The Structural Basis for the Immune Recognition of Factor VIII

摘要/摘要（项目1） 该项目的中心假设是，fVIII的内在结构和动力学特征负责 其免疫原性。这一假设的基础是与其他免疫原性相比，FVIII具有显著的免疫原性。 proteins.此外，在对fVIII的早期免疫应答期间，fVIII免疫复合物可扩增免疫原性。 通过Fc受体或B细胞受体（BCR）、抗原呈递或其它机制免疫应答。到 为了解决这一假设，更好地了解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。目的一是发展X射线晶体学 fVIII和fVIII免疫复合物的结构。发展fVIII的原子级分辨率X射线结构， 活化的fVIII和fVIII免疫复合物是更好地理解免疫应答的基础， fVIII.可用的fVIII X射线结构不是原子级分辨率。免疫复合物的结构 可获得FVIII C2结构域。然而，没有与全长或B的免疫复合物的结构 结构域缺失fVIII.我们已经开发了一个高表达平台，可以产生亚克数量的FVIII， 命名为HP47的构建体用于晶体学试验。在初步研究中，我们有HP 47 fVIII的晶体， 分辨率超过3.2 μ m，并正在完善结构。目的2是表征FVIII抗体表位 通过氢氘交换质谱法（HDX MS），电子探针， 显微镜（EM）和表面等离子体共振光谱。这些方法都是最先进的 用于表征表位的互补方法。C1和C2结构域的HDX MS图谱研究 由我们组执行。我们现在已经获得了~ 250-kDa全长fVIII分子的HDX覆盖， 其将用于绘制整个FVIII分子的免疫显性表位。目的3是表征FVIII 免疫复合物的沉降速度分析超滤（SV AUC）。IgG免疫复合物 可以增强对同源抗原的免疫应答。然而，fVIII免疫复合物在免疫系统中的作用是不确定的。 对FVIII的免疫应答尚未被探索。两种或多种IgG分子的组合， 假设非重叠表位可以形成大的免疫复合物， 兆道尔顿。SV AUC是鉴定和表征大蛋白质-蛋白质复合物的首选方法。 在初步研究中，我们确定了二元fVIII/抗A2 MAb 4A 4和fVIII/抗C2 MAb 4A 4的化学计量。 MAb 3D 12复合物和三元fVIII/4A 4/3D 12复合物。这些研究将扩展到表征 由另外重要类别的抗-fVIII MAb形成的免疫复合物的化学计量。x射线 结合晶体学、HDX MS、EM和SV AUC结果，开发fVIII IgG免疫模型 配合物此外，将在细胞和体内测试目标3中表征的免疫复合物 与项目2合作的系统可能对FVIII的免疫原性有贡献。

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.