Structural determination of the IdeS-IgG complex to guide therapeutic development of IdeS

IDES是一种由化脓性链球菌产生的免疫球蛋白抗体降解酶，具有广阔的应用前景 治疗免疫球蛋白介导的自身免疫性疾病和预防器官移植的候选药物 拒绝。IDES在人类中的治疗应用受到先前存在的对化脓性链球菌和 艾迪斯。拟议的研究将从结构上表征IDES-Ig G复合体，以获得对 IDES的功能以及它是如何实现如此高的抗体特异性的。从这项工作中收集到的结构性信息 将被用来引导带有聚乙二醇链的IDE的重新表面处理以降低免疫原性 并在保持免疫球蛋白结合和切割活性的同时改善药代动力学特性。具体目标1： 测定IDES-Ig G复合体的分子组成。我们将使用尺寸排斥层析法 用多角度静态光散射(SEC-MALS)和负染色电子显微镜(EM)测试 假设IDES以二聚体的形式与免疫球蛋白结合。SEC-MALS将得到IDES-Ig G复合体的分子量 由此我们可以确定该络合物的化学计量组成。我们将使用负染EM来 想象这个复合体，以表征它的整体构象和组成。结果将有助于确定 IDES是否以2：1或1：1的比例结合免疫球蛋白。具体目标2：IDES-Ig G复合体的结构特征。 我们将使用氢-氚交换(HDX)与质谱仪和X射线结晶学相结合的方法来 阐明IDES-免疫球蛋白复合体的结构，并验证IDES与免疫球蛋白中的外位体结合的假设。 我们将确定IDE和免疫球蛋白中参与相互作用的残基，并告知IDE是如何发挥作用的 对免疫球蛋白有极高的特异性。具体目标3：表面重建和体内试验。我们将检验这一假设 用聚乙二醇化聚乙二醇酯(即聚乙二醇化聚乙二醇酯)重新出现IDES将通过减少 免疫原性和增加其循环半衰期。我们将使用目标2中的结构数据来指导突变 在免疫球蛋白结合所必需的或接近结合界面以防止聚乙二醇化的IDES中赖氨酸残基的含量。突变 将通过使用Rosetta对残留物进行计算分析，然后确定这些 突变是使用与相关酶的序列同源性在进化上表示的。我们建议这一点 合理的方法将产生以保持其酶活性的聚乙二醇重新出现的IDES。一种实时FP 将开发酶试验来验证非聚乙二醇化和聚乙二醇化的IDES变异体的活性。三明治 使用兔多克隆IDES抗体的ELISA将在体外测试聚乙二醇化阻断免疫原性的能力。 体内切割活性和聚乙二醇化IDES候选构建体的清除将通过以下方法在小鼠身上进行检测 测量一段时间内血清免疫球蛋白水平的变化。

IdeS, an IgG-antibody degrading enzyme produced by Streptococcus pyogenes bacteria, is a promising therapeutic candidate for treating IgG-mediated autoimmune disorders and preventing organ transplant rejection. The therapeutic use of IdeS in humans is limited by pre-existing immunity against S. pyogenes and IdeS. The proposed research will structurally characterize the IdeS-IgG complex to gain mechanistic insight into IdeS’s function and how it achieves such high specificity for IgG. Structural information gleaned from this work will be leveraged to guide resurfacing of IdeS with polyethylene glycol (PEG) chains to reduce immunogenicity and improve pharmacokinetic properties while maintaining IgG binding and cleavage activity. Specific Aim 1: Determine the molecular composition of the IdeS–IgG complex. We will use size exclusion chromatography with multi-angle static light scattering (SEC-MALS) and negative stain electron microscopy (EM) to test the hypothesis that IdeS binds as a dimer to IgG. SEC-MALS will yield the molecular weight of the IdeS-IgG complex from which we can determine the stoichiometric composition of the complex. We will use negative-stain EM to visualize this complex to characterize its overall conformation and composition. The results will help to determine whether IdeS binds IgG in a 2:1 or 1:1 ratio. Specific Aim 2: Structurally characterize the IdeS-IgG complex. We will use hydrogen-deuterium exchange (HDX) coupled to mass spectrometry and X-ray crystallography to elucidate the structure of the IdeS-IgG complex and test the hypothesis that IdeS binds to an exosite in IgG. We will identify residues in IdeS and IgG that participate in their interaction and inform on how IdeS is capable of extreme specificity for IgG. Specific Aim 3: Resurface IdeS and test in vivo. We will test the hypothesis that resurfacing IdeS with PEG (i.g., PEGylation) will enhance its therapeutic potential by reducing immunogenicity and increasing its circulatory half-life. We will use structural data from Aim 2 to guide mutation of Lys residues in IdeS necessary for IgG binding or close to binding interfaces to prevent PEGylation. Mutations will be chosen by computational analysis of the residues using Rosetta and then determining whether these mutations are evolutionarily represented using sequence homology to related enzymes. We propose that this rational approach will yield IdeS resurfaced with PEG that maintains its enzymatic activity. A real-time FP enzyme assay will be developed to validate activity of non-PEGylated and PEGylated IdeS variants. Sandwich ELISAs using rabbit polyclonal IdeS antibodies will test the ability of PEGylation to block immunogenicity in vitro. In vivo cleavage activity and clearance of a PEGylated IdeS candidate construct will be assayed in mice by measuring changes in IgG serum levels over time.