Basic studies for development of inhibitor to target nuclear inflammatory cytokine related with sepsis

本研究的目的是设计针对被认为是引起脓毒症的因素的HMGB-1（高迁移率族蛋白-1）的抗体或肽抑制剂，以阐明HMGB-1在脓毒症的炎症、恶化和致死中的作用。利用我们构建的噬菌体抗体库，我们分离到了两株抗HMGB-1的单链抗体。这些抗体以30和47 nM的Kd值与HMGB-1的B结构域结合，并且这些scFv中的一个形成称为双抗体的二聚体。通过LPS刺激的THP-1细胞IL-8释放实验，进一步评价了该单链抗体的抗炎活性。出乎意料的是，这些scFv不能抑制IL-8的释放，但双抗体反而增强了IL-8的释放。这些结果表明，分离的HMGB-1特异性的两个scFv不具有抑制炎症的抑制活性，这是通过从THP-1释放IL-8来估计的，并且双抗体可能通过HMGB-1的二聚化而具有增强IL-8释放的潜力，尽管其机制尚不清楚。通过常规的生物淘选从随机肽库中分离HMGB-1特异性肽被证明是非常困难的，这是因为肽与HMGB-1的非特异性结合。

The purpose of this search is the design of antibody or peptide inhibitor against HMGB-1(High Mobility Group Box Protein-1) which is considered to be a factor causing the sepsis to elucidate the role of the HMGB-1 in inflammation, exacerbation and lethality in sepsis. By using antibody phage display library constipated by us, we isolated two single chain Fv (scFv) antibodies specific to HMGB-1. These antibodies bound to B-domain of HMGB-1 with Kd values of 30 and 47nM, and one of these scFv formed the dimer called diabody. We further estimated the inflammation-blocking activity of the isolated scFv by using IL-8 releasing assay on THP-1 cell which was stimulated with LPS. Unexpectedly, these scFv could not inhibit the IL-8 release but the diabody rather enhanced it. These results indicate that the isolated HMGB-1-specific two scFv don't possesses the inhibitory activity to repress the inflammation which is estimated by IL-8 release from THP-1 and that the diabody has the potential to enhance the IL-8 release probably through the dimerization of HMGB-1, although its mechanism has been still unknown. The isolation of the HMGB-1-specific peptide by the conventional biopanning from the random peptide library was proven to be very difficult, because of the non-specific binding of the phages to HMGB-1.