⁸⁹Zr-Labeled Anti-PD-L1 Antibody PET Monitors Gemcitabine Therapy-Induced Modulation of Tumor PD-L1 Expression.

Abstract

We developed an ⁸⁹Zr-labeled anti-programmed death ligand 1 (anti-PD-L1) immune PET that can monitor chemotherapy-mediated modulation of tumor PD-L1 expression in living subjects. <b>Methods:</b> Anti-PD-L1 underwent sulfohydryl moiety-specific conjugation with maleimide-deferoxamine followed by ⁸⁹Zr radiolabeling. CT26 colon cancer cells and PD-L1-overexpressing CT26/PD-L1 cells underwent binding assays, flow cytometry, and Western blotting. In vivo pharmacokinetics, biodistribution, and PET imaging were evaluated in mice. <b>Results:</b>⁸⁹Zr-anti-PD-L1 synthesis was straightforward and efficient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that reduction produced half-antibody fragments, and matrix-assisted laser desorption ionization time-of-flight analysis estimated 2.18 conjugations per antibody, indicating specific conjugation at the hinge-region disulfide bonds. CT26/PD-L1 cells showed 102.2 ± 6.7-fold greater ⁸⁹Zr-anti-PD-L1 binding than that of weakly expressing CT26 cells. Excellent target specificity was confirmed by a drastic reduction in binding by excess cold antibody. Intravenous ⁸⁹Zr-anti-PD-L1 followed biexponential blood clearance. PET/CT image analysis demonstrated decreases in major organ activity over 7 d, whereas high CT26/PD-L1 tumor activity was maintained. Again, this was suppressed by excess cold antibody. Treatment of CT26 cells with gemcitabine for 24 h augmented PD-L1 protein to 592.4% ± 114.2% of the control level and increased ⁸⁹Zr-anti-PD-L1 binding, accompanied by increased AKT (protein kinase B) activation and reduced phosphatase and tensin homolog (PTEN). In CT26 tumor-bearing mice, gemcitabine treatment substantially increased tumor uptake from 1.56% ± 0.48% to 6.24% ± 0.37% injected dose per gram (tumor-to-blood ratio, 34.7). Immunoblots revealed significant increases in tumor PD-L1 and activated AKT and a decrease in PTEN. <b>Conclusion:</b>⁸⁹Zr-anti-PD-L1 showed specific targeting with favorable imaging properties. Gemcitabine treatment upregulated cancer cell and tumor PD-L1 expression and increased ⁸⁹Zr-anti-PD-L1 uptake. ⁸⁹Zr-anti-PD-L1 PET may thus be useful for monitoring chemotherapy-mediated tumor PD-L1 modulation in living subjects.