A novel PET tracer for noninvasive imaging the checkpoints expression of innate and adaptive immunity in tumors by simultaneously targeting CD24 and PD-L1

The success of tumor immunotherapy depends on the innate and adaptive immune responses, with CD24 and PD-L1 being key targets. ^DBP1 peptide is a novel bispecific D-peptide, targeting both CD24 and PD-L1 simultaneously. In this study, by radiolabeling ^DBP1 peptide, we developed a novel PET modality to noninvasively evaluate CD24 and PD-L1 expressions in tumors. To enhance the solubility of ^DBP1, a hydrophilic lysine was added into C-terminal residue of the peptide, which was then modified with a chelator NOTA to produce the radiotracer precursor NOTA-^DBP1k. NOTA-^DBP1k showed high affinity for CD24 (K_D = 10.70 ± 0.70 nM) and PD-L1 (K_D = 5.40 ± 0.61 nM). [⁶⁸Ga]Ga-NOTA-^DBP1k was synthesized with a high radiochemical yield (71 ± 3.0 %) and exhibited high hydrophilicity and stability. [⁶⁸Ga]Ga-NOTA-^DBP1k showed higher uptake in high CD24/PD-L1 expressed MCF-7 cells than that in low CD24/PD-L1 expressed U-87MG cells in vitro. In vivo, [⁶⁸Ga]Ga-NOTA-^DBP1k showed high uptake in MCF-7 tumors and had favorable tumor-to-background ratios by microPET imaging. On the contrary, low uptake was found in U-87MG tumors, which was significantly lower than that in MCF-7 tumors (0.42 ± 0.02 %ID/g vs. 1.01 ± 0.06 %ID/g, p < 0.05). The biodistribution study was consistent with the findings of microPET imaging results. These results demonstrated that [⁶⁸Ga]Ga-NOTA-^DBP1k can noninvasively image the CD24 and PD-L1 checkpoint expression of innate and adaptive immunity in tumors and may be helpful for guiding the CD24/PD-L1 dual-checkpoints blockage immunotherapy.