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首页> 美国卫生研究院文献>Iranian Journal of Pharmaceutical Research : IJPR >Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha
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Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha

机译：抗肿瘤坏死因子α的新型人源化单链可变片段抗体的设计与构建

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The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of diseases such as Crohn’s disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some inflammatory diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized single chain fragment variable mAb against human TNF-α (hD2). The subsequent cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in-vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies.
机译：促炎性细胞因子TNF-α在克罗恩病，牛皮癣，银屑病关节炎和类风湿性关节炎等疾病的发展和持久性中起主要作用，是使用抗TNF-α治疗的基础。 TNF-α的中和或其与相应受体的结合的阻断主要用作针对某些炎性疾病的治疗策略。这项研究旨在研究针对TNF-α的人源化单链抗体（scFv）的产生。因此，选择鼠单克隆抗体D2 mAb通过互补决定区（CDR）移植方法进行人源化。简而言之，用特定的人单链支架置换来自D2 mAb的CDR导致了针对人TNF-α（hD2）的新型人源化单链片段可变mAb的产生。随后将hD2克隆到合适的表达载体pGEX-6P-1中，导致52 kDa GST融合蛋白在大肠杆菌中表达，大部分以包涵体形式存在。通过添加4 M尿素并随后进行透析以回收可溶性形式的融合蛋白，可以实现GST-hD2包涵体的溶解和重折叠。然后通过固定的谷胱甘肽通过亲和色谱法纯化可溶性GST-hD2。 GST下拉实验显示GST-hD2与TNF-α蛋白之间存在正相互作用。此外，MTT分析的结果表明，纯化的GST-hD2具有TNF-α中和活性（Kd为1.03nM），因此hD2具有被开发为治疗剂的潜力。但是，与其他抗TNF-α抗体相比，需要更多的研究来阐明hD2的体内TNF-α中和活性的潜力。

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