Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors.
Affitoxin--a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors.
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DOI：
10.1097/cji.0b013e3181ad4d5d
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发表时间：
2009-10
期刊：
影响因子：
--
通讯作者：
Capala J
中科院分区：
文献类型：
--
作者：
Zielinski R;Lyakhov I;Jacobs A;Chertov O;Kramer-Marek G;Francella N;Stephen A;Fisher R;Blumenthal R;Capala J
人表皮生长因子受体2（HER 2）的表达在25 - 30%的乳腺癌中扩增，并且与不利的预后相关。在这里，我们报告了Affitoxin的构建，纯化和表征-一种新型的HER 2特异性细胞毒性分子，结合HER 2特异性Affibody分子作为靶向部分和PE 38 KDEL，这是假单胞菌外毒素A（PE）的截短版本，作为细胞杀伤剂。它是高度可溶的，并且在其纯化期间不需要额外的重折叠、氧化或还原步骤。使用表面等离子体共振（SPR）技术和竞争性结合试验，我们已经表明，阿菲毒素特异性结合HER 2与纳摩尔亲和力。我们还观察到HER 2受体表达与结合至细胞表面的阿菲毒素的保留之间的高度相关性。Affitoxin结合和内化之后是翻译延伸因子2（eEF 2）的PE活性结构域介导的ADP-核糖基化，因此抑制蛋白质合成，如用Affitoxin处理的HER 2阳性细胞的蛋白质表达分析所示。测定的HER 2阴性细胞MDA-MB 468的IC 50值（65±2.63 pM）比低HER 2水平表达的MCF 7细胞的值（2.56±0.1 pM）高20倍以上，并且其HER 2过表达衍生物MCF 7/HER 2（62.7±5.9 fM）几乎高三个数量级。这些研究表明，Affitoxin是一种有吸引力的基于PE 38的候选药物，用于治疗HER 2阳性肿瘤。
Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25 – 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin – a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A (PE), as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using Surface Plasmon Resonance (SPR) technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 receptor expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by PE activity domain-mediated ADP-ribosylation of translation elongation factor 2 (eEF2) and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65±2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56±0.1 pM), and almost three orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7±5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.