Radiolabeling and Imaging of Anti-IFNγ Antibody

Radiochemistry of anti-mouse-IFNγ (clone AN18, Thermo Fisher Scientific) and IgG1 isotype control anti-horseradish peroxidase (clone HRPN, BE0088, BioXCell) was performed as described previously (22 (link), 26 (link)). All antibodies were conjugated to p-SCN-Bn-Desferrioxamine (DFO) with a 1:5 mole ratio of mAb : DFO in saline at pH ~9 for 1hr at 37°C. Unbound DFO was removed via spin column centrifugation (MWCO: 30 kDa, GE Vivaspin 500). ⁸⁹Zr (3D Imaging) was incubated with the mAb-DFO conjugates at pH ~ 7.2-7.4 at room temperature for 1hr. Unbound ⁸⁹Zr was removed via spin column centrifugation (MWCO: 30 kDa, GE Vivaspin 500) using saline as eluent buffer. [⁸⁹Zr]Zr-DFO-anti-IFNγ and [⁸⁹Zr]Zr-DFO-IgG were each labeled at a specific activity of ~5 mCi/mg. Radiochemical yields of both constructs were >95% as determined via radio-instant thin layer chromatography (iTLC, Eckert & Ziegler). Tumor-bearing animals used for imaging were injected i.v. with radiolabeled antibodies (189 ± 31 µCi) in ~150 µL sterile saline. PET images were acquired 72 hrs post-injection on a Bruker Albira SI microPET/CT system. Images were decay corrected and analyzed in PMOD version 4.304. Volume of Interest (VOI) measurements within tumors were used to determine the uptake of the radiotracer, which is expressed as the maximum injected dose per mL (%ID/mL).