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

Abstract

Blocking the interaction of the immune checkpoint molecules programmed cell death protein-1 (PD-1) and its ligand, PD-L1, using specific antibodies has been a major breakthrough for immune oncology. Whole-body PD-L1 expression positron emission tomography (PET) imaging may potentially allow for a better prediction of response to PD-1 targeted therapies. Imaging of PD-L1 expression is feasible by PET with the Adnectin protein ¹⁸F-BMS-986192. However, radiofluorination of proteins, such as BMS-986192 remains complex and labelling yields are low. The goal of this study was therefore the development and preclinical evaluation of a ⁶⁸Ga-labeled Adnectin protein (⁶⁸Ga- BMS-986192) to facilitate clinical trials. Methods: ⁶⁸Ga-labeling of DOTA-conjugated Adnectin (BXA-206362) was carried out in NaOAc-buffer at pH 5.5 (50°C, 15min). In vitro stability in human serum at 37°C was analyzed using Radio-thin layer chromatography (Radio-TLC) and Radio-high performance liquid chromatography (Radio-HPLC). 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 negative control. Immunohistochemical staining studies, biodistribution and small animal PET studies of ⁶⁸Ga-BMS-986192 were carried out using PD-L1-positive and negative U-698-M-bearing NSG mice. Results: ⁶⁸Ga-BMS-986192 was obtained with quantitative radiochemical yields (RCYs) >97% and with high radiochemical purity (RCP). In vitro stability in human serum was ≥ 95% after 4h of incubation. High and specific binding of ⁶⁸Ga-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 IHC staining. In vivo, ⁶⁸Ga-BMS-986192 uptake was high in PD-L1+ tumors (9.0±2.1%ID/g at 1hp.i.) and kidneys (56.9±9.2% ID/g at 1hp.i.) with negligible uptake in other tissues. PD-L1 negative tumors demonstrated only background uptake of radioactivity (0.6±0.1% ID/g). Co-injection of an excess of unlabelled Adnectin reduced tumor uptake of PD-L1 by more than 80%. Conclusion: ⁶⁸Ga-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 demonstrate the feasibility of ⁶⁸Ga-BMS-986192 for imaging of PD-L1 expression in tumors and is encouraging for further clinical applications of PD-L1 ligands.