Evaluation of Chelator-to-Antibody Ratio on Development of Zr-iPET Tracer for Imaging of PD-L1 Expression on Tumor

Zr-iPET has been widely used for preclinical and clinical immunotherapy studies to predict patient stratification or evaluate therapeutic efficacy. In this study, we prepared and evaluated Zr-DFO-anti-PD-L1-mAb tracers with varying chelator-to-antibody ratios (CARs), including Zr-DFO-anti-PD-L1-mAb_3X (), Zr-DFO-anti-PD-L1-mAb_10X (), and Zr-DFO-anti-PD-L1-mAb_20X (). The DFO-anti-PD-L1-mAb conjugates with varying CARs were prepared using a random conjugation method and then subjected to quality control. The conjugates were radiolabeled with Zr and evaluated in a PD-L1-expressing CT26 tumor-bearing mouse model. Next, iPET imaging, biodistribution, pharmacokinetics, and ex vivo pathological and immunohistochemical examinations were conducted. LC-MS analysis revealed that DFO-anti-PD-L1-mAb conjugates were prepared with CARs ranging from 0.4 to 2.0. Radiochemical purity for all tracer groups was >99% after purification. The specific activity levels of , , and were 2.2 ± 0.6, 8.2 ± 0.6, and 10.5 ± 1.6 μCi/μg, respectively. Zr-iPET imaging showed evident tumor uptake in all tracer groups and reached the maximum uptake value at 24 h postinjection (p.i.). Biodistribution data at 168 h p.i. revealed that the tumor-to-liver, tumor-to-muscle, and tumor-to-blood uptake ratios for , , and were 0.46 ± 0.14, 0.58 ± 0.33, and 1.54 ± 0.51; 4.7 ± 1.3, 7.1 ± 3.9, and 14.7 ± 1.1; and 13.1 ± 5.8, 19.4 ± 13.8, and 41.3 ± 10.6, respectively. Significant differences were observed between and in the aforementioned uptake ratios at 168 h p.i. The mean residence time and elimination half-life for , , and were 25.4 ± 4.9, 24.2 ± 6.1, and 25.8 ± 3.3 h and 11.8 ± 0.5, 11.1 ± 0.7, and 11.7 ± 0.6 h, respectively. No statistical differences were found between-tracer in the aforementioned pharmacokinetic parameters. In conclusion, Zr-DFO-anti-PD-L1-mAb tracers with a CAR of 1.4-2.0 may be better at imaging PD-L1 expression in tumors than are traditional low-CAR Zr-iPET tracers.