Development of a ⁶⁸ Ga-Labeled Peptide Radiotracer for Noninvasive Imaging of PD-L1 Expression in Tumors

Programmed death-ligand 1 (PD-L1) is a key immune checkpoint protein that facilitates tumor immune escape and correlates with response to immune checkpoint inhibitor therapy. However, noninvasive, real-time assessment of dynamic PD-L1 expression remains challenging due to tumor heterogeneity and limitations of tissue biopsies. In this study, we rationally designed and evaluated a novel ⁶⁸Ga-labeled peptide-based radiotracer, ⁶⁸Ga-DOTA-P6, for PET imaging of PD-L1. The PD-L1-targeting peptide P6 was identified via a high-throughput OBOC peptide library and validated using SPR and cellular fluorescence assays. The radiotracer demonstrated high radiochemical purity (>95%) and excellent stability in saline, PBS, and fetal bovine serum (>90% intact after 2 h). In vivo micro-PET/CT imaging in H1975 and MDA-MB-231 tumor-bearing mice revealed rapid, PD-L1-dependent tumor accumulation. Co-injection of unlabeled P6 reduced tumor uptake by 69%, confirming specificity. Biodistribution studies showed highest accumulation in kidneys and significant tumor uptake. Immunohistochemistry confirmed PD-L1 overexpression in H1975 tumors, with a strong correlation between SUVmax and PD-L1 levels (<i>r</i> = 0.8060, <i>p</i> < 0.001). Together, these findings demonstrate the promise of ⁶⁸Ga-DOTA-P6 for noninvasive imaging of PD-L1 expression and potential prediction of immunotherapy response.