The Structural Basis for the Immune Recognition of Factor VIII

摘要/摘要(项目1) 这个项目的中心假设是FVIII的内在结构和动态特征负责 它的免疫原性。这一假说的基础是FVIII与其他疫苗相比具有显著的免疫原性 蛋白质。此外，在对FVIII的早期免疫反应中，FVIII免疫复合体可能会放大 免疫反应通过Fc受体或B细胞受体(BCR)、抗原提呈或其他机制实现。至 解决这一假说，更好地理解FVIII和FVIII免疫的结构和动力学 复合体是必需的。这个项目有三个具体目标。目标1是发展X射线结晶学 FVIII和FVIII免疫复合物的结构。FVIII原子能级分辨X射线结构的发展， 激活的FVIII和FVIII免疫复合体是更好地理解免疫反应的基础 Fviii.可用的FVIII X射线结构不是原子级别的分辨率。沙门氏菌免疫复合物的结构 FVIII C2域可用。然而，没有全长或B的免疫复合物的结构 域已删除Fviii。我们已经开发了一种高表达平台，可以产生亚克量的FVIII 构建用于结晶学实验的HP47。在初步研究中，我们发现了HP47FVIII的晶体 衍射率超过3.2？分辨率，并且正在改进结构。目的2是鉴定FVIII抗体表位 和FVIII免疫络合物的氢氚交换质谱学(HDX MS)，电子 显微镜(EM)和表面等离子体共振谱。这些方法是最先进的。 表位特征的互补方法。C_1和C_2结构域的HDX MS作图研究 都是由我们团队表演的。我们现在已经获得了~250 kDa全长FVIII分子的HDX覆盖， 它将被用于绘制整个FVIII分子的免疫优势表位图。目标3是描述FVIII的特征 沉降速度分析超速离心法免疫复合物(SV AUC)。免疫球蛋白免疫复合物 可以放大对同源抗原的免疫反应。然而，FVIII免疫复合体在人类免疫系统中的作用 对FVIII的免疫应答还没有被探索。识别两个或多个免疫球蛋白G分子的组合 假设不重叠的表位可以形成大的免疫复合体，其分子量可以延伸到 百万吨级。SV AUC是识别和表征大型蛋白质-蛋白质复合体的首选方法。 在初步研究中，我们测定了FVIII/抗A2单抗4A4和FVIII/抗C2的化学计量比 单抗3D12和FVIII/4A4/3D12三元配合物。这些研究将扩展到表征 由其他重要的抗FVIII单抗形成的免疫复合体的化学计量比。X射线 结晶学、HDX-MS、EM和SV AUC结果相结合建立FVIII免疫球蛋白模型 复合体。此外，将在细胞和体内测试AIM 3中表征的免疫复合体 与项目2合作，为FVIII的免疫原性做出可能的贡献。

Summary/Abstract (Project 1) The central hypothesis of this project is that intrinsic structural and dynamic features of fVIII are responsible for its immunogenicity. The basis for this hypothesis is the remarkable immunogenicity of fVIII compared to other proteins. Additionally, during the early immune response to fVIII, fVIII immune complexes may amplify the immune response through Fc receptor or B cell receptor (BCR), antigen presentation or other mechanisms. To address this hypothesis, a better understanding of the structures and dynamics of fVIII and fVIII immune complexes is required. There are three Specific Aims in this project. Aim 1 is to develop X-ray crystallographic structures of fVIII and fVIII immune complexes. Development of atomic level resolution X-ray structures of fVIII, activated fVIII and fVIII immune complexes is fundamental to a better understanding of the immune response to fVIII. The available fVIII X-ray structures are not at atomic level resolution. Structures of immune complexes of the fVIII C2 domain are available. However, there are no structures of immune complexes with full-length or B domain-deleted fVIII. We have developed a high-expression platform that yields sub-gram quantities of a fVIII construct designated HP47 for crystallographic trials. In preliminary studies, we have crystals of HP47 fVIII that diffract beyond 3.2 Å resolution and are refining the structure. Aim 2 is to characterize fVIII antibody epitopes and fVIII immune complexes by hydrogen deuterium exchange mass spectrometry (HDX MS), electron microscopy (EM) and surface plasmon resonance spectroscopy. These methods are state-of-the-art complementary methods to characterize epitopes. HDX MS mapping studies of the C1 and C2 domain have been performed by our group. We have now obtained HDX coverage of the ~ 250-kDa full-length fVIII molecule, which will be used to map immunodominant epitopes throughout the fVIII molecule. Aim 3 is to characterize fVIII immune complexes by sedimentation velocity analytical ultracentrifugation (SV AUC). IgG immune complexes can amplify the immune response to the cognate antigen. However, the role of fVIII immune complexes in the immune response to fVIII has not been explored. A combination of two or more IgG molecules that recognize non-overlapping epitopes hypothetically can form large immune complexes with molecular weights extending to megadaltons. SV AUC is the method of choice for identifying and characterizing large protein-protein complexes. In preliminary studies, we have determined the stoichiometry of binary fVIII/anti-A2 MAb 4A4 and fVIII/anti-C2 MAb 3D12 complexes and the ternary fVIII/4A4/3D12 complex. These studies will be extended to characterize the stoichiometries of immune complexes formed by additional important classes of anti-fVIII MAbs. X-ray crystallographic, HDX MS, EM and SV AUC results be combined to develop models of fVIII IgG immune complexes. Additionally, the immune complexes characterized in Aim 3 will be tested in cellular and in vivo systems in collaboration with Project 2 for their possible contribution to the immunogenicity of fVIII.