A kit formulated under good manufacturing practices (GMP) for preparing [89Zr]Zr-Bn-DFO-Pembrolizumab injection for human administration and associated preclinical imaging, biodistribution, dosimetry and toxicity studies

PET with [ 89 Zr]Zr-Bn-DFO-pembrolizumab is a potential imaging biomarker to assess delivery of pembrolizumab to tumours and predict response to PD-1 checkpoint immunotherapy. Clinical quality [ 89 Zr]Zr-Bn-DFO-pembrolizumab injection has been prepared under Good Manufacturing Practices (GMP) through multistep radiolabeling processes but no kit formulation has been reported. Our aim was to formulate a kit for preparation of [ 89 Zr]Zr-Bn-DFO-pembrolizumab injection under GMP conditions and conduct translational bridge studies to support a clinical trial of imaging delivery of pembrolizumab to lung cancer brain metastases using PET. Three sequential lots of unit-dose kits containing 2 mg of Bn-DFO-pembrolizumab in 1.0 mL of 0.1 M NaHCO 3 buffer, pH 7.4 were formulated. All lots met specifications for protein concentration (1.8–2.2 mg/mL), pH (7.0–7.5), purity and homogeneity (SDS-PAGE), Bn-DFO conjugation (3.0–6.0 Bn-DFO), PD-1 binding affinity (K D = 1–35 pM) and sterility (USP Sterility Test) and endotoxins (USP Bacterial Endotoxins Test). The labeling efficiency with 37 MBq of 89 Zr was 98.8–99.4%. Kits were stable up to 5 months while [ 89 Zr]Zr-Bn-DFO-pembrolizumab injection was stable for 24 h at 2–6 °C. PET specifically imaged PD-1-positive MOLT-4 human T-cell lymphoblastic leukemia tumours in NRG mice at 72 h p.i. of [ 89 Zr]Zr-Bn-DFO-pembrolizumab prepared from the kit. Biodistribution studies in non-tumour bearing BALB/c mice revealed slow elimination from the blood and the highest normal organ uptake was in the lungs, liver, kidneys, spleen and bone. There was no uptake in the brain. The highest normal organ equivalent doses projected for adult humans injected with 37 MBq of [ 89 Zr]Zr-Bn-DFO-pembrolizumab were in the kidneys, liver, lungs, red marrow, osteogenic cells and spleen. The predicted whole body dose was 9–10 mSv, which is comparable to PET with [ 18 F]F-2-fluorodeoxyglucose (10.5 mSv). Administration of multiples of the human administered activity and mass per kilogram to healthy BALB/c mice showed no decrease in body weight or blood cell counts or increased liver transaminases or creatinine over 14 days, indicating no toxicity. A kit was formulated under GMP conditions to prepare [ 89 Zr]Zr-Bn-DFO-pembrolizumab injection in a quality suitable for patient administration. Preclinical translational bridge studies predicted that the radiopharmaceutical would be safe and effective for PET imaging in humans and provided an equivalent whole body dose comparable to a PET scan with 18 F-FDG.