Optimizing conditions to minimize antibody requirements for ⁸⁹Zr radiolabeling

Objectives Due to its attractive physical and chemical properties, the positron emitter ⁸⁹Zr is well-suited to radiolabel monoclonal antibodies (mAb) for immuno-PET imaging, from mouse to human. Established protocols for radiolabeling with ⁸⁹Zr require significant quantities of mAb. This increases the cost of preclinical experiments performed using commercial research mAb, and slows down the evaluation of novel custom mAb by requiring production scale-up to conduct such studies. The purpose of this study was to optimize conjugation and radiolabeling conditions using microgram quantities of mAb with p-SCN-Bn-desferoxamine (DFO).

Methods ⁸⁹Zr was produced by proton irradiation of a 99.9% yttrium target (⁸⁹Y disc: 13 mm Ø, 0.254 mm thick, total mass of ⁸⁹Y: 152 mg) at 10 µA for one hour. Trastuzumab was conjugated with DFO at room temperature over night with DFO:mAb ratios of 3:1, 6:1 and 10:1 in PBS pH 8.9-9.1. The purified conjugated mAb was then radiolabeled at room temperature with varying quantities and concentration of ⁸⁹Zr and antibodies for one hour, followed by purification using PD-10 and molecular weight filter spin columns. Radiolabeling yields (RCY) and specific activities were determined by ITLC chromatography. The immunoreactivity fraction was estimated according to the Lindmo method using SKOV-3 cells. The number of chelates was evaluated via an isotopic dilution assay.

Results After conjugation, the average numbers of chelates per antibody were 3.1±0.4, 5.1±0.3 and 2.5±0.2 for DFO:mAb ratios of 3:1, 6:1 and 10:1 respectively. To radiolabel 200 µg of mAb an optimal specific activity of 123.6 kBq/µg was achieved using a ratio of 6:1. The radiochemical purity of the purified radiolabeled Ab was consistently greater than 96%. For DFO:mAb of 3:1 and 6:1, the immunoreactivity was close to 100%, whereas a result of 52% was obtained for 10:1. Decreasing amounts of mAb were radiolabeled using consistent mAb and radioactivity concentrations. RCY were 33, 24, 19 and 11 % for 200, 50, 10 and 2 µg of Trastuzumab-DFO. A 2 fold decrease in ⁸⁹Zr activity did not improve the specific activity for 200 and 2 µg, but increased RCY to 77 and 61 % which corresponded to 20.1 and 18.4 kBq/µg for 50 and 10 µg of mAb respectively. Increasing the activity concentration with 10 µg of mAb and 2.2 MBq of ⁸⁹Zr led to a RCY of 36% and a specific activity of 77.7 kBq/µg.

Conclusions We defined satisfying conditions for 200 µg of Trastuzumab radiolabeling with ⁸⁹Zr for PET imaging of HER2 positive tumor-bearing mice using a ratio of 6:1 for DFO:mAb without decreasing binding efficiency. Decreasing the scale is challenging with ⁸⁹Zr, and this is likely affected by potential trace impurities related to the target material, such as iron. Smaller quantities of mAb labeled with ⁸⁹Zr maintained similar specific activities by decreasing the ⁸⁹Zr activity used for radiolabeling down to 10 µg of mAb. Further experiments are underway to determine if labeling of mAb in microgram quantities with ⁸⁹Zr can be further improved by reducing the iron content or increasing the integrated beam current when producing ⁸⁹Zr.