Side-by-Side Comparison of Five Chelators for 89Zr-Labeling of Biomolecules: Investigation of Chemical/Radiochemical Properties and Complex Stability

Simple Summary The positron emitter 89Zr4+ is an important radionuclide for the preparation of radiolabeled antibodies, being applied in highly specific and sensitive positron emission tomography (PET) imaging of malignancies. The introduction of 89Zr4+ into biomolecules is performed using chelating agents, wrapping up the radiometal and preventing its release from the antibody by forming so-called complexes. Desferrioxamine B (DFO) is the clinical gold standard chelator for the preparation of 89Zr antibodies despite its known inability to stably encapsulate the radiometal, resulting in 89Zr release and associated challenges such as decreased image quality and radiation dose to healthy tissues. Therefore, several research groups have been working to develop new chelating agents able to stably encapsulate the 89Zr4+ ion. However, there are no data available directly comparing the stability of the formed 89Zr complexes of the most promising chelating agents developed so far. Here, we report on the comparison of five different chelators with high potential for stable complexation of 89Zr and determined two of them—DFO* and 3,4,3-(LI-1,2-HOPO)—to be highly interesting for the preparation of 89Zr-based radiolabeled agents and routine clinical application. Abstract In this work, five different chelating agents, namely DFO, CTH-36, DFO*, 3,4,3-(LI-1,2-HOPO) and DOTA-GA, were compared with regard to the relative kinetic inertness of their corresponding 89Zr complexes to evaluate their potential for in vivo application and stable 89Zr complexation. The chelators were identically functionalized with tetrazines, enabling a fully comparable, efficient, chemoselective and biorthogonal conjugation chemistry for the modification of any complementarily derivatized biomolecules of interest. A small model peptide of clinical relevance (TCO-c(RGDfK)) was derivatized via iEDDA click reaction with the developed chelating agents (TCO = trans-cyclooctene and iEDDA = inverse electron demand Diels-Alder). The bioconjugates were labeled with 89Zr4+, and their radiochemical properties (labeling conditions and efficiency), logD(7.4), as well as the relative kinetic inertness of the formed complexes, were compared. Furthermore, density functional theory (DFT) calculations were conducted to identify potential influences of chelator modification on complex formation and geometry. The results of the DFT studies showed—apart from the DOTA-GA derivative—no significant influence of chelator backbone functionalization or the conjugation of the chelator tetrazines by iEDDA. All tetrazines could be efficiently introduced into c(RGDfK), demonstrating the high suitability of the agents for efficient and chemoselective bioconjugation. The DFO-, CTH-36- and DFO*-modified c(RGDfK) peptides showed a high radiolabeling efficiency under mild reaction conditions and complete 89Zr incorporation within 1 h, yielding the 89Zr-labeled analogs as homogenous products. In contrast, 3,4,3-(LI-1,2-HOPO)-c(RGDfK) required considerably prolonged reaction times of 5 h for complete radiometal incorporation and yielded several different 89Zr-labeled species. The labeling of the DOTA-GA-modified peptide was not successful at all. Compared to [89Zr]Zr-DFO-, [89Zr]Zr-CTH-36- and [89Zr]Zr-DFO*-c(RGDfK), the corresponding [89Zr]Zr-3,4,3-(LI-1,2-HOPO) peptide showed a strongly increased lipophilicity. Finally, the relative stability of the 89Zr complexes against the EDTA challenge was investigated. The [89Zr]Zr-DFO complex showed—as expected—a low kinetic inertness. Unexpectedly, also, the [89Zr]Zr-CTH-36 complex demonstrated a high susceptibility against the challenge, limiting the usefulness of CTH-36 for stable 89Zr complexation. Only the [89Zr]Zr-DFO* and the [89Zr]Zr-3,4,3-(LI-1,2-HOPO) complexes demonstrated a high inertness, qualifying them for further comparative in vivo investigation to determine the most appropriate alternative to DFO for clinical application.