Optimizing conditions to minimize antibody requirements for Zr-89 radiolabeling

1097 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 O, 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.