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Structure and Stability Studies of Factor VIII to Overcome the Immune Response

克服免疫反应的因子 VIII 的结构和稳定性研究

基本信息

批准号：
7939168
负责人：
Paul Clinton Spiegel
金额：
$ 38.93万
依托单位：
WESTERN WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-16 至 2013-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7939168
关键词：
Affect Antibodies Antibody Formation Autoantibodies Binding Blocking Antibodies Blood Circulation Blood Platelets Blood coagulation C2 Domain Comparative Study Complex Complication Computer Analysis Crystallography Data Development Disease Engineering Epitopes Factor IXa Factor VIII Factor Xa Family suidae Genes Genetic Glycoproteins Goals Half-Life Hemophilia A Hemorrhage Human Immune response Incidence Individual Infusion procedures Investigation Lead Life Limb structure Membrane Mutagenesis Nature Pathogenicity Patients Plasma Property Protein Engineering Proteins Recombinant DNA Recombinants Regulation Replacement Therapy Roentgen Rays Serine Protease Structure Surface Therapeutic Thermodynamics Thrombin Treatment Factor Ursidae Family antibody inhibitor base cofactor comparative design dosage human F8 protein improved insight male patient population public health relevance release factor response von Willebrand Factor

项目摘要

DESCRIPTION (provided by applicant): Blood coagulation factor VIII is a non-enzymatic glycoprotein cofactor that is essential for proper regulation of the blood coagulation cascade. Genetic deficiencies in factor VIII cause hemophilia A, which affects 1 in 5,000 males worldwide. Treatment for hemophilia A consists of repeated infusions of recombinant or plasma-derived factor VIII, which can result in the development of inhibitor antibodies that arise in approximately one-third of the hemophilia A patient population that receives therapy. The C2 domain of factor VIII is essential for proper interactions with von Willebrand factor and the membrane surface of activated platelets, and is a major epitope that is recognized by inhibitory antibodies. Recent studies indicate that a high incidence of the antibody response result in antibodies that block the activation of factor VIII by the serine proteases, thrombin or factor Xa. These "non-classical" antibodies possess a high level of pathogenicity and display positive cooperativity with "classical" antibodies, resulting in a dramatic decrease in the level of factor VIII activity. Much of the potency of the pathogenic response may correspond to the marginal stability of the human factor VIII protein. Indeed, factor VIII constructs that have increased stability, such as porcine factor VIII, can overcome the immune response and result in a modest level of factor VIII activity in circulation. This proposal aims to use structure-based computational protein design and x-ray crystallography to better understand the stability of the factor VIII C2 domain and the nature of its interactions with "nonclassical" antibody inhibitors. Additionally, we propose to understand the structural basis for cooperativity between the "classical" and "nonclassical" antibody types that target the factor VIII C2 domain. The specific aims of this project are: (1) Comparative study of the thermodynamic and functional properties of human and porcine FVIII C2 domains; (2) Structure-based computational protein design to engineer a thermo-stabilized FVIII C2 domain; (3) X-ray crystal structure of a human FVIII C2 domain/nonclassical antibody complex; and (4) X-ray crystal structure of a C2 domain/classical/nonclassical antibody ternary complex. PUBLIC HEALTH RELEVANCE: Hemophilia A is a bleeding disorder that affects 1 in 5,000 males worldwide. Upon treatment with factor VIII, inhibitor antibodies develop which often worsens their condition and makes therapy more difficult and expensive. This proposal aims to provide a better understanding of how these antibodies become pathogenic and will lead to the development of better treatments for hemophilia A patients. Hemophilia A is caused by the disruption of the gene encoding blood coagulation factor VIII and is the most common form of hemophilia, affecting 1 in 5,000 males worldwide. The most significant complication in the management of hemophilia A is the development of an inhibitory immune response to therapeutically infused factor VIII. Studying the structural basis of antibody recognition of factor VIII as well as using protein engineering to improve the stability of factor VIII may lead to improved therapies for hemophiliacs worldwide.

描述（由申请方提供）：凝血因子VIII是一种非酶促糖蛋白辅因子，对凝血级联反应的适当调节至关重要。因子VIII的遗传缺陷导致血友病A，其影响全世界5，000名男性中的1名。血友病A的治疗包括重复输注重组或血浆源性因子VIII，这可能导致抑制剂抗体的产生，约三分之一接受治疗的血友病A患者人群中会出现抑制剂抗体。因子VIII的C2结构域对于与血管性血友病因子和活化血小板的膜表面的适当相互作用是必需的，并且是被抑制性抗体识别的主要表位。最近的研究表明，抗体应答的高发生率导致抗体阻断丝氨酸蛋白酶、凝血酶或因子Xa对因子VIII的活化。这些“非经典”抗体具有高水平的致病性，并显示出与“经典”抗体的正协同性，导致因子VIII活性水平的显著降低。致病反应的大部分效力可能对应于人因子VIII蛋白的边缘稳定性。事实上，具有增加的稳定性的因子VIII构建体，例如猪因子VIII，可以克服免疫应答并导致循环中适度水平的因子VIII活性。该提案旨在使用基于结构的计算蛋白质设计和X射线晶体学来更好地了解因子VIII C2结构域的稳定性及其与“非经典”抗体抑制剂相互作用的性质。此外，我们建议了解的“经典”和“非经典”的抗体类型，靶向因子VIII C2结构域之间的协同性的结构基础。本项目的具体目标是：（1）人和猪FVIII C2结构域的热力学和功能特性的比较研究;（2）基于结构的计算蛋白质设计以工程化热稳定的FVIII C2结构域;（3）人FVIII C2结构域/非经典抗体复合物的X射线晶体结构;（4）人FVIII C2结构域/非经典抗体复合物的X射线晶体结构。和（4）C2结构域/经典/非经典抗体三元复合物的X射线晶体结构。公共卫生相关性：血友病A是一种出血性疾病，影响全球5,000名男性中的1名。在用因子VIII治疗时，抑制剂抗体产生，这通常使他们的病情恶化，并使治疗更加困难和昂贵。该提案旨在更好地了解这些抗体如何致病，并将导致开发更好的血友病A患者治疗方法。血友病A是由编码凝血因子VIII的基因破坏引起的，是血友病的最常见形式，影响全世界每5，000名男性中的1名。血友病A管理中最显著的并发症是对治疗性输注的因子VIII产生抑制性免疫应答。研究抗体识别因子VIII的结构基础以及使用蛋白质工程来提高因子VIII的稳定性可能会导致改善全球血友病患者的治疗。