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

Abstract

The goal of this study was to develop an ⁸⁹Zr-labeled anti-PD-L1 immune PET technique that can quantitatively monitor chemotherapy-mediated modulation of tumor PD-L1 expression in living subjects. Methods: Anti-PD-L1 antibodies underwent sulfohydryl moiety-specific conjugation with maleimide-deferoxamine (DFO) followed by ⁸⁹Zr radiolabeling. ⁸⁹Zr-PD-L1 IgG was evaluated for radiochemical purity, specific activity and radiolabel stability. Parental CT26 colon cancer cells and CT26/PD-L1 cells engineered to stably overexpress PD-L1 underwent binding assays, flow cytometry, and Western blotting. In vivo pharmacokinetics was evaluated and tumor-bearing mice underwent biodistribution and PET studies after ⁸⁹Zr-PD-L1 IgG injection. Results: ⁸⁹Zr-PD-L1 IgG synthesis was straightforward and efficient. SDS PAGE showed that reduction with TCEP produced half-antibody fragments and MALDI-TOF analysis estimated 2.18 DFOs conjugated per antibody, indicting site-specific conjugation at the hinge region disulfide bonds. High expressing CT26/PD-L1 cells showed 102.2 ± 6.7-fold greater ⁸⁹Zr-PD-L1 IgG binding compared to weak expressing CT26 cells. Excellent target specificity was confirmed by a drastic reduction of CT26/PD-L1 cell binding to 3.0 ± 0.8% by excess cold antibody. Intravenous 89Zr-PD-L1 IgG followed bi-exponential blood clearance. Tissue activity assessed by PET/CT demonstrated decreases in major organ activity over 7 days, 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 controls and increased ⁸⁹Zr-PD-L1 IgG binding. This was accompanied by increased AKT activation and reduced PTEN. In CT26 tumor mice, gemcitabine treatment substantially increased tumor uptake from 1.56 ± 0.48 to 6.24 ± 0.37 %ID/g (tumor/blood ratio: 34.7). Immunoblots revealed significant increases in tumor PD-L1 and activated AKT and a decrease of PTEN. Conclusion: ⁸⁹Zr-PD-L1 IgG showed specific targeting with favorable pharmacokinetic and PET imaging properties. Gemcitabine treatment upregulated cancer cell and tumor PD-L1 expression and increased ⁸⁹Zr-PD-L1 IgG uptake. ⁸⁹Zr-PD-L1 IgG PET may thus be useful for monitoring chemotherapy-mediated tumor PD-L1 modulation in living subjects.