A single-chain TNF receptor antagonist is an effective inhibitor of TNF mediated cytotoxicity.
A single-chain TNF receptor antagonist is an effective inhibitor of TNF mediated cytotoxicity.
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单链 TNF 受体拮抗剂是 TNF 介导的细胞毒性的有效抑制剂。
DOI：
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
K. Pfizenmaier
中科院分区：
文献类型：
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作者：
D. Moosmayer;S. Dübel;B. Brocks;H. Watzka;C. Hampp;P. Scheurich;M. Little;K. Pfizenmaier
肿瘤坏死因子（TNF）是免疫和炎症反应的重要介质，已被认为是多种自身免疫性和炎症性疾病的主要致病因子。TNF受体TR60在信号传导TNF的致病活性中起关键作用。我们在这里描述的分子克隆和细菌生产的单链抗体（scFv H398）针对TR60具有拮抗活性。通过PCR从鼠杂交瘤细胞系H398分离VH和VL编码序列，克隆到scFv表达载体中并在大肠杆菌中表达。重组抗体（Ab）片段被发现作为一个活跃的可溶性蛋白的周质，但也形成包涵体。从包涵体中纯化的重折叠scFv H398显示出在37 ℃下具有功能性和稳定性，半衰期为50小时。scFv与亲本酶促产生的Fab H398的抗原结合特性的比较揭示，两种Ab片段具有相同的表位特异性和1.5nM的相同抗原结合亲和力。在体外测定中，证明scFv H398是TNF介导的细胞毒性的有效抑制剂，其IC50为22 nM，这与天然Fab H398的拮抗活性相当，其IC50为12 nM。由于scFv H398具有亲本Ab H398的高亲和力TR60结合和受体拮抗活性，但预期在人中抗原性较低，因此它为开发针对TNF介导的疾病的新型治疗试剂提供了有价值的工具。
Tumour necrosis factor (TNF) is an important mediator of immune and inflammatory responses and has been recognized as a major pathogenic factor in several autoimmune and inflammatory diseases. TNF receptor TR60 plays a critical role in signalling the pathogenic activities of TNF. We here describe molecular cloning and bacterial production of a single-chain antibody (scFv H398) directed against TR60 which possesses antagonistic activity. VH and VL encoding sequences were isolated by PCR from the murine hybridoma cell line H398, cloned into a scFv expression vector and expressed in Escherichia coli. The recombinant antibody (Ab) fragment was found as an active soluble protein in the periplasm but also formed inclusion bodies. Re-folded scFv H398 purified from inclusion bodies was shown to be functional and stable at 37 degrees C with a half-life of 50 h. Comparison of the antigen binding characteristics of scFv with the parental enzymatically produced Fab H398 revealed that both Ab fragments have the same epitope specificity and an identical antigen binding affinity of 1.5 nM. In an in vitro assay it was demonstrated that scFv H398 is an efficient inhibitor of TNF mediated cytotoxicity with an IC50 of 22 nM, which is comparable to the antagonistic activity of natural Fab H398 with an IC50 of 12 nM. As scFv H398 possesses the high affinity TR60 binding and receptor antagonistic activity of the parental Ab H398 but is expected to be less antigenic in man, it provides a valuable tool for the development of novel therapeutic reagents against TNF mediated diseases.