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

本研究的目的是设计抗HMGB-1(高迁移率组盒蛋白-1)的抗体或多肽抑制剂，以阐明HMGB-1在脓毒症的炎症、恶化和致死性中的作用。利用自制的噬菌体抗体库，我们分离到了两株针对HMGB-1的单链抗体。这些抗体与HMGB-1的B区结合，Kd值分别为30和47 nM，其中一种单链抗体形成二聚体，称为Diabody。进一步用IL-8释放法检测单链抗体对内毒素刺激的THP-1细胞的抗炎活性。出乎意料的是，这些单链抗体不能抑制IL-8的释放，但糖尿病却增强了IL-8的释放。这些结果表明，分离的HMGB-1特异的两个单链抗体不具有通过THP-1释放IL-8来估计的抑制炎症的活性，Diabody可能通过HMGB-1的二聚化来促进IL-8的释放，尽管其机制尚不清楚。由于噬菌体与HMGB-1的非特异性结合，用常规的生物扫描方法从随机多肽文库中分离HMGB-1特异性多肽是非常困难的。

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.