determined in vitro. Next, their in vivo biodistribution was evaluated using PET/CT imaging in a subcutaneous tumor mouse model. Despite their high structural similarity, notable differences in pharmacokinetics were observed in vivo, more specifically in the tumor and liver signal. Tracer [68Ga]Ga-NODAGA-Sq-NbCEA was found to be the best performer in our study, with a tumor signal 1.6-fold higher than [68Ga]Ga-NODAGA-NbCEA and a tumor-to-liver ratio 1.5-fold and 1.7-fold higher than that of [68Ga]Ga-NOTA-Bn-NCS-NbCEA and [68Ga]Ga-NODAGA-Bn-NCS-NbCEA, respectively. By comparing this tracer to the negative control [68Ga]Ga-NODAGA-Sq-R3b23, specific tumor targeting was demonstrated.