Barth S, Huhn M, Matthey B, Schnell R, Tawadros S, Schinköthe T, Lorenzen J, Diehl V, Engert A
Department I of Internal Medicine, University of Cologne, Cologne, Germany.
Int J Cancer. 2000 Jun 1;86(5):718-24. doi: 10.1002/(sici)1097-0215(20000601)86:5<718::aid-ijc18>3.0.co;2-n.
Since clinical phase-I/II trials in patients with resistant Hodgkin's lymphoma treated with the chemically linked anti-CD25 ricin-A-chain immunotoxin RFT5-SMPT-dgA indicate promising results for patients with minimal residual disease, we constructed a new immunotoxin by fusing the RFT5 single-chain variable fragment to a deletion mutant of Pseudomonas exotoxin A (ETA'). The recombinant protein was directed into the periplasmic space of E. coli by means of the pET-derived expression vector pBM1.1 and our newly developed expression/purification method. Biologically active RFT5(scFv)-ETA' was isolated by freezing/thawing and purified by immobilized metal-ion affinity and molecular-size-chromatography. RFT5(scFv)-ETA' was subsequently used for the treatment of disseminated human Hodgkin's lymphoma in a SCID-mouse model. The mean survival time (MST) of L540rec-challenged SCID mice was 38.1 days. A single i.v. injection of 40 microg recombinant immunotoxin (rIT) 1 day after tumor inoculation resulted in 100% tumor-free mice, extending the MST to more than 220 days (p < 0.0001). The blood-distribution time T(1/2)alpha was 39.65 min, the serum elimination time T(1/2)alpha, 756.6 min. All animals were assessed for soluble interleukin-2 receptor alpha, which is directly correlated to tumor burden. Soluble CD25 was not detectable in mice treated with the rIT. Our findings, concerning potent anti-tumor effects of a recombinant anti-CD25 immunotoxin against disseminated Hodgkin's lymphoma in SCID mice reported here demonstrate that RFT5(scFv)-ETA' might be suitable for further evaluation against Hodgkin's lymphoma in humans.
由于用化学连接的抗CD25蓖麻毒素A链免疫毒素RFT5-SMPT-dgA治疗难治性霍奇金淋巴瘤患者的临床I/II期试验表明，对微小残留病患者有良好效果，我们通过将RFT5单链可变片段与铜绿假单胞菌外毒素A（ETA'）的缺失突变体融合构建了一种新的免疫毒素。借助pET衍生的表达载体pBM1.1和我们新开发的表达/纯化方法，将重组蛋白导入大肠杆菌的周质空间。通过冻融法分离出具有生物活性的RFT5（scFv）-ETA'，并通过固定化金属离子亲和色谱和分子大小色谱进行纯化。随后，RFT5（scFv）-ETA'用于SCID小鼠模型中播散性人类霍奇金淋巴瘤的治疗。接种L540rec的SCID小鼠的平均生存时间（MST）为38.1天。肿瘤接种后1天单次静脉注射40μg重组免疫毒素（rIT），可使小鼠无瘤率达到100%，MST延长至220天以上（p<0.0001）。血液分布时间T（1/2）α为39.65分钟，血清消除时间T（1/2）β为756.6分钟。对所有动物检测了与肿瘤负荷直接相关的可溶性白细胞介素-2受体α。在用rIT治疗的小鼠中未检测到可溶性CD25。我们在此报道的关于重组抗CD25免疫毒素对SCID小鼠播散性霍奇金淋巴瘤具有强大抗肿瘤作用的研究结果表明，RFT（scFv）-ETA'可能适合进一步用于人类霍奇金淋巴瘤的评估。
