A Novel Small Cyclic Peptide-Based ⁶⁸Ga-Radiotracer For PET Imaging Of PD-L1 Expression In Tumors

Objective:In the tumor microenvironment,programmed death protein1/programmed death protein ligand 1（PD-L1）signaling pathways help tumors to escape the immune system.We designed a gallium-68(⁶⁸Ga)-labeled small-molecule peptide targeting PD-L1 and used positron emission tomography/computed tomography（PET/CT）to detect and dynamically monitor the expression level of PD-L1 in tumors.Methods:S-Cyclo（ETSK）-SF-NH2（SETSKSF）is a cyclic peptide inhibitor comprising seven amino acid residues.It was connected with the chelating agent DOTA,labeled DOTA-SETSKSF,with the positron nuclide ⁶⁸Ga.The radiochemical purity of a in normal saline（NS）,phosphate buffered saline（PBS）and fetal bovine serum（FBS）was measured at 37℃for 4 hours at multiple time points,and the in vitro stability data of ⁶⁸Ga-DOTA-SETSKSF.In addition,blood samples were taken from mice injected with the tracer at different time points to measure the radioactivity count to plot the time radioactivity curve and obtain the pharmacokinetic data.Using human recombinant protein PD-L1,we conducted affinity test of ⁶⁸Ga-DOTA-SETSKSF for PD-L1,and calculated the immune binding fraction of ⁶⁸Ga-DOTA-SETSKSF and PD-L1.Western blot（WB）was used to determine the expression level of PD-L1 on the surface of non-small cell lung cancer cells（NCI-H1975 and A549）.We stained NCI-H1975 and A549 cells by immunofluorescence reaction,and observed the expression of PD-L1 on the cell membrane of the two types of cells by laser scanning confocal microscope.In addition,we also conducted cell specific uptake experiments,time binding experiments on NCI-H1975,A549 and B16-F10 cells,and saturated binding experiments on NCI-H1975 cells to confirm the expression and localization of PD-L1 at the cell level and determine the uptake.Biodistribution and imaging experiments were performed using the NCI-H1975 and B16-F10tumor models.Finally,we took tumor tissues of experimental mice and sliced them for immunohistochemical analysis to verify the expression of PD-L1 in tumor tissues of mice.Results:⁶⁸Ga-DOTA-SETSKSF was successfully synthesized,and the radiochemical purity was>99%after purification.The in vitro stability of 68Ga-DOTA-SETSKSF was>95%in NS,PBS,and FBS at 37°C after 4 h of incubation.The immunoreactive fraction was 89.21%（R²>0.99）,suggesting that the binding of ⁶⁸Ga-DOTA-SETSKSF to PD-L1 was indeed specific.The counts per minute（CPM）in the H1975,B16F10,and A549 cells in the ⁶⁸Ga-DOTA-SETSKSF experimental groups were 13913±643,7388±784,and 3006±182,respectively.The number of H1975 and B16F10 cells was significantly higher than that of A549 cells（t=28.246,P=0.000;t=9.422,P<0.05）,whereas that in the corresponding three blocking groups indicates lower activity,with values of 1679±1090,884±84,and 762±68,respectively.Cell-binding experiments confirmed that ⁶⁸Ga-DOTA-SETSKSF exhibited high uptake in NCI-H1975 tumors with high PD-L1 expression,moderate uptake in B16-F10tumors with moderate PD-L1 expression and low uptake in A549 tumors with low PD-L1 expression.The specific binding curve of the H1975 cells showed a typical sigmoidal shape,with high binding affinity（KD=82.53±7.54 n Mol/L）.The receptor density（Bmax）was 14408±492.The imaging agent was injected into the mice and quickly cleared from the bloodstream,and the clear half-life(T_1/2)of ⁶⁸Ga-DOTA-SETSKSF from the blood was 14.48±3.26 min.After the imaging agent was removed from the blood,it quickly entered the tumor tissue and remained there for a long time.The percentages of the injected dose per gram of tissue（%ID/g）for NCI-H1975 and A549 tumors were 5.29±0.21 and 0.89±0.10 at 1 h after injection,respectively.The NCI-H1975 tumor-to-muscle and tumor-to-blood ratios were 41.79±5.81 and 4.75±0.19 at 4 h,respectively.Apart from the NCI-H1975 tumor,the kidney and bladder also showed high accumulation suggesting that ⁶⁸Ga-DOTA-SETSKSF was excreted primarily through the urinary system.PET/CT images 1 hour after injection in male C57BL/6 tumor mice aged 5-8 weeks showed high accumulation of ⁶⁸Ga-DOTA-SETSKSF in NCI-H1975 tumors and low uptake in A549 tumors（%ID/g values were 5.29±0.21 and 0.89±0.10,respectively）,which was consistent with the results of biodistribution experiments.No significant uptake was observed in H1975 tumors in blocking dose mice,underlining the specificity of ⁶⁸Ga-DOTA-SETSKSF for PD-L1 binding.Immunohistochemical analysis for PD-L1expression demonstrating high,moderate,and low immunoreactivity in H1975,B16F10,and A549 tumors,respectively.Conclusions:⁶⁸Ga-DOTA-SETSKSF has high radiochemical purity and stability,and ⁶⁸Ga-DOTA-SETSKSF PET/CT can be used to monitor the expression of PD-L1 in tumor-bearing mice,which has potential clinical application value.