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

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

  • Mol Imaging Biol. 2020 Aug;22(4):842-853. doi: 10.1007/s11307-019-01441-3.
Lin Qiu 1 Hui Tan 1 Qingyu Lin 1 Zhan Si 1 Wujian Mao 1 Tingting Wang 1 Zhequan Fu 1 Dengfeng Cheng 2 Hongcheng Shi 3
Affiliations

Affiliations

  • 1 Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Xuhui District, Shanghai, 200032, China.
  • 2 Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Xuhui District, Shanghai, 200032, China. cheng.dengfeng@zs-hospital.sh.cn.
  • 3 Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Xuhui District, Shanghai, 200032, China. shihongcheng163@163.com.
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.

Keywords

Atezolizumab; Click chemistry; PD-L1; Pretargeted imaging.

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