A Preclinical Study of [⁶⁴Cu]Cu-NOTA-KN035 for Molecular Imaging of PD-L1 in Tumors

The programmed cell death protein-1/programmed cell death ligand-1 (PD-1/PD-L1) axis plays a central role in tumor immune regulation, with PD-L1 expression serving as a critical biomarker for patient stratification and response prediction. Accurate, noninvasive assessment of PD-L1 expression is, therefore, essential for guiding clinical decision-making. KN035 is an ∼79.6 kDa fusion protein comprising a humanized single-domain antibody linked to an Fc fragment, offering a smaller molecular size than conventional monoclonal antibodies. In this study, KN035 was conjugated with p-SCN-Bn-NOTA and radiolabeled with ⁶⁴Cu to generate [⁶⁴Cu]Cu-NOTA-KN035 for PET imaging of PD-L1. The tracer showed high radiochemical purity (>95%) and strong binding specificity in vitro. In vivo PET imaging and biodistribution studies were performed in H1975 (high PD-L1 expression) and A549 (low PD-L1 expression) nonsmall cell lung cancer (NSCLC) xenograft models. Clear tumor visualization was achieved at 4 h postinjection (5.62 ± 0.55%ID/g in H1975; 4.16 ± 0.18%ID/g in A549), with peak uptake at 48 h (12.32 ± 0.66 and 5.72 ± 0.21%ID/g, respectively). Tumor uptake decreased significantly after blocking with excess KN035, confirming the specificity. These results demonstrate the high PD-L1-targeting specificity of [⁶⁴Cu]Cu-NOTA-KN035, suggesting its great potential as a noninvasive diagnostic tool for immunotherapy-based treatments in the future.