Bruell Daniela, Stöcker Michael, Huhn Michael, Redding Nicole, Küpper Michael, Schumacher Petra, Paetz Antje, Bruns Christiane J, Haisma Hidde J, Fischer Rainer, Finnern Ricarda, Barth Stefan
Department of Molecular Biotechnology, University Aachen, Aachen, Germany.
Int J Oncol. 2003 Oct;23(4):1179-86. doi: 10.3892/ijo.23.4.1179.
Pancreatic carcinoma still has the highest mortality rate in comparison to any other malignancy. Major reasons are late detection of disease, highly aggressive tumor growth and the early formation of metastases. Thus, novel effective therapies are urgently needed to improve the outcome of the patients. Overexpression of the epidermal growth factor receptor (EGFR) and its ligands has been implicated in the oncogenesis of pancreatic carcinoma and associated with an unfavorable prognosis. Consequently, the EGFR represents a specific target antigen suitable for immunotherapy. We generated a recombinant immunotoxin by fusing the anti-EGFR single chain fragment 425(scFv) to a truncated mutant of Pseudomonas Exotoxin A (ETA'). Using the expression vector pBM1.1, functional 425(scFv)-ETA' was periplasmically expressed under osmotic stress conditions in the presence of compatible solutes. The 72 kDa His10-tagged fusion protein was purified by a combination of metal-ion affinity and molecular size chromatography. Binding activity and specificity of the immunotoxin to the EGFR-positive pancreatic carcinoma cell line L3.6pl was confirmed by flow cytometry and ELISA. Finally, 425(scFv)-ETA' showed significant toxicity toward this cell line reaching 50% inhibition of cell proliferation at a concentration (IC50) of 7.5 ng/ml. This is the first report documenting the specific cytotoxicity of a recombinant immunotoxin towards metastatic pancreatic carcinoma cells, suggesting that EGFR-specific antibody toxins may become valuable therapeutic reagents for the treatment of pancreatic carcinoma.
与其他任何恶性肿瘤相比，胰腺癌的死亡率仍然最高。主要原因是疾病发现较晚、肿瘤生长高度侵袭性以及早期转移的形成。因此，迫切需要新的有效疗法来改善患者的治疗效果。表皮生长因子受体（EGFR）及其配体的过表达与胰腺癌的肿瘤发生有关，并与不良预后相关。因此，EGFR是一种适合免疫治疗的特异性靶抗原。我们通过将抗EGFR单链片段425（scFv）与铜绿假单胞菌外毒素A（ETA'）的截短突变体融合，构建了一种重组免疫毒素。使用表达载体pBM1.1，在存在相容性溶质的渗透胁迫条件下，功能性425（scFv）-ETA'在周质中表达。通过金属离子亲和色谱和分子大小色谱相结合的方法纯化了72 kDa的His10标签融合蛋白。通过流式细胞术和ELISA证实了免疫毒素对EGFR阳性胰腺癌细胞系L3.6pl的结合活性和特异性。最后，425（scFv）-ETA'对该细胞系显示出显著的毒性，在浓度为7.5 ng/ml时达到50%的细胞增殖抑制率（IC50）。这是第一份记录重组免疫毒素对转移性胰腺癌细胞具有特异性细胞毒性的报告，表明EGFR特异性抗体毒素可能成为治疗胰腺癌的有价值的治疗试剂。
