Barth S, Huhn M, Wels W, Diehl V, Engert A
Medizinische Klinik I der Universitat zu Koln, Labor fur Immunotherapie, D-50924 Koln, Germany.
Int J Mol Med. 1998 Jan;1(1):249-56. doi: 10.3892/ijmm.1.1.249.
The data of a closed phase I/II trial in patients with resistant Hodgkin's lymphoma indicate promising results using a chemically linked anti-CD25 ricin-A immunotoxin (IT) (RFT5-SMPT-dgA). This IT is based on the high-affinity moab RFT5. Since recombinant DNA technology permits the readier production of large amounts of ITs, we constructed a new RFT5-based fusion toxin [RFT5(scFv)-ETA']. We isolated mRNA from the hybridoma cell line RFT5, synthesized first strand cDNA and performed RT-PCR. Amplified coding regions of the light and heavy chain variable domains were joined together with a synthetic (Gly4-Ser)3 linker. The resulting single chain variable fragment (scFv) was fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-induced expression in Escherichia coli, the 70 kDa His-tagged fusion protein [RFT5(scFv)-ETA'] was isolated by osmotic shock and sonication under denaturing conditions. The recombinant toxin was purified on a Ni2+-NTA chelating sepharose and eluted with 250 mM imidazole. Pooled protein was renatured, dialyzed and concentrated by precipitation. Binding properties of RFT5(scFv)-ETA' were assessed on the CD25-expressing cell line L540cy by ELISA, immunohistochemistry and FACS analysis. CD25-specific binding was confirmed by immunoprecipitation experiments with recombinant human IL-2 receptor alpha. The in vitro toxicity of the chimeric protein was tested on the Hodgkin-derived cell lines L540cy, L428, L1236, a monocyte cell line U937 and a Burkitt lymphoma cell line BL38. RFT5(scFv)-ETA' inhibited protein biosynthesis of L540cy and L428 cells by 50% at concentrations (IC50) of 18 and 12 ng/ml, respectively. CD25-specific toxicity was confirmed by competitive toxicity assays. These data confirm for the first time binding specificity and toxicity of a recombinant anti-CD25 immunotoxin, against Hodgkin-derived cell lines; its applicability on Hodgkin's lymphoma needs yet to be evaluated in vivo.
一项针对难治性霍奇金淋巴瘤患者的封闭性I/II期试验数据表明，使用化学连接的抗CD25蓖麻毒素A免疫毒素（IT）（RFT5-SMPT-dgA）取得了有前景的结果。这种IT基于高亲和力单克隆抗体RFT5。由于重组DNA技术使大量IT的生产更加容易，我们构建了一种新的基于RFT5的融合毒素[RFT5（scFv）-ETA']。我们从杂交瘤细胞系RFT5中分离mRNA，合成第一链cDNA并进行逆转录聚合酶链反应。轻链和重链可变区的扩增编码区通过合成的（Gly4-Ser）3接头连接在一起。所得的单链可变片段（scFv）与缺乏细胞结合结构域I的修饰铜绿假单胞菌外毒素A（ETA'）融合。在大肠杆菌中经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达后，通过渗透休克和变性条件下的超声处理分离出70 kDa的带有His标签的融合蛋白[RFT5（scFv）-ETA']。重组毒素在Ni2+-NTA螯合琼脂糖上纯化，并用250 mM咪唑洗脱。合并的蛋白经复性、透析并通过沉淀浓缩。通过酶联免疫吸附测定（ELISA）、免疫组织化学和荧光激活细胞分选（FACS）分析评估RFT5（scFv）-ETA'在表达CD25的细胞系L540cy上的结合特性。用重组人白细胞介素-2受体α进行免疫沉淀实验证实了CD25特异性结合。在霍奇金来源的细胞系L540cy、L428、L1236、单核细胞系U937和伯基特淋巴瘤细胞系BL38上测试了嵌合蛋白的体外毒性。RFT5（scFv）-ETA'在浓度（半数抑制浓度，IC50）分别为18和12 ng/ml时，对L540cy和L428细胞的蛋白质生物合成抑制率达50%。通过竞争性毒性试验证实了CD25特异性毒性。这些数据首次证实了重组抗CD25免疫毒素对霍奇金来源细胞系的结合特异性和毒性；其在霍奇金淋巴瘤上的适用性尚需在体内进行评估。
