Moosmayer D, Dübel S, Brocks B, Watzka H, Hampp C, Scheurich P, Little M, Pfizenmaier K
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Ther Immunol. 1995 Feb;2(1):31-40.
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.
肿瘤坏死因子（TNF）是免疫和炎症反应的重要介质，已被公认为多种自身免疫性和炎症性疾病的主要致病因素。TNF受体TR60在传递TNF的致病活性信号中起关键作用。我们在此描述了一种针对TR60的单链抗体（scFv H398）的分子克隆和细菌生产，该抗体具有拮抗活性。通过PCR从小鼠杂交瘤细胞系H398中分离出编码VH和VL的序列，克隆到scFv表达载体中并在大肠杆菌中表达。重组抗体（Ab）片段在周质中以活性可溶性蛋白形式存在，但也形成包涵体。从包涵体中纯化并重新折叠的scFv H398在37℃下具有功能且稳定，半衰期为50小时。将scFv与亲本酶促产生的Fab H398的抗原结合特性进行比较，结果表明两种Ab片段具有相同的表位特异性和1.5 nM的相同抗原结合亲和力。在体外试验中证明，scFv H398是TNF介导的细胞毒性的有效抑制剂，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，但预计在人体内抗原性较低，因此它为开发针对TNF介导疾病的新型治疗试剂提供了有价值工具。
