Lindberg Hanna, Hofström Camilla, Altai Mohamed, Honorvar Hadis, Wållberg Helena, Orlova Anna, Ståhl Stefan, Gräslund Torbjörn, Tolmachev Vladimir
Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.
Tumour Biol. 2012 Jun;33(3):641-51. doi: 10.1007/s13277-011-0305-z. Epub 2012 Jan 17.
Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide (99m)Tc. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with (99m)Tc. (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, (99m)Tc-Z(HER2:2395)-VDC and (99m)Tc-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of (99m)Tc-Z(HER2:2395)-VDC, but it was substantially higher than uptake of (99m)Tc-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of (99m)Tc was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of (99m)Tc labelling due to a partial loss of site-specificity of nuclide chelation.
亲和体分子是一类小的(约7 kDa)稳定支架蛋白,在体内作为放射性核素分子成像的示踪剂具有很高的潜力。在C末端掺入含半胱氨酸的肽基螯合剂为用放射性核素(99m)Tc进行稳定标记提供了机会。在C末端使用GGGC螯合剂,在先前研究的基于肽的螯合剂中,肾脏放射性保留最低。此前还证明,用带负电荷的组氨酸-谷氨酸-组氨酸-谷氨酸-组氨酸-谷氨酸(HEHEHE)标签取代His(6)标签,可以通过固定金属离子亲和色谱(IMAC)纯化亲和体分子,并使使用几种不同核素在C末端进行位点特异性标记的缀合物的肝脏放射性积累较低。我们推测,在亲和体分子的N末端使用HEHEHE标签和在C末端使用GGGC螯合剂的组合将是一种有利的形式,既允许IMAC纯化,又能使排泄器官摄取较低。为了研究这一假设,生成了一种(HE)(3)-Z(HER2:342)-GGGC亲和体分子。它可以通过IMAC有效纯化,并用(99m)Tc稳定标记。(99m)Tc-(HE)(3)-Z(HER2:342)-GGGC保留了与HER2表达细胞的特异性结合。在NMRI小鼠中,(99m)Tc-(HE)(3)-Z(HER2:342)-GGGC的肝脏摄取低于对照亲和体分子(99m)Tc-Z(HER2:2395)-VDC和(99m)Tc-Z(HER2:342)-GGGC的摄取。在注射后1小时和4小时,(99m)Tc-(HE)(3)-Z(HER2:342)-GGGC的肾脏摄取比(99m)Tc-Z(HER2:2395)-VDC的摄取低2至3倍,但明显高于(99m)Tc-Z(HER2:342)-GGGC的摄取。进一步研究表明,一部分(99m)Tc被HEHEHE标签螯合,这导致肾脏中放射性积累更高。因此,在(99m)Tc标记的情况下,由于核素螯合的位点特异性部分丧失,发现在靶向支架蛋白中使用HEHEHE标签和GGGC螯合剂的组合是不可取的。