101 [89Zr]Zr-DFO-durvalumab PET/CT before durvalumab treatment in R/M SCCHN patients Conjugation, radiolabeling and quality control of [89Zr]Zr-DFO-durvalumab [89Zr]Zr-DFO-durvalumab was produced under good manufacturing practice (GMP) conditions. Recently, one study has reported the conjugation and 89Zr labeling of DFO-durvalumab as performed for the individual patient22. Due to the larger sample size of the PINCH study, we performed the conjugation of DFO-durvalumab as a bulk at the Radboudumc. For the process of 89Zr labeling, durvalumab (AstraZeneca, Cambridge, England) was conjugated with the bifunctional chelator tetrafluorophenol-N-succinyldesferal-Fe(III) (TFP-N-SucDf or DFO; ABX, Radeberg, Germany) using 200 mg durvalumab and a 2-fold molar excess of DFO23. To remove unconjugated DFO, the conjugated DFO-durvalumab was purified using disposable gel permeation columns (PD10, GE Healthcare Life sciences, Eindhoven, The Netherlands). Subsequent dilution with NaCl 0.9% resulted in a final concentration 2.5 mg/ml which was filtered through 0.22 μm Millex GV filter and dispensed. Radiolabeling of DFO-durvalumab with 89Zr was performed under GMP conditions at Radboudumc and transported to participating centers. The radiolabeling process is performed at pH 7.2. To achieve the desired pH value, oxalic acid, sodium carbonate and HEPES buffer (adjusted to pH 7.3 by use of sodium hydroxide solution) are added. After addition of the conjugated monoclonal antibody DFO-Durvalumab and HEPES buffer, radiolabeling is allowed to take place during 30 minutes at room temperature. After radiolabeling, purification is performed to remove the oxalic acid and HEPES. This purification takes place by use of a disposable PD10 column and NaCl 0.9% as eluent. The final radiolabeled product contains a total volume of 10 ml with a concentration of durvalumab ([89Zr]Zr-DFOdurvalumab + unlabeled non-conjugated durvalumab) according to the assigned dose cohort. The unlabeled antibody is added to prevent possible splenic uptake of the radiolabeled antibody24. The final product contains ~37 MBq [89Zr]Zr-DFO-durvalumab at the time of injection. The radiochemical purity (as checked by thin layer chromatography and high-performance liquid chromatography) was ≥95%. Specifically bound fraction of radiolabeled DFO-durvalumab was determined during validation using a binding assay performed in triplicate with a fixed MDA-MB231 cell concentration, including controls for non-specific binding. The ratio of total bound activity divided by the non-specifically bound activity was always ≥2. 5
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