Evaluation of Hy3ADA as a bifunctional 89Zr-chelator for PET imaging applications.

Introduction: PET imaging with ⁸⁹Zr labeled antibodies have provided images with high specificity and sensitivity and could provide insight into in clinical treatment with targeted therapies. The current gold standard chelator, deferoxamine (DFO) has been shown to bind Zr⁴⁺ in six of the eight coordination sites leaving space for two additional water molecules. This leaves DFO complexes susceptible to deprotonation/decomplexation, resulting in uptake of ⁸⁹Zr in bone tissue. To form a more stable complex, we investigated the use of a 6-amino-1,4-diazepane with three hydroxamate sidechains (Hy₃ADA⁵) to utilize a more rigid backbone while still applying the use of hydroxamate binding moieties for binding.

Methods: The Hy₃ADA⁵ chelator was coupled to trastuzumab (Tras) through a squaric acid linker in a 8:1 molar ratio (Hy₃ADA⁵-SA:Tras) and the immunoreactivity was evaluated via the Lindmo assay using BT-474 cells. Stability of the Hy₃ADA⁵-Tras complex was evaluated in PBS and human serum. Mice were implanted with estrogen pellets 7 days before implantation of BT-474 cells into the shoulder 1 month before the study. Three groups of mice were injected with 100 µCi (50 µg) of labeled Hy₃ADA⁵-Tras, Hy₃ADA⁵-Tras with a cold mass of antibody, or DFO-Tras. Mice were imaged with PET/CT 1d, 3d, 5d, and 7d post injection. Biodistribution analysis was performed following the 7d image collection.

Results: Hy₃ADA⁵-Tras was labeled with ⁸⁹Zr at a specific activity of 2 µCi/µg. Conjugation of the chelator did not alter the antibody’s specificity. ⁸⁹Zr-Hy₃ADA⁵-Tras was 100% stable at 37°C in both PBS and human serum for 7 days. PET imaging showed both ⁸⁹Zr-Hy₃ADA⁵-Tras and DFO-Tras had a maximum tumor uptake after 5 days (mean SUV values of 8.67 ± 2.07 and 7.17 ± 1.17 respectively). Specificity was confirmed with low uptake in the mice that received a blocking dose (mean SUV values of 2.89 ± 0.48). Biodistribution results showed similar mean tumor uptake for the Hy₃ADA⁵-Tras and DFO-Tras groups (41.0 ± 9.5 vs. 41.2 ± 19.9 %ID/g). Similar mean femur uptake was also observed in both groups (10.93 ± 2.24 vs 10.01 ± 6.22 %ID/g).

Conclusions: ⁸⁹Zr-Hy₃ADA⁵-Tras is stable complex that accumulates within tumors at the same rate as the currently used DFO-Tras, showing promise for the scaffold for future modifications.

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