Synthesis and Preclinical Evaluation of a 68Ga-Labeled Adnectin, 68Ga-BMS-986192, as a PET Agent for Imaging PD-L1 Expression

Blocking the interaction of the immune checkpoint molecule programmed cell death protein-1 and its ligand, PD-L1, using specific antibodies has been a major breakthrough for immune oncology. Whole-body PD-L1 expression PET imaging may potentially allow for a better prediction of response to programmed cell death protein-1-targeted therapies. Imaging of PD-L1 expression is feasible by PET with the adnectin protein F-18-BMS-986192. However, radiofluorination of proteins such as BMS-986192 remains complex and labeling yields are low. The goal of this study was therefore the development and preclinical evaluation of a Ga-68-labeled adnectin protein (Ga-68-BMS986192) to facilitate clinical trials. Methods: 68 Ga labeling of DOTA-conjugated adnectin (BXA-206362) was performed in NaOAc-buffer at pH 5.5 (50 degrees C, 15 min). In vitro stability in human serum at 37 degrees C was analyzed using radio-thin layer chromatography and radio-high-performance liquid chromatography. PD-L1 binding assays were performed using the transduced PD-L1-expressing lymphoma cell line U-698-M and wild-type U-698-M cells as a negative control. Immunohistochemical staining studies, biodistribution studies, and small-animal PET studies of Ga-68-BMS-986192 were performed using PD-L1-positive and PD-L1-negative U-698-M-bearing NSG mice. Results: Ga-68-BMS-986192 was obtained with quantitative radiochemical yields of more than 97% and with high radiochemical purity. In vitro stability in human serum was at least 95% after 4 h of incubation. High and specific binding of Ga-68-BMS-986192 to human PD-L1-expressing cancer cells was confirmed, which closely correlates with the respective PD-L1 expression level determined by flow cytometry and immunohistochemistry staining. In vivo, Ga-68-BMS-986192 uptake was high at 1 h after injection in PD-Li-positive tumors (9.0 +/- 2.1 percentage injected dose [56 ID]/g) and kidneys (56.9 +/- 9.2 %ID]/g), with negligible uptake in other tissues. PD-L1-negative tumors demonstrated only background uptake of radioactivity (0.6 +/- 0.1 %ID/g). Coinjection of an excess of unlabeled adnectin reduced tumor uptake of PD-L1 by more than 80%. Conclusion: Ga-68-BMS-986192 enables easy radiosynthesis and shows excellent in vitro and in vivo PD-L1-targeting characteristics. The high tumor uptake combined with low background accumulation at early imaging time points demonstrates the feasibility of Ga-68-BMS-986192 for imaging of PD-L1 expression in tumors and is encouraging for further clinical applications of PD-L1 ligands.