Objective: The immune checkpoint PD-L1, expressed on a wide variety of tumors, is positively correlated with tumor progression and is influenced by microenvironment alteration or chemotherapy. This underlines the need for biomarkers to quantify changes in PD-L1 expression that can influence immunotherapy response. We thus developed an immuno-PET technique based on site-specific 89Zr-labeled PD-L1 antibodies that can image changes in tumor PD-L1 expression. We further identified the mechanisms underlying PD-L1 upregulation by gemcitabine treatment.
Methods: Anti-PD-L1 antibodies reduced with tris(2-carboxyethyl)phosphine underwent sulfohydryl moiety-specific conjugation with maleimide-deferroxamine and were radiolabeled with 89Zr. Cultured CT26 colon cancer cells and cells engineered to constitutively overexpress PD-L1 (CT26/PD-L1 cells) with or without treatment were compared for cell binding assays, flow cytometry, and Western blotting. Balb/c mice bearing CT26 or CT26/PD-L1 tumors were prepared and underwent in vivo PET imaging for up to 7 day after 89Zr PD-L1 antibody injection.
Results: PD-L1 antibodies were efficiently labeled with 89Zr in a site-specific manner, and radiolabeling efficiency reached 80%. The radio-probe showed low binding to CT26 cells that had weak PD-L1 expression at baseline. CT26/PD-L1 cells had high PD-L1 expression and showed 102.2 ± 6.7-fold higher levels of 89Zr PD-L1 antibody binding. Competitive binding assay confirmed dramatically reduced cell binding to 3.0 ± 0.8% of uninhibited controls in the presence of excess cold antibody. Treatment of CT26 cells with gemcitabine or olaparib for 24 h resulted in dose-dependent increases of 89Zr PD-L1 antibody binding. Immunoblots revealed that treatment with 50 nM gemcitabine remarkably increased PD-L1 expression and 10 μM olaparib also increased PD-L1 expression to 592.4 ± 114.2% and 224.8 ± 155.9% of controls, respectively. Increased activation of AKT and loss of PTEN was found to accompany PD-L1 upregulation. PET/CT imaging displayed clear 89Zr-PD-L1 IgG accumulation in CT26 and CT26/PD-L1 tumors by 96 h post-injection. Preinjection of excess antibody inhibited CT26/PD-L1 tumor uptake at 6 days to 39.6 ± 22.8% of uninhibited level. 89Zr-PD-L1 IgG uptake in CT26 tumors at 6 days post-injection was significantly increased to 6.24 ± 0.37 %ID/g for gemcitabine-treated animals (tumor-to-blood ratio, 34.7), compared to only 1.56 ± 0.48 %ID/g for untreated mice.
Conclusion: Cancer cells’ PD-L1 expression was increased by gemcitabine treatment in a manner accompanied by increased binding of 89Zr PD-L1 antibodies. Furthermore, PET imaging of murine models demonstrated increased radio-probe accumulation in tumors that had upregulated PD-L1 expression by gemcitabine treatment. Thus, 89Zr PD-L1 antibody immune-PET may be useful for noninvasive monitoring of tumor PD-L1 modulation in living subjects.
Citation Format: Kyung-Ho Jung, Jin Won Park, Jin Hee Lee, Young Seok Cho, Kyung-Han Lee. Imaging of PD-L1 modulation by gemcitabine using 89Zr labeled anti-PD-L1 antibody PET [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B11.