Abstract：
IL-23 is a double-subunit cytokine that plays an important role in shaping the immune response. IL-23 was found to be associated with several autoinflammatory diseases by generating sustained inflammatory loops that lead to tissue damage. Antibody neutralization of IL-23 was proven to be effective in ameliorating associated diseases. However, antibodies as large proteins have limited tissue penetration and tend to elicit anti-drug antibodies. Additionally, anti-IL-23 antibodies target only one subunit of IL-23 leaving the other one unneutralized. Here, we attempted to isolate a recycling single domain antibody by phage display. One of IL-23 subunits, p19, was expressed in E. coli fused to Gamillus protein to stabilize the α-helix-only p19. To remove Gamillus binders, two biopanning methods were investigated, first, preselection with Gamillus and second, challenge with IL-23 then on the subsequent round challenge with p19-Gam. The isolation of calcium-dependent and pH-dependent recycling binders was performed with EDTA and citrate buffers respectively. Both methods of panning failed to isolate high-affinity and specific p19 recycling binders, while from the second panning method, a high affinity and specific p19 standard binder, namely H11, was successfully isolated. H11 significantly inhibited the gene expression of IL-17 and IL-22 in IL-23-challenged PBMCs indicating H11 specificity and neutralizing ability for IL-23. The new binder due to its small size can overcome antibodies limitations, also, it can be further engineered in the future for antigen clearance such as fusing it to cell penetrating peptides, granting H11 the ability to clear excess IL-23 and enhancing its potential therapeutic effect.
摘要：
IL-23是一种在形成免疫应答中起重要作用的双亚基细胞因子。发现IL-23通过产生导致组织损伤的持续炎症回路而与几种自身炎症疾病相关。IL-23的抗体中和被证明可有效改善相关疾病。然而,作为大蛋白质的抗体具有有限的组织渗透，并倾向于引发抗药物抗体。此外,抗IL-23抗体仅靶向IL-23的一个亚基,而另一个未中和。这里,我们试图通过噬菌体展示分离出再循环单域抗体。IL-23亚基之一,p19在大肠杆菌中表达,该大肠杆菌融合于Gamillus蛋白以稳定仅有α-螺旋的p19。为了去除γ-菌结合剂,研究了两种生物淘选方法，首先,与Gamillus和第二预选，用IL-23挑战，然后用p19-Gam进行下一轮挑战。钙依赖性和pH依赖性再循环结合剂的分离分别用EDTA和柠檬酸盐缓冲液进行。两种淘选方法都未能分离出高亲和力和特异性的p19回收结合剂，而从第二种平移方法来看，高亲和力和特异性的P19标准粘合剂，即H11被成功分离。H11显著抑制IL-23攻击的PBMC中IL-17和IL-22的基因表达,表明H11特异性和对IL-23的中和能力。新的粘合剂由于其体积小，可以克服抗体的局限性，还,它可以在将来进一步工程用于抗原清除，例如将其与细胞穿透肽融合，给予H11清除过量IL-23的能力并增强其潜在的治疗效果。
