据预测，在锆 89 (89Zr) 免疫正电子发射断层扫描 (89Zr- immunoPET）可以增强 89Zr 放射免疫缀合物的体内稳定性。然而，将 [89Zr]Zr-DOTA 与单克隆抗体 (mAb) 缀合仍然是一个挑战，因为 [89Zr]Zr-DOTA 螯合所需的热处理可能会导致 mAb 部分的热变性。我们开发了一种基于四嗪 (Tz) 共轭双功能 DOTA 衍生物 2,2',2″-(10-(1-(4-(1,2,4, 5-四嗪-3-基)苯基)-3,21,26-三氧代-6,9,12,15,18-五氧杂-29-羧基-2,22,25-三氮杂二十六烷-29-基)-1, 4,7,10-四氮杂环十二烷-1,4,7-三基）三乙酸 (DOTAGA-Tz) 和反电子需求 Diels-Alder (IEDDA) 点击化学反应，其中反式环辛烯修饰的 mAb 与 [89Zr 缀合]Zr-DOTAGA，无需受热。通过体外、离体和体内测试以及与传统去铁胺 (DFO) 对应物 [89Zr]Zr-DFO-曲妥珠单抗的比较分析，证实了 IEDDA 衍生的 [89Zr]Zr-DOTAGA-曲妥珠单抗的稳定性。使用 [89Zr]Zr-DOTAGA-曲妥珠单抗的体内免疫 PET 成像清晰地显示了鼠异种移植模型中的人表皮生长因子受体 2 阳性恶性肿瘤。与 [89Zr]Zr-DFO-曲妥珠单抗相比，[89Zr]Zr-DFO-曲妥珠单抗在 72 小时延迟扫描中观察到更大的肿瘤对比度。这些发现表明我们的 IEDDA 连接方法可以成为合成 [89Zr]Zr-DOTA-mAb 的有效方法，并且可以增强 89Zr-immunoPET 在 DOTA 介导的放射免疫治疗中的治疗潜力。

There have been predictions that the use of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) in zirconium-89 (89Zr) immuno-positron emission tomography (89Zr-immunoPET) could enhance the in vivo stability of 89Zr radioimmunoconjugates. However, conjugating [89Zr]Zr-DOTA to a monoclonal antibody (mAb) remains a challenge as the heat treatment required for [89Zr]Zr-DOTA chelation can lead to thermal denaturation of the mAb moieties. We developed a method for synthesizing [89Zr]Zr-DOTA-mAb based on a tetrazine (Tz)-conjugated bifunctional DOTA derivative 2,2',2″-(10-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,21,26-trioxo-6,9,12,15,18-pentaoxa-29-carboxy-2,22,25-triazanonacosane-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-Tz) and the inverse electron-demand Diels-Alder (IEDDA) click chemistry reaction where trans-cyclooctene-modified mAbs are conjugated to [89Zr]Zr-DOTAGA without being exposed to heat. The stability of IEDDA-derived [89Zr]Zr-DOTAGA-trastuzumab was confirmed by in vitro, ex vivo, and in vivo testing and comparative analysis against the conventional deferoxamine (DFO) counterpart [89Zr]Zr-DFO-trastuzumab. The in vivo immunoPET imaging using [89Zr]Zr-DOTAGA-trastuzumab clearly visualized human epidermal growth factor receptor 2-positive malignancies in murine xenograft models. Greater tumor contrast was observed from [89Zr]Zr-DOTAGA-trastuzumab at a 72-h delayed scan compared with [89Zr]Zr-DFO-trastuzumab. These findings suggest that our IEDDA ligation approach can be an effective means of synthesizing [89Zr]Zr-DOTA-mAb and can enhance the theranostic potential of 89Zr-immunoPET in DOTA-mediated radioimmunotherapy.