PDF(Pubmed)
Abstract：
The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.
摘要：
2007年，将治疗性抗C5抗体引入临床实践，激发了补体靶向疗法的发展。这导致了最近批准的C3抑制肽，针对C1s的抗体和临床前和临床开发中的几种补体抑制剂的完整管道。然而,没有有效抑制所有三种补体起始途径和靶向宿主细胞表面标志物以及补体调理素的抑制剂。为了克服这一点，我们设计了一种新的融合蛋白，结合了三种天然补体调节蛋白衰变加速因子（DAF）的选定结构域，因子H(FH)和补体受体1(CR1)。这种三重融合补体抑制剂(TriFu)与多种变体及其结构单元一起被重组表达和纯化。我们通过表面等离子体共振分析了这些蛋白质的配体结合亲和力和衰变加速活性。此外,我们使用标准的经典和替代途径限制性溶血试验,在PNH红细胞溶血试验之后,在几种体外/离体试验中进行了补体抑制试验.建立了一种新型的替代途径疾病C3肾小球病（C3G）的体外模型，以评估抑制剂阻止C3在内皮细胞上沉积的潜力。除了以类似酶的方式灭活补体转化酶的新型工程三重融合变体之外，测试靶向C3、C5、因子B和因子D的化学计量补体抑制剂作为比较物。三重融合方法产生了有效的补体抑制剂,其有效地抑制所有三种补体起始途径,同时靶向表面标志物。
