The Structural Basis for the Immune Recognition of Factor VIII

因子 VIII 免疫识别的结构基础

• 批准号: 10406902
• 负责人: John S. Lollar
• 金额: $ 38.16万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406902
• 关键词: Address; Antibodies; Antigen Presentation; Antigen-Antibody Complex; Antigens; B-Cell Antigen Receptor; Binding; C2 Domain; Collaborations; Complex; Crystallization; DAG/PE-Binding Domain; Deuterium; Development; Electron Microscopy; Electrons; Epitopes; F8 gene; Fab Immunoglobulins; Factor VIII; Fc Receptor; Human; Hydrogen; Immune; Immune response; Immunodominant Epitopes; Immunoglobulin G; Length; Maps; Mass Spectrum Analysis; Methods; Modeling; Molecular Weight; Negative Staining; Protein Analysis; Proteins; Receptor Cell; Resolution; Roentgen Rays; Role; Spectrum Analysis; Structure; Surface Plasmon Resonance; System; Testing; analytical ultracentrifugation; experience; immunogenicity; in vivo; inhibitor; particle; protein complex; protein protein interaction; sedimentation velocity; skill acquisition; stoichiometry; von Willebrand Factor

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.

摘要/摘要（项目1） 该项目的中心假设是FVIII的内在结构和动态特征是负责的 它的免疫原性。该假设的基础是FVIII的显着免疫原性 蛋白质。另外，在早期对FVIII的免疫反应期间，FVIII免疫复合物可能会放大 通过FC受体或B细胞受体（BCR），抗原表现或其他机制的免疫反应。到 解决这一假设，更好地理解FVIII和FVIII免疫的结构和动力学 需要复合物。该项目有三个具体的目标。目标1是开发X射线晶体学 FVIII和FVIII免疫复合物的结构。 FVIII的原子水平分辨率X射线结构的开发， 活化的FVIII和FVIII免疫复合物是更好地理解对免疫反应的基础 FVIII。可用的FVIII X射线结构不是原子水平分辨率。免疫复合物的结构 FVIII C2域可用。但是，没有具有全长或B的免疫复合物结构 域删除的FVIII。我们已经开发了一个高表达平台，该平台产生了fviii的亚克格数量 构建指定的HP47用于晶体学试验。在初步研究中，我们有HP47 FVIII的晶体 衍射超过3.2Å分辨率，正在完善结构。 AIM 2是表征FVIII抗体表位 通过氢氘交换质谱法（HDX MS），电子，电子复合物和FVIII免疫复合物 显微镜（EM）和表面等离子体共振光谱。这些方法是最先进的 完全表征表位的方法。 HDX MS映射研究C1和C2结构域具有 现在，我们获得了〜250-kDa全长FVIII分子的HDX覆盖率， 它将用于绘制整个FVIII分子的免疫主导表位。 AIM 3是表征FVIII 通过沉积速度分析超速离心（SV AUC）通过沉积速度进行免疫复合物。 IgG免疫复合物 可以扩增对同源抗原的免疫响应。但是，FVIII免疫复合物在 尚未探讨对FVIII的免疫反应。识别两个或多个IgG分子的组合 假设的非重叠表位可以形成大型免疫复合物，分子量延伸至 Megadaltons。 SV AUC是识别和表征大蛋白质蛋白复合物的首选方法。 在初步研究中，我们确定了二元FVIII/抗A2 MAB 4A4和FVIII/anti-C2的化学计量法 MAB 3D12复合物和三元FVIII/4A4/3D12复合物。这些研究将扩展以表征 由其他重要类别的抗FVIII mAb形成的免疫复合物的石化元素法。 X射线 晶体学，HDX MS，EM和SV AUC结果合并以开发FVIII IgG免疫模型 复合物。此外，在AIM 3中特征的免疫复合物将在细胞和体内测试 系统与Project 2合作，可能对FVIII的免疫原性做出贡献。