A highly specific hydrophilic peptide for PET imaging of PD-L1 expression in tumors

5 Objectives: . The immune checkpoint protein programmed death ligand 1 (PD-L1) is expressed in many cancers as a means to evade the immune system. Immunohistochemical (IHC) methods for detecting and quantifying PD-L1 in tumors are important to guide therapy. IHC, however, has several shortcomings, including sampling error and discordance between primary and metastatic histology. Precision PET radiotracers can provide dynamic in vivoassessment of PD-L1 expression in all lesions concurrently. PD-L1 imaging agents that fit within standard clinical workflow have been few. Here we evaluate a hydrophilic, copper-64 (64Cu) radiolabeled, human PD-L1 specific peptide (DK222, Mw: 2.31 kDa) to quantify PD-L1 expression in tumors. Methods. DK222 was conjugated with NODAGA, labeled with 64Cu, and purified by HPLC. We determined the affinities of DK222 and its analogs for PD-L1 by in vitro competitive inhibition. We tested the specificity of [64Cu]DK222 in vitro and in vivo. Those studies were performed using six cell lines and tumors with varying levels of PD-L1 expression and were derived from breast (MDAMB231, SUM149), lung (HCC827 and H226) and ovarian (CHO-hPD-L1 and CHO) cancers. We performed PET imaging and biodistribution studies to determine [64Cu]DK222 pharmacokinetics and blocking studies to confirm PD-L1 specificity. We assessed PD-L1 expression in the tumors by IHC to correlate [64Cu]DK222 uptake with PD-L1 levels. Results. DK222 and its copper-bound analog demonstrated high affinities for PD-L1 ( 95% purity and in high specific activity. In vitro studies proved that cell uptake of [64Cu]DK222 was reflective of PD-L1 expression detected by flow cytometry (Fig. 1B). High contrast PET images were obtained within 60 min and as early as 10 min after [64Cu]DK222 administration (Fig. 1C & E). PET imaging demonstrated the highest accumulation of radioactivity in the bladder, kidneys, and tumor, with minimal uptake in other organs (Fig. 1C & D). Imaging and biodistribution studies proved that [64Cu]DK222 uptake in the tumors correlated with PD-L1 expression detected by IHC as follows: CHO-hPD-L1 (%ID/g, 25.9±4.0) > MDAMB231 (15.9±0.8) > HCC827 (4.2±0.8) > SUM149 (1.7±0.1) > CHO (1.3±0.2) tumors (Fig. 1C-G). Confirming PD-L1 specificity, [64Cu]DK222 uptake in the PD-L1 positive tumors was significantly inhibited in mice co-administered with blocking dose (50 mg/kg) of parent peptide (Fig. 1C-E). Conclusions. Our results establish that [64Cu]DK222 demonstrates features desired for clinical translation of a targeted molecular imaging agent: 1) high affinity and specificity to PD-L1; 2) low normal tissue uptake, allowing use across many tumor types; and, 3) suitable image contrast within 60 min of radiotracer administration to fit within the standard clinical workflow. Research Support. Allegheny Health Network-Johns Hopkins Cancer Research Fund, NIHR01CA16631, NIH P30 CA006973 and P41 EB024495.