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A Pretargeted Imaging Strategy for Immune Checkpoint Ligand PD-L1 Expression in Tumor Based on Bioorthogonal Diels-Alder Click Chemistry

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Abstract

Purpose

The use of antibodies as tracers requires labeling with isotopes with long half-lives due to their slow pharmacokinetics, which creates prohibitively high radiation dose to non-target organs. Pretargeted methodology could avoid the high radiation exposure due to the slow pharmacokinetics of antibodies. In this investigation, we reported the development of a novel pretargeted single photon emission computed tomography (SPECT) imaging strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) for evaluating immune checkpoint ligand PD-L1 expression in tumor based on bioorthogonal Diels-Alder click chemistry.

Procedures

The radioligand [99mTc]HYNIC-PEG11-Tz was achieved by the synthesis of a 6-hydrazinonicotinc acid (HYNIC) modified 1,2,4,5-tetrazine (Tz) and subsequently radiolabeled with technetium-99m (Tc-99m). The stability of [99mTc]HYNIC-PEG11-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. Atezolizumab was modified with trans-cyclooctene (TCO). The [99mTc]HYNIC-PEG11-Tz and atezolizumab-TCO interaction was tested in vitro. Pretargeted H1975 cell immunoreactivity binding and saturation binding assays were evaluated. Pretargeted biodistribution and SPECT imaging experiments were performed in H1975 and A549 tumor-bearing modal mice to evaluate the PD-L1 expression level.

Results

[99mTc]HYNIC-PEG11-Tz was successfully radiosynthesized with a specific activity of 9.25 MBq/μg and a radiochemical purity above 95 % as confirmed by reversed-phase HPLC (RP-HPLC). [99mTc]HYNIC-PEG11-Tz showed favorable stability in NS, PBS, and FBS and rapid blood clearance in mice. The atezolizumab was modified with TCO-NHS ester to produce a conjugate with an average 6.4 TCO moieties as confirmed by liquid chromatograph-mass spectrometer (LC-MS). Size exclusion HPLC revealed almost complete reaction between atezolizumab-TCO and [99mTc]HYNIC-PEG11-Tz in vitro, with the 1:1 Tz-to-mAb reaction providing a conversion yield of 88.65 ± 1.22 %. Pretargeted cell immunoreactivity binding and saturation binding assays showed high affinity to H1975 cells. After allowing 48 h for accumulation of atezolizumab-TCO in H1975 tumor, pretargeted in vivo biodistribution revealed high uptake of the radiotracer in the tumor with a tumor-to-muscle ratio of 27.51 and tumor-to-blood ratio of 1.91. Pretargeted SPECT imaging delineated the H1975 tumor clearly. Pretargeted biodistribution and SPECT imaging in control groups demonstrated a significantly reduced tracer accumulation in the A549 tumor.

Conclusions

We have developed a HYNIC-modified Tz derivative, and the HYNIC-PEG11-Tz was labeled with Tc-99m with a high specific activity and radiochemical purity. [99mTc]HYNIC-PEG11-Tz reacted rapidly and almost completely towards atezolizumab-TCO in vitro with the 1:1 Tz-to-mAb reaction. SPECT imaging using the pretargeted strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) demonstrated high-contrast images for high PD-L1 expression H1975 tumor and a low background accumulation of the probe. The pretargeted imaging strategy is a powerful tool for evaluating PD-L1 expression in xenograft mice tumor models and a potential candidate for translational clinical application.

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Acknowledgments

We want to thank for the technical supports from Prof. Yingjian Zhang and Dr. Jianping Zhang from Center for Biomedical Imaging, Fudan University, and Shanghai Engineering Research Center of Molecular Imaging Probes.

Funding

This study was funded by The National Nature Science Foundation of China (11875114, 81671735, and 81871407) and Open Large Infrastructure Research of Chinese Academy of Science.

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Authors and Affiliations

  1. Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Xuhui District, Shanghai, 200032, China

    Lin Qiu, Hui Tan, Qingyu Lin, Zhan Si, Wujian Mao, Tingting Wang, Zhequan Fu, Dengfeng Cheng & Hongcheng Shi

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  1. Lin Qiu
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  2. Hui Tan
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  3. Qingyu Lin
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  4. Zhan Si
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  9. Hongcheng Shi
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Correspondence to Dengfeng Cheng or Hongcheng Shi.

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All applicable institutional and/or national guidelines for the care and use of animals were followed.

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Qiu, L., Tan, H., Lin, Q. et al. A Pretargeted Imaging Strategy for Immune Checkpoint Ligand PD-L1 Expression in Tumor Based on Bioorthogonal Diels-Alder Click Chemistry. Mol Imaging Biol 22, 842–853 (2020). https://doi.org/10.1007/s11307-019-01441-3

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