Preparation of WL-12-HBED-CC for PD-L1 targetedimaging and preliminary application in healthy volunteers

Introduction: The cyclic peptide WL-12 is of a high KD value (~23 nM) with PD-L1 antigen, and has been designed as PD-L1 targeted imaging tracers, such as ⁶⁸Ga-DOTA-WL-12, ⁶⁴Cu-DOTAGA-WL-12 and ¹⁸F-FPy-WL-12. Linker and chelating agent will affect the labeling efficiency and in vivo metabolism, so a more suitable chelating agent for Ga-68 labeling, HBED-CC, was modified to WL-12 and in vivo metabolism was evaluated with PET/MR.

Methods: cyclo[AcTyr-NMeAla-Asn-Pro-His-Leu-Hyp-Trp-Ser-Trp(Me)-NMeNle-NMeNle-Orn(X)-Cys]-Gly-NH₂ was synthesized, where X stands for HBED-CC, and then characterized with LC-MS. WL-12-HBED-CC was labeled with fresh ⁶⁸Ga³⁺ eluent in 0.05 M HCl at pH = 4 for 10 min. Labeling rate was evaluated via C-18 cartridge separation. Five healthy volunteers (3 male/2 female) were injected with WL-12-HBED-CC-⁶⁸Ga at 1 MBq/kg body weight, and scanned at 15, 30, 60, 120, 180 and 240 min post injection. Scan protocols included PET scan, T1 MR and T2 MR.

Results: WL-12-HBED-CC was synthesized with purity of 97.03% and directly used as the precursor. Ga-68 was labeled to WL-12-HBED-CC with labeling rate > 99%, and was of a good in vitro stability in saline. There was not a difference on distribution between male and female volunteers. Different with the reported ⁶⁸Ga-NOTA-WL-12 that was mainly metabolized via liver and kidney, WL-12-HBED-CC-⁶⁸Ga was mainly metabolized via the enterohepatic circulation. Gallbladder was also of high uptake due to the biliary secretion resulted from fasting state. The relative low liver and intestines background at 60 min means an ideal imaging time window to detect PD-L1-expressed tissues and lesions. In addition, compared with the reported ⁸⁹Zr-Atezolizumab that has been testified preclinically, the fast metabolism of WL-12-HBED-CC-⁶⁸Ga showed a feasibility of clinical transformation. A series of WL-12-HBED-CC-⁶⁸Ga PET/MR images of a volunteer was exhibited in Figure 1, showing the dynamic changes of enterohepatic circulation of WL-12-HBED-CC-⁶⁸Ga.

Conclusions: WL-12-HBED-CC was successfully synthesized, and easily labeled with Ga-68. The preliminary application in healthy volunteers proved the feasibility of clinical transformation in PD-L1 targeted imaging. Acknowledgement: The research was supported by the "234 Discipline Climbing Plan" of Changhai Hospital of Naval Medical University (the Second Military Medical University).Figure 1. WL-12-HBED-CC-⁶⁸Ga PET/MR of a healthy volunteer (famale, 30 years old) at different time points post injection.

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