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IgG抗体降解酶， 用于治疗IgG介导的自身免疫疾病和预防器官移植的治疗候选物 排斥反应IdeS在人类中的治疗用途受到预先存在的针对S的免疫的限制。化脓性和 IdeS.拟议的研究将对IdeS-IgG复合物进行结构表征，以深入了解其机制 IdeS的功能以及它如何实现对IgG的高度特异性。从这项工作中收集的结构信息 将用于指导IdeS与聚乙二醇（PEG）链的表面置换，以降低免疫原性 并改善药代动力学性质，同时保持IgG结合和切割活性。具体目标1： 测定IdeS-IgG复合物的分子组成。我们将使用尺寸排阻色谱法 用多角度静态光散射（SEC-MALS）和负染色电子显微镜（EM）检测 IdeS作为二聚体与IgG结合的假设。SEC-MALS将产生IdeS-IgG复合物的分子量 由此我们可以确定络合物的化学计量组成。我们将使用负染色EM， 可视化该复合物以表征其整体构象和组成。结果将有助于确定 IdeS是否以2：1或1：1的比例结合IgG。具体目的2：对IdeS-IgG复合物进行结构表征。 我们将使用氢氘交换（HDX）结合质谱和X射线晶体学， 阐明IdeS-IgG复合物的结构并测试IdeS结合IgG中的外部位点的假设。 我们将确定IdeS和IgG中参与其相互作用的残基，并告知IdeS如何能够 对免疫球蛋白G的特异性。具体目标3：IdeS表面再处理和体内测试。我们将检验这个假设 用PEG（即，聚乙二醇化）将通过减少 免疫原性和增加其循环半衰期。我们将使用来自Aim 2的结构数据来指导突变 IdeS中IgG结合所必需的或接近结合界面以防止PEG化的Lys残基。突变 将通过使用Rosetta对残差进行计算分析来选择，然后确定这些 使用与相关酶的序列同源性在进化上表示突变。我们建议， 合理的方法将产生用保持其酶活性的PEG重新表面化的IdeS。实时FP 将开发酶测定以验证非PEG化和PEG化IdeS变体的活性。三明治 使用兔多克隆IdeS抗体的ELISA将测试PEG化在体外阻断免疫原性的能力。 PEG化的IdeS候选构建体的体内切割活性和清除将通过以下方法在小鼠中测定： 测量IgG血清水平随时间的变化。

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.