⁸⁹Zr-labeled anti-PD-1 antibody for in vivo monitoring of infiltrating PD-1⁺ T lymphocytes in a pancreatic tumor mouse model

Objectives: Pancreatic cancer has the lowest survival rate of all cancers, only 3-6% of those diagnosed survive for five years. Survival has improved for most cancers over the last decades, but not pancreatic cancer. Recently, the rise of immunotherapy has been one of the most startling and promising developments in cancer research. Especially, the drugs called “immune checkpoint inhibitors” were used to lift the natural brakes that restrain immune effector cells, allowing them to go to town on tumors. With such immunotherapy, cancer patients who were given months to live are still here years later. Unfortunately, not all patients respond to immune checkpoint blockade. One of the main reason for the non-responding patients are that there are not enough infiltrated cytotoxic T lymphocytes inside the tumor microenvironment. The objective of this study is to develop radiolabeled anti-PD-1 antibody for evaluating levels of the infiltrating cytotoxic T lymphocytes in a pancreatic tumor mouse model.

Methods: Anti-PD-1 antibody can specifically bind to activated T cell surface PD-1. This specific recognition between the antibody and antigen provides a unique opportunity for in vivo PET imaging of PD-1 levels (PD-1⁺ cells). In this study, we first synthesized the DFO (deferoxamine) modified anti-mouse PD-1 antibody. Then we radiolabeled it with ⁸⁹Zr to prepare the ⁸⁹Zr-anti-PD-1. KPC ( LSL-Kras^G12D/+;LSL-Trp53^R172H/+;Pdx-1-Cre) The orthotopic model (use of KPC cell line derived from KPC mice) was prepared by injecting 0.2 million KPC cells orthotopically into the pancreas of the mouse, and the model was ready in two weeks. The distribution of ⁸⁹Zr-anti-PD-1 in the KPC mice was performed by PET/CT imaging of KPC tumor-bearing mice 2 and 24 h post-dose injection. Wild type mice without tumor were also injected and imaged for comparison. An immune stimulant, particulate beta-glucan (WGP), was injected intraperitoneally before radioactive dosing to priming the immune system, and the resulting changes of PD-1⁺ cell distribution was monitored with PET/CT imaging.

Results: We have successfully prepared the ⁸⁹Zr-labeled anti-PD-1 antibody by conjugating a DFO chelator onto the antibody molecule, and radiolabeling it with ⁸⁹Zr. We then successfully used this PD-1-targeting agent to monitor the infiltrating PD-1⁺ T lymphocytes in a PKC pancreatic tumor mouse model. Tumor-infiltrating T lymphocytes were clearly visualized by ⁸⁹Zr-anti-PD-1 at 2 and 24 h post-dose injection with a tumor uptake of 15.22 %ID/g at 24 h time point. Accumulation of radioactive signals at the naïve pancreas in wild type mice were not visualized. Pre-injection of WGP in PKC mice didn’t specifically increase the infiltrated T lymphocytes for the tumor at 24 h post dose injection (16.16 vs 15.22 %ID/g for w/ and w/o WGP, respectively), but the ⁸⁹Zr-anti-PD-1 levels were significantly higher in the blood (33.56 vs 2.97 %ID/g), at the meantime with much lower uptake in the lung (10.91 vs 18.08 %ID/g), liver (16.34 vs 47.14 %ID/g), spleen (17.1 vs 62.09 %ID/g), and kidney (15.23 vs 70.84 %ID/g). Conclusion: We have successfully synthesized the ⁸⁹Zr-anti-PD-1and used it for monitoring the in vivo PD-1⁺ T lymphocytes trafficking in a pancreatic tumor mouse model. The promising imaging data suggest its potential for in vivo PET imaging of PD-1 levels in a pancreatic tumor mouse model.