Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Xuhui District, Shanghai, 200032, China.
Mol Imaging Biol. 2020 Aug;22(4):842-853. doi: 10.1007/s11307-019-01441-3.
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.
The radioligand [Tc]HYNIC-PEG-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 [Tc]HYNIC-PEG-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. Atezolizumab was modified with trans-cyclooctene (TCO). The [Tc]HYNIC-PEG-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.
[Tc]HYNIC-PEG-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). [Tc]HYNIC-PEG-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 [Tc]HYNIC-PEG-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.
We have developed a HYNIC-modified Tz derivative, and the HYNIC-PEG-Tz was labeled with Tc-99m with a high specific activity and radiochemical purity. [Tc]HYNIC-PEG-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/[Tc]HYNIC-PEG-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.
由于抗体的药代动力学缓慢，作为示踪剂使用的抗体需要用半衰期长的同位素进行标记，这会导致非靶器官的放射性剂量过高。预靶向方法可以避免由于抗体的药代动力学缓慢而导致的高辐射暴露。在这项研究中，我们报道了一种新的基于生物正交 Diels-Alder 点击化学的预靶向单光子发射计算机断层扫描（SPECT）成像策略（atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz）的发展，用于评估肿瘤中免疫检查点配体 PD-L1 的表达。
通过合成 6-肼基烟酸（HYNIC）修饰的 1,2,4,5-四嗪（Tz），随后用锝-99m（Tc-99m）标记，得到放射性配体[Tc]HYNIC-PEG-Tz。在体外评估[Tc]HYNIC-PEG-Tz 的稳定性，并在体内进行血液药代动力学测试。阿特珠单抗用反式环辛烯（TCO）修饰。在体外测试[Tc]HYNIC-PEG-Tz 和阿特珠单抗-TCO 的相互作用。评估了 H1975 细胞预靶向免疫反应结合和饱和结合测定。在 H1975 和 A549 荷瘤模型小鼠中进行预靶向生物分布和 SPECT 成像实验，以评估 PD-L1 的表达水平。
[Tc]HYNIC-PEG-Tz 通过反相高效液相色谱法（RP-HPLC）证实，成功地以 9.25MBq/μg 的比活度和 95%以上的放射化学纯度进行了放射性合成。[Tc]HYNIC-PEG-Tz 在 NS、PBS 和 FBS 中表现出良好的稳定性，在小鼠体内血液清除迅速。阿特珠单抗用 TCO-NHS 酯修饰，生成平均 6.4 个 TCO 部分的缀合物，通过液相色谱-质谱（LC-MS）证实。排阻高效液相色谱法显示阿特珠单抗-TCO 和[Tc]HYNIC-PEG-Tz 之间几乎完全反应，1：1 Tz-单抗反应的转化率为 88.65±1.22%。预靶向细胞免疫反应结合和饱和结合测定显示对 H1975 细胞具有高亲和力。在 H1975 肿瘤中积累阿特珠单抗-TCO 48 小时后，进行预靶向体内生物分布，结果显示放射性示踪剂在肿瘤中摄取量高，肿瘤与肌肉的比值为 27.51，肿瘤与血液的比值为 1.91。预靶向 SPECT 成像清晰地描绘了 H1975 肿瘤。对照组的预靶向生物分布和 SPECT 成像显示，A549 肿瘤中的示踪剂积累明显减少。
我们开发了一种 HYNIC 修饰的 Tz 衍生物，并用 Tc-99m 对 HYNIC-PEG-Tz 进行了高比活度和高放射化学纯度的标记。[Tc]HYNIC-PEG-Tz 与阿特珠单抗-TCO 在体外快速且几乎完全反应，1：1 Tz-单抗反应的转化率为 88.65±1.22%。使用预靶向策略（阿特珠单抗-TCO/[Tc]HYNIC-PEG-Tz）进行 SPECT 成像，为高 PD-L1 表达的 H1975 肿瘤和探针低背景积累提供了高对比度的图像。预靶向成像策略是评估异种移植小鼠肿瘤模型中 PD-L1 表达的有力工具，也是潜在的转化临床应用候选者。
