Division of Biomedical Radiation Sciences, Department of Radiology, Oncology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, 75185, Uppsala, Sweden.
Amino Acids. 2012 May;42(5):1975-85. doi: 10.1007/s00726-011-0927-x. Epub 2011 May 15.
Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide 99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of 99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied 99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of 99mTc. The biodistribution of all 99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of 99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.
亲和体分子是一类新型的分子展示亲和力蛋白，基于非免疫球蛋白支架筛选。临床前研究和初步临床数据表明，亲和体分子在注射后不久即可提供肿瘤相关分子靶标的高对比度成像。在重组亲和体分子的 C 末端使用含有半胱氨酸的肽基螯合剂，使放射性核素 99mTc 能够进行位点特异性标记。早期研究表明，肽基螯合剂中的氨基酸位置、组成和顺序会影响亲和体分子的标记稳定性、细胞处理和生物分布。为了研究氨基酸顺序的影响，制备并表征了一系列含有 GSGC、GEGC 和 GKGC 螯合剂的抗 HER2 亲和体分子。研究了它们与 HER2 的亲和力、99mTc 标记亲和体分子的细胞处理及其生物分布。并将这些性质与之前研究的含有 GGSC、GGEC 和 GGKC 螯合剂的 99mTc 标记亲和体分子进行了比较。所有变体对 HER2 的亲和力均为皮摩尔级。在螯合剂中替换单个氨基酸对 99mTc 的细胞处理有明显影响。所有 99mTc 标记亲和体分子的生物分布总体上相当，主要区别在于放射性在排泄器官中的摄取和保留。放射性在肝脏中的积累对于赖氨酸含有的螯合剂更高，而肾脏对 99mTc 的保留则受到螯合剂氨基酸组成的显著影响。氨基酸顺序会影响一些缀合物在注射后 1 小时时对肾脏的摄取，但在以后的时间点差异会减小。这些信息对于开发其他支架蛋白为基础的成像和治疗性放射性标记缀合物可能会有所帮助。
