@misc{oai:repo.qst.go.jp:00082856,
 author = {Kuan, Hu and Lin, Xie and Masayuki, Hanyu and Zhang, Yiding and Zhang, Ming-Rong and Kuan, Hu and Lin, Xie and Masayuki, Hanyu and Zhang, Yiding and Zhang, Ming-Rong},
 month = {May},
 note = {Objectives: D-peptides are hyper-resistant to proteolytic degradation in L-homochiral living
subjects, making them remarkedly increased gut, blood plasma, and intracellular half-lives.
D-peptides are theoretically ideal drug candidates, however, the potential of D-peptides in
radiopharmaceuticals is rarely explored. Recently, a D-dodecapeptide antagonist (DPA) identified
by mirror-image phage display revealed high efficacy to block the programmed death-ligand 1
(PD-L1)/programmed death 1 (PD-1) interaction in animal models. In this work, we report the
radiolabeling of DPA with 68Ga and study its metabolism and target engagement in tumor-bearing
mice using PET. Methods: The PD-L1 protein expression in the human glioblastoma U87MG cell
line was analyzed by flow cytometry and immunofluorescence imaging. The stability of [68Ga]DPA
in saline and mouse serum was examined by radio HPLC and TLC. The cellular uptake of
[68Ga]DPA into U87MG cells was measured at 15, 30, 60, and 120 min after co-incubation. For
PET imaging, we inoculated 1×106 U87MG cells to the left flank of Balb/c nude mice
subcutaneously. When the tumors grow to ̴100 mm3 at 7 days after inoculation, mice were injected
with [68Ga]DPA (0.5 mCi per mouse) intravenously. Dynamic PET was performed from 0-60 min
postinjection. To comprehensively profile the in vivo distribution of the tracer, ex vivo distribution
was carried out in Balb/c nude mice bearing U87MG tumors. Mice were sacrificed at 5, 30, 60, and
120 min postinjection of the tracer (0.05 mCi per mouse). The tumor and organs were harvested and
subjected to gamma counter measurement. Based on the ex vivo biodistribution data, we calculated
the tumor to blood, tumor to liver, and tumor to muscle ratios. As a PD-L1 negative control, we also
performed PET imaging in Bon-1 tumor-bearing Balb/c nude mice. Finally, the PD-L1 expression
level in U87MG and Bon-1 tumor tissue was immunohistologically stained and compared. Results:
The U87G cells were detected with approximately 60% subpopulation that express PD-L1 protein.
The uptake of the tracer by U87MG cells showed a time-dependent manner and increased from
2%AD at 15 min of co-incubation to 4%AD at 120 min of co-incubation. Whole-body PET imaging
showed substantial [68Ga]DPA accumulation in U87MG tumors at 30 and 60 min postinjection. The
highest radioactivity appeared in the kidney and bladder, and non-striking radioactivity was noticed
in other tissues, such as the liver and lung. Ex vivo biodistribution data verified the observation in
the PET study. The washout of the tracers from nonspecific organs such as blood, liver, and muscle
was significantly faster than that in the tumor, indicating PD-L1 specific retention in the U87MG
tumor. The optimum image contrast was achieved at 60 min postinjection. Immunohistological
analysis revealed obvious PD-L1 expression in U87MG tumors but rare of that in Bon-1 tumors.
Conclusions: We have successfully labeled the DPA with 68Ga. The in vivo profile of [68Ga]DPA
suggested it a potential radiotracer for PD-L1 visualization. Our study paves the way for exploiting
D-peptides as radiopharmaceuticals to manage cancer, both as PET tracers and radiotheranostics
agents. Acknowledgement: We thank the financial support from the JSPS KAKENHI grant no.
19K17156., eSRS},
 title = {PET imaging of PD-L1 overexpressing tumors with a 68Ga labeled D-dodecapeptide},
 year = {2021}
}