Development and in vitro evaluation of new bifunctional Zr-chelators based on the 6-amino-1,4-diazepane scaffold for immuno-PET applications.

Introduction: Combination of hydroxamate bearing side chains with the 6-amino-1,4-diazepane scaffold provides a promising strategy for fast and stable Zr-labeling of antibodies. Following this approach, we hereby present the development, labeling kinetics and in vitro complex stability of three resulting bifunctional chelator derivatives both stand-alone and coupled to a model protein in comparison to different linear deferoxamine (DFO) derivatives.

Methods: The novel Zr-chelator HyADA was prepared via amide-coupling of separately synthesized 6-amino-1,4-diazepane-6-pentanoic acid and hydroxamate-containing side chains. Two further bifunctional derivatives were synthesized by extending the resulting system with either a squaramide- or p-isothiocyanatophenyl moiety for simplified binding to proteins. After coupling to a model antibody and purification, the resulting immunoconjugates as well as the unbound chelator derivatives were Zr-labeled at room temperature (RT) and neutral pH. For comparison, different DFO derivatives were analogously coupled, purified and radiolabeled. In vitro complex stability of the resulting radioconjugates was investigated in phosphate buffered saline (PBS) and human serum at 37 °C over a period of 7 days.

Results: Zr-labeling of the novel unbound HyADA derivatives indicated rapid complexation kinetics resulting in high radiochemical conversions (RCC) of 84-94% after 90 min. Similar or even faster radiolabeling with slightly increased maximum yields was obtained using the DFO-analogues. Initially, [Zr]Zr-DFO*-p-Ph-NCS showed a delayed formation, nevertheless reaching almost quantitative complexation. Radiolabeling of the corresponding immunoconjugates HyADA-SA-mAb and HyADA-p-Ph-NCS-mAb resulted in 82.0 ± 1.1 and 89.2 ± 0.7% RCC, respectively after 90 min representing high but slightly lower labeling efficiency compared to the DFO- and DFO*-functionalized analogues. All examined radioimmunoconjugates showed very high in vitro complex stability both in human serum and PBS, providing no significant release of the radiometal. In the case of unbound chelators, however, the p-Ph-NCS-functionalized derivatives indicated considerable instability in human serum already after 1 h.

Conclusion: The novel chelator derivatives based on hydroxamate-functionalized 6-amino-1,4-diazepane revealed fast and high yielding Zr-labeling kinetics as well as high in vitro complex stability both stand-alone and coupled to an antibody. Therefore, HyADA represents a promising tool for radiolabeling of biomolecules such as antibodies at mild conditions for immuno-PET applications.