The aim of this study was to design a novel tracer targeting programmed cell death-ligand 2 (PD-L2) to dynamically monitor PD-L2 expression and perform preclinical screening to identify patients who may benefit from immune checkpoint inhibitor therapy (ICI) therapy.

⁸⁹Zr labelling of DFO-conjugated PD-L2 antibody (ATL2) was carried out in Na₂CO₃ buffer at pH 7 (37 °C, 1 h). In vitro stability was analysed using radio-thin layer chromatography (radio-TLC). The affinity of [⁸⁹Zr]Zr-DFO-ATL2 was evaluated by radio-ELISA. Cell uptake, pharmacokinetic, and biodistribution experiments were used to evaluate the biological properties. Micro-PET/CT imaging with [⁸⁹Zr]Zr-DFO-ATL2 was conducted at different time points. Immunohistochemical and HE staining studies were carried out using tumour tissues from tumour-bearing mice.

The radiochemical yield of [⁸⁹Zr]Zr-DFO-ATL2 was 65.6 ± 3.9%, and the radiochemical purity (RCP) of the tracer was greater than 99%. The tracer maintained relatively high stability and had a high affinity for the PD-L2 protein (Kd = 31.85 nM, R² = 0.94). The uptake of [⁸⁹Zr]Zr-DFO-ATL2 in A549-PD-L2 cells was higher than that in A549 cells at each time point. Micro-PET/CT showed significant uptake in the tumour region of mice bearing tumours derived from A549-PD-L2 (SUVmax = 3.53 ± 0.09 at 96 h) and H2228 (SUVmax = 2.30 ± 0.12 at 48 h) cells.

The high tumour uptake at early imaging time points demonstrates the feasibility of applying [⁸⁹Zr]Zr-DFO-ATL2 to image PD-L2 expression in tumours and is encouraging for further clinical application in the screening of patients who may benefit from ICI therapy.