Visualization of cancer-associated alterations of molecular phenotype using radionuclide imaging is a noninvasive approach to stratifying patients for targeted therapies. The engineered albumin-binding domain–derived affinity protein (ADAPT) is a promising tracer for radionuclide molecular imaging because of its small size (6.5 kDa), which satisfies the precondition for efficient tumor penetration and rapid clearance. Previous studies demonstrated that the human epidermal growth factor receptor type 2 (HER2)–targeting ADAPT6 labeled with radiometals at the N terminus is able to image HER2 expression in xenografts a few hours after injection. The aim of this study was to evaluate whether the use of a nonresidualizing label or placement of the labels at the C terminus would further improve the targeting properties of ADAPT6. Methods: Two constructs, Cys²-ADAPT6 and Cys⁵⁹-ADAPT6, having the (HE)₃DANS sequence at the N terminus were produced and site-specifically labeled using ¹¹¹In-DOTA or ¹²⁵I-iodo-((4-hydroxyphenyl)ethyl) maleimide (HPEM). The conjugates were compared in vitro and in vivo. HER2-targeting properties and biodistribution were evaluated in BALB/C nu/nu mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Results: Specific HER2 binding and high affinity were preserved after labeling. Both Cys²-ADAPT6 and Cys⁵⁹-ADAPT6 were internalized slowly by HER2-expressing cancer cells. Depending on the label position, uptake at 4 h after injection varied from 10% to 22% of the injected dose per gram of tumor tissue. Regardless of terminus position, the ¹²⁵I-HPEM label provided more than 140-fold lower renal uptake than the ¹¹¹In-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the ¹¹¹In-DOTA–labeled ADAPT variants in other organs. Tumor-to-blood ratios for ¹¹¹In-labeled Cys²-ADAPT6 and Cys⁵⁹-ADAPT6 did not differ significantly (250–280), but ¹¹¹In-DOTA-Cys⁵⁹-ADAPT6 provided significantly higher tumor-to-lung, tumor-to-liver, tumor-to-spleen, and tumor-to-muscle ratios. Radioiodinated variants had similar tumor-to-organ ratios, but ¹²⁵I-HPEM-Cys⁵⁹-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. Conclusion: Residualizing properties of the label strongly influence the targeting properties of ADAPT6. The position of the radiolabel influences targeting as well, although to a lesser extent. Placement of a label at the C terminus yields the best biodistribution features for both radiometal and radiohalogen labels. Low renal retention of the radioiodine label creates a precondition for radionuclide therapy using ¹³¹I-labeled HPEM-Cys⁵⁹-ADAPT6.

使用放射性核素成像可视化与癌症相关的分子表型改变是对患者进行靶向治疗的一种非侵入性方法。工程化的白蛋白结合域衍生的亲和蛋白（ADAPT）体积小（6.5 kDa），是放射性核素分子成像的有希望的示踪剂，它满足有效的肿瘤穿透和快速清除的前提。先前的研究表明，在N末端用放射性金属标记的靶向人类表皮生长因子2型（HER2）的ADAPT6能够在注射后数小时内成像异种移植物中HER2的表达。这项研究的目的是评估使用非残基标签或将标签放置在C末端是否会进一步改善ADAPT6的靶向特性。方法：制备了两个在N端具有（HE）₃ DANS序列的Cys ² -ADAPT6和Cys ⁵⁹ -ADAPT6构建体，并使用¹¹¹ In-DOTA或¹²⁵ I-碘-（（4-羟苯基）进行了位点特异性标记。）乙基）马来酰亚胺（HPEM）。在体外和体内比较缀合物。在带有卵巢癌细胞（SKOV-3）异种移植物的BALB / C nu / nu小鼠中评估了HER2靶向特性和生物分布。结果：标记后保留了特异性HER2结合和高亲和力。半胱氨酸² -ADAPT6和半胱氨酸⁵⁹-ADAPT6被表达HER2的癌细胞缓慢内在化。取决于标记物的位置，注射后4 h的摄取量为每克肿瘤组织注射剂量的10％至22％。不论终点位置如何，注射后第4天¹²⁵ I-HPEM标签的肾脏摄取量都比¹¹¹ In-DOTA标签低140倍以上。相反，其他器官中^111种In-DOTA标记的ADAPT变体的肿瘤与器官的比率均较高。¹¹¹ In标记的Cys ² -ADAPT6和Cys ⁵⁹ -ADAPT6的肿瘤血比没有显着差异（250-280），但¹¹¹ In-DOTA-Cys ⁵⁹-ADAPT6可显着提高肿瘤与肺，肿瘤与肝脏，肿瘤与脾的比例以及肿瘤与肌肉的比例。放射性碘标记的变体具有相似的肿瘤-器官比率，但是¹²⁵ I-HPEM-Cys ⁵⁹ -ADAPT6具有显着更高的肿瘤摄取和更高的肿瘤-肾脏比率。结论：标签的残留特性强烈影响ADAPT6的靶向特性。放射性标记的位置也影响靶向，尽管程度较小。将标记放置在C末端可产生放射性金属和放射性卤素标记的最佳生物分布特征。放射性碘标记物的低肾脏保留为使用¹³¹ I标记的HPEM-Cys ⁵⁹进行放射性核素治疗创造了先决条件-ADAPT6。