School of Biotechnology, Division of Protein Technology, KTH Royal Institute of Technology, Stockholm, Sweden.
Institute for Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and.
J Nucl Med. 2018 Jan;59(1):93-99. doi: 10.2967/jnumed.117.197202. Epub 2017 Sep 1.
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. Two constructs, Cys-ADAPT6 and Cys-ADAPT6, having the (HE)DANS sequence at the N terminus were produced and site-specifically labeled using In-DOTA or I-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 mice bearing ovarian carcinoma cell (SKOV-3) xenografts. Specific HER2 binding and high affinity were preserved after labeling. Both Cys-ADAPT6 and Cys-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 I-HPEM label provided more than 140-fold lower renal uptake than the In-DOTA label at 4 after injection. The tumor-to-organ ratios were, in contrast, higher for both of the In-DOTA-labeled ADAPT variants in other organs. Tumor-to-blood ratios for In-labeled Cys-ADAPT6 and Cys-ADAPT6 did not differ significantly (250-280), but In-DOTA-Cys-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 I-HPEM-Cys-ADAPT6 had significantly higher tumor uptake and a higher tumor-to-kidney ratio. 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 I-labeled HPEM-Cys-ADAPT6.
使用放射性核素成像可视化癌症相关的分子表型改变是一种用于对靶向治疗进行分层的非侵入性方法。工程化的白蛋白结合域衍生的亲和蛋白（ADAPT）是一种有前途的放射性核素分子成像示踪剂，因为其分子量小（6.5 kDa），满足了高效肿瘤穿透和快速清除的前提条件。以前的研究表明，靶向人表皮生长因子受体 2（HER2）的 ADAPT6 通过放射性金属在 N 末端标记，能够在注射后几个小时内对异种移植中的 HER2 表达进行成像。本研究的目的是评估使用非残留标记或在 C 末端放置标记是否会进一步改善 ADAPT6 的靶向特性。 生成了具有 N 末端（HE）DANS 序列的两种构建体，Cys-ADAPT6 和 Cys-ADAPT6，并使用 In-DOTA 或 I-碘代-（4-羟基苯基）乙基马来酰亚胺（HPEM）进行了特异性标记。在体外和体内比较了这些缀合物。在携带卵巢癌细胞（SKOV-3）异种移植的 BALB/C 小鼠中评估了 HER2 靶向特性和生物分布。 标记后保留了特异性的 HER2 结合和高亲和力。HER2 表达的癌细胞缓慢内化 Cys-ADAPT6 和 Cys-ADAPT6。根据标记位置的不同，注射后 4 小时时肿瘤组织每克的摄取量在注射剂量的 10%至 22%之间变化。无论末端位置如何，与 In-DOTA 标记物相比，I-HPEM 标记物在注射后 4 小时时提供的肾脏摄取量低 140 多倍。相比之下，在其他器官中，两种 In-DOTA 标记的 ADAPT 变体的肿瘤与器官比值均较高。In 标记的 Cys-ADAPT6 和 Cys-ADAPT6 的肿瘤与血液比值无显著差异（250-280），但 In-DOTA-Cys-ADAPT6 提供了更高的肿瘤与肺、肿瘤与肝、肿瘤与脾和肿瘤与肌肉比值。放射性碘变体具有相似的肿瘤与器官比值，但 I-HPEM-Cys-ADAPT6 的肿瘤摄取量更高，肿瘤与肾脏比值更高。 标记的残留特性强烈影响 ADAPT6 的靶向特性。放射性标记的位置也会影响靶向，尽管影响较小。在 C 末端放置标记物可产生最佳的放射性金属和放射性卤化物标记物的生物分布特征。放射性碘标记物的低肾脏保留为使用 I 标记的 HPEM-Cys-ADAPT6 进行放射性核素治疗创造了条件。
