Pretargeted imaging has emerged as an effective multistep strategy aiming to improve imaging contrast and reduce patient radiation exposure through decoupling of the radioactivity from the targeting vector. The inverse electron-demand Diels–Alder (IEDDA) reaction between a trans-cyclooctene (TCO)-conjugated antibody and a labeled tetrazine holds great promise for pretargeted imaging applications due to its bioorthogonality, rapid kinetics under mild conditions, and formation of stable products. Herein, we describe the use of functionalized carbonylacrylic reagents for site-specific incorporation of TCO onto a human epidermal growth factor receptor 2 (HER2) antibody (THIOMAB) containing an engineered unpaired cysteine residue, generating homogeneous conjugates. Precise labeling of THIOMAB–TCO with a fluorescent or radiolabeled tetrazine revealed the potential of the TCO-functionalized antibody for imaging the HER2 after pretargeting in a cellular context in a HER2 positive breast cancer cell line. Control studies with MDA–MD-231 cells, which do not express HER2, further confirmed the target specificity of the modified antibody. THIOMAB–TCO was also evaluated in vivo after pretargeting and subsequent administration of an ¹¹¹In-labeled tetrazine. Biodistribution studies in breast cancer tumor-bearing mice showed a significant activity accumulation on HER2⁺ tumors, which was 2.6-fold higher than in HER2^– tumors. Additionally, biodistribution studies with THIOMAB without the TCO handle also resulted in a decreased uptake of ¹¹¹In–DOTA–Tz on HER2⁺ tumors. Altogether, these results clearly indicate the occurrence of the click reaction at the tumor site, i.e., pretargeting of SK-BR-3 HER2-expressing cells with THIOMAB–TCO and reaction through the TCO moiety present in the antibody. The combined advantages of site-selectivity and stability of TCO tagged-antibodies could allow application of biorthogonal chemistry strategies for pretargeting imaging with minimal side-reactions and background.

中文翻译：

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基于半胱氨​​酸选择性抗体修饰和 IEDDA 生物正交手柄的体内预靶向点击化学

预靶向成像已成为一种有效的多步骤策略，旨在通过将放射性与靶向矢量解耦来提高成像对比度并减少患者的辐射暴露。一个之间的逆电子需求狄尔斯-阿尔德（IEDDA）反应反环辛烯 (TCO) 偶联的抗体和标记的四嗪由于其生物正交性、温和条件下的快速动力学以及稳定产物的形成，在预靶向成像应用中具有广阔的前景。在此，我们描述了使用功能化羰基丙烯酸试剂将 TCO 定点掺入到含有工程化未配对半胱氨酸残基的人表皮生长因子受体 2 (HER2) 抗体 (THIOMAB) 上，从而产生均质偶联物。用荧光或放射性标记的四嗪对 THIOMAB-TCO 进行精确标记，揭示了 TCO 功能化抗体在 HER2 阳性乳腺癌细胞系的细胞环境中成像后对 HER2 成像的潜力。使用不表达 HER2 的 MDA-MD-231 细胞进行对照研究，进一步证实了修饰抗体的靶标特异性。THIOMAB-TCO 在预靶向和后续给药后也进行了体内评估。¹¹¹ In-标记的四嗪。乳腺癌荷瘤小鼠的生物分布研究表明，HER2 ⁺肿瘤具有显着的活性积累，比 HER2 ^-肿瘤高 2.6 倍。此外，使用不含 TCO 手柄的 THIOMAB 进行的生物分布研究也导致HER2 ⁺上¹¹¹ In-DOTA-Tz 的摄取减少肿瘤。总之，这些结果清楚地表明在肿瘤部位发生了点击反应，即表达 SK-BR-3 HER2 的细胞与 THIOMAB-TCO 变黑，并通过抗体中存在的 TCO 部分发生反应。TCO 标记抗体的位点选择性和稳定性的综合优势可以应用双正交化学策略进行预靶向成像，同时将副反应和背景降至最低。