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

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.

Toward Optimized 89Zr-Immuno-PET: Side-by-Side Comparison of [89Zr]Zr-DFO-, [89Zr]Zr-3,4,3-(LI-1,2-HOPO)- and [89Zr]Zr-DFO*-Cetuximab for Tumor Imaging: Which Chelator Is the Most Suitable?

“…For the subsequent modification of cetuximab-TCO 2 with DFO, DFO* and 3,4,3-(LI-1,2-HOPO) by iEDDA, being highly efficient at ambient temperature and physiological pH [ 22 , 23 ], tetrazine-modified analogs of the chelators ( 4 – 6 , Figure 3 A) were prepared as described before [ 9 ] and applied in the following cetuximab bioconjugation reactions. So far, these chelator tetrazines were only used for conjugation to one small peptide, the subsequent 89 Zr-radiolabeling of the conjugates and comparative testing of the relative kinetic inertnesses of the complexes against challenge, 89 Zr 4+ liberation and transchelation.…”

“…All other chemicals and solvents were obtained from Carl Roth (Karlsruhe, Germany), Sigma Aldrich (Taufkirchen, Germany), TCI Deutschland GmbH (Eschborn, Germany) and Thermo Fisher GmbH (Kandel, Germany). The chelator tetrazines 4 – 6 were synthesized as described before [ 9 ]. [ 89 Zr]Zr-oxalate solution in 1.0 M oxalic acid was purchased from PerkinElmer (NEZ308000MC, Rodgau, Germany).…”

“…Some recent complex challenge experiments and in vivo PET imaging studies on 89 Zr-labeled antibodies in tumor-bearing mice compared relative stabilities of 89 Zr-complexes with different chelators have been developed over the last years. These studies revealed some of these new chelating agents to be of high interest for further testing, and potential clinical translation, whereas several others could be excluded due to a relatively lower or similarly low kinetic inertness of their 89 Zr-complexes compared to [ 89 Zr]Zr-DFO [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ].…”

“…Especially [ 89 Zr]Zr-DFO* ( Figure 1 B) demonstrated a superior kinetic inertness compared to [ 89 Zr]Zr-DFO and many other newly developed chelators [ 9 , 11 , 12 , 14 ]. Thus, DFO* seems to be the superior chelating agent for stable 89 Zr-introduction into antibodies and 89 Zr-based immuno-PET imaging.…”

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz

“…Significant efforts have been dedicated to overcome these obstacles through the development of engineered antibody variants with faster pharmacokinetics and pretargeted approaches for radiolabeling the antibodies in vivo after their administration and peak accumulation to the target site . Recently, in vivo click reactions based on the bio-orthogonal inverse electron demand Diels–Alder ligation (IEDDA) between dienophile-functionalized antibodies and small-molecule radioligands based on tetrazine structures have obtained high interest. − Pretargeted immuno-PET imaging would bring significant advantages: reducing the radioactive exposure of the patients and allowing the use of the short half-live radionuclides for imaging purposes (Figure ). , The preclinical proof of concept of the two-step pretargeted immuno-PET imaging and radioimmunotherapy with IEDDA have been successfully achieved by several research groups. ,− …”