Radiolabeling and validation of a new acyclic chelator bearing N-hydroxy-N-methyl succinamide pendant arms for Zirconium-89 radionuclide, superior performance in comparison with DFO

Objectives: Long half-life Zirconium-89 (⁸⁹Zr) matches very well with the biological half-life of monoclonal antibody (mAb) for optimal molecular imaging. To date, radiolabeling of mAbs with ⁸⁹Zr is solely achieved through desferrioxamine (DFO), a siderophore with 6 coordination sites but concerns remain regarding the in vivo stability of this complex. Recently, our group has developed a new chelator with four N-hydroxy-N-methyl succinamide (4HMS) pendant arms, for ⁶⁸Ga-PET and we propose to study its complexation properties with ⁸⁹Zr radiometal.

Methods: The radiolabeling efficiency and the stability of ⁸⁹Zr-chelator are examined and compared with that of ⁸⁹Zr-DFO. Effective specific activity (ESA), radiometals challenging, DTPA challenging, biodistribution and imaging studies in balb/C mice were also performed.

Results: Preliminary radiolabeling experiments demonstrated quantitative radiolabeling of both chelators with ⁸⁹Zr within 10-15 min at room temperature at pH 7. ESA of our ⁸⁹Zr-chelator is approximately 16 fold higher compared to ⁸⁹Zr-DFO (7259 ± 997 versus 452 ± 107 GBq/μmol respectively). No transmetalation was observed for the ⁸⁹Zr-4HMS chelator in presence of 10 equivalents of iron chloride; ⁸⁹Zr-complexes remained intact at ~95% and ~86% for 4HMS ligand and DFO after 7 days. In presence of 1000 equivalents of DTPA, ⁸⁹Zr-DFO was not stable but ⁸⁹Zr-4HMSA remained intact at 92% and 98% at pH 7 and 8.5 respectively. ⁸⁹Zr-4HMS chelator also showed high stability in mouse plasma over 7 days. After 1h injection in healthy mice, the activity was largely found in kidneys for ⁸⁹Zr-4HMS chelator (9.41 ± 5.00 %ID/g) and ⁸⁹Zr-DFO (6.94 ± 0.46 %ID/g) suggesting renal excretion. At 24 h post-injection (p.i.) most of the ⁸⁹Zr-4HMS chelator was cleared from all organs and the low amount of activity in kidneys and bone (0.91±0.10 and 0.02 ± 0.01 %ID/g) is consistent with the clearance of the intact complex. While ⁸⁹Zr-DFO accumulated in kidneys, liver, bone and spleen (3.02 ± 1.98, 0.22 ± 0.07, 0.17 ± 0.13 and 0.12 ± 0.1 %ID/g) at 24 h. The PET images match the trends observed in the biodistribution results.

Conclusion: In terms of Zr⁴⁺ chelation and stability, 4HMS ligand has proven to be a superior chelator compared to DFO. Validation of the corresponding 4HMS bifunctional chelator and its conjugation to an mAb is currently underway. Research Support: Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2014-04354).