Radiosynthesis and preclinical evaluation of [ ^99m Tc]Tc-HYNIC–durvalumab for immuno-SPECT imaging of PD-L1 expression in tumor models

Abstract Accurate, non-invasive measurement of programmed death-ligand 1 (PD-L1) expression is crucial for the optimization of immune checkpoint inhibitor cancer treatments. We describe the radiosynthesis and preclinical assessment of a new technetium-99m ( 99m Tc) conjugated immuno-SPECT agent, [ 99m Tc]Tc-HYNIC–durvalumab, for PD-L1 imaging in vivo . Durvalumab was conjugated with hydrazinonicotinamide (HYNIC) through lysine residues using an N-hydroxysuccinimide (NHS) ester-based method and subsequently radiolabeled with technetium-99m ( 99m Tc) in the presence of tricine and ethylenediamine-N,N′-diacetic acid (EDDA) as co-ligands. Radiochemical purity, physicochemical stability in physiologically relevant media, and immunoreactivity were determined. Specific binding affinity and kinetics of internalization were studied in PD-L1-positive A549 cells. Biodistribution and SPECT imaging were carried out in A549 xenograft-bearing mice and rabbits. Specificity was confirmed by co-injection with an excess of unlabeled durvalumab. 99m Tc-HYNIC-Durvalumab had 98.14 ± 0.27 % radiochemical purity and good stability (>90 % intact at 4 h in serum). The tracer showed high PD-L1 affinity and extensive receptor-mediated binding (28.54 ± 0.65 % at 4 h). In vivo , tumor uptake at 4 h post-injection was 6.42 ± 0.11 %ID/g, significantly reduced by co-administration of excess unlabeled antibody (1.56 ± 0.05 %ID/g, p < 0.01). Tumor-to-background ratio was 3.84, allowing for high-contrast SPECT imaging that accurately defined PD-L1-expressing tumors. These findings support 99m Tc-HYNIC-Durvalumab to be a highly specific, stable, and clinically translatable SPECT radiotracer for quantitative PD-L1 expression imaging. This agent has great promise to enable patient stratification and therapeutic monitoring in immuno-oncology. Further studies toward clinical translation is warranted.