Tran Thuy, Engfeldt Torun, Orlova Anna, Widström Charles, Bruskin Alexander, Tolmachev Vladimir, Karlström Amelie Eriksson
Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Sweden.
Bioconjug Chem. 2007 Mar-Apr;18(2):549-58. doi: 10.1021/bc060291m. Epub 2007 Mar 2.
Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (ZHER2:342) with a dissociation constant (Kd) of 22 pM for binding to the HER2 receptor has been reported earlier. Preclinical and pilot clinical studies have demonstrated the utility of radiolabeled ZHER2:342 in imaging of HER2-expressing tumors. The small size and cysteine-free structure of Affibody molecules enable complete peptide synthesis and direct incorporation of radionuclide chelators. The goal of this study was to evaluate if incorporation of the natural peptide sequences cysteine-diglycine (CGG) and cysteine-triglycine (CGGG) sequences would enable labeling of Affibody molecules with 99mTc. In a model monomeric form, the chelating sequences were incorporated by peptide synthesis. The HER2-binding affinity was 280 and 250 pM for CGG-ZHER2:342 and CGGG-ZHER2:342, respectively. Conjugates were directly labeled with 99mTc with 90% efficiency and preserved the capacity to bind specifically to HER2-expressing cells. The biodistribution in normal mice showed a rapid clearance from the blood and the majority of organs (except kidneys). In the mice bearing SKOV-3 xenografts, tumor uptake of 99mTc-CGG-ZHER2:342 was HER2-specific and a tumor-to-blood ratio of 9.2 was obtained at 6 h postinjection. Gamma-camera imaging with 99mTc-CGG-ZHER2:342 clearly visualized tumors at 6 h postinjection. The results show that the use of a cysteine-based chelator enables 99mTc-labeling of Affibody molecules for imaging.
亲和体分子是一类新型的亲和蛋白，它利用一种基于源自A蛋白的58个氨基酸结构域的支架。小尺寸（7 kDa）的亲和体分子可被选择用于以高亲和力结合细胞表面靶点。此前已报道一种与HER2受体结合的解离常数（Kd）为22 pM的亲和体分子（ZHER2:342）。临床前和先导临床研究已证明放射性标记的ZHER2:342在HER2表达肿瘤成像中的效用。亲和体分子的小尺寸和无半胱氨酸结构使得能够进行完整的肽合成并直接掺入放射性核素螯合剂。本研究的目的是评估掺入天然肽序列半胱氨酸 - 二甘氨酸（CGG）和半胱氨酸 - 三甘氨酸（CGGG）序列是否能使亲和体分子用99mTc进行标记。在模型单体形式中，通过肽合成掺入螯合序列。CGG - ZHER2:342和CGGG - ZHER2:342与HER2的结合亲和力分别为280和250 pM。偶联物用99mTc直接标记，效率为90%，并保留了特异性结合HER2表达细胞的能力。在正常小鼠中的生物分布显示从血液和大多数器官（肾脏除外）快速清除。在携带SKOV - 3异种移植瘤的小鼠中，99mTc - CGG - ZHER2:342的肿瘤摄取是HER2特异性的，注射后6小时肿瘤与血液的比率为9.2。注射99mTc - CGG - ZHER2:342后6小时，γ相机成像清晰地显示出肿瘤。结果表明，使用基于半胱氨酸的螯合剂能够使亲和体分子用99mTc标记用于成像。
