Quality Control, Metabolism Analysis and Micro-PET imaging of ^sup 64^Cu-NOTA-Trastuzumab in Gastric Cancer Patient-Derived Xenograft Mouse Model

Objectives: Abstract The purpose of this study was to establish the quality control, and quantify the 64Cu-NOTA-Trastuzumab in gastric cancer patient-derived xenografts (PDX) mouse models by applying the molecular imaging technique. Methods: Trastuzumab was labeled with 64Cu using NOTA-NCS as bi-functional chelator, and hIgG1 was labeled with same procedures as a negative control agent. Immunoreactivity of NOTA conjugated and radiolabeled antibodies were tested by ELISA analysis and NCI-N87 cell lines over-epxressing HER2, respectively. Healthy Balb/c mice were applied for dose-dependent pharmacokinetics analysis of radio-antibodies which were without or co-injected with 2 mg Trastuzumab or hIgG1. HER2-positive (case 176, n = 12) and HER2-negative (case 168, n=3) PDX mice were established and validated by western blot, DNA amplification and immunohistochemistry (IHC). Both models were conducted for Micro-PET imaging by tail injection of 18.5 MBq of 64Cu-NOTA-Trastuzumab or 64Cu-NOTA-hIgG1 as negative controls. In the protein dose escalation study, 0.5 mg, 2.0 mg trastuzumab and 2.0 mg hIgG1 co-injection with 18.5 MBq 64Cu-NOTA-Trastuzumab. Radio-probes uptake in tumor and main organs were quantified by region of interested (ROI) analysis of the micro-PET images and auto-radiography. Result: NOTA-Trastuzumab was efficiently radiolabeled with 64Cu over a 99% radio-chemical purity and 17.5 GBq/µmol specific activities. The immune-activity were preserved as the non-modified antibody, and the radiopharmaceutical proved to be stable for up to 5 physical half-life of 64Cu both in-vitro and in-vivo. Two serials of PDX gastric cancer models were successfully established: case 176 for HER2 positive and case 168 for HER2 negative. In Micro-PET imaging studies, 64Cu-NOTA-Trastuzumab exhibits a significant higher tumor uptake (11.45 ± 0.42 ID%/g) compared with 64Cu-NOTA-IgG1 (3.25 ± 0.28 ID%/g, n = 5, p = 0.0004) and 18F-FDG in HER2-positive PDX tumor models (n = 3, p < 0.001 ) at 36 h and 2h after intravenous injections, respectively. Lower level uptake of 64Cu-NOTA-Trastuzumab (6.35 ± 0.48 ID%/g) in HER2-negative PDX tumor models further confirmed specific binding of the radio-probe. Interestingly, the co-injection of 2.0 mg Trastuzumab (15.52 ± 1.97 ID%/g) or 2.0 mg hIgG1 (15.64 ± 3.54 ID%/g) increased the 64Cu-NOTA-Trastuzumab tumor uptake in PDX tumor (HER2+) model compared with 64Cu-NOTA-Trastuzumab alone (p < 0.05) at 36 h post injection. There were good correla