Factor VIII Immunogenicity-Biology and Structure: Project 4

项目4-摘要 我们的方法的生物化学和结构基础的研究抑制免疫反应， FVIII，从我们广泛的生物化学背景中得出一个新的观点， 以及相关的凝血蛋白FV。我们将首次系统地讨论 人类患者数据表明，对人FVIII的抗体应答在性质和强度上根本不同， 与人FV的相似。使用注射到野生型小鼠中的两种蛋白质的可比较的B结构域缺失变体， 我们建议比较总的抑制性抗体应答。差分响应的进一步映射将 使用FVIII或FV的嵌合变体进行，所述嵌合变体含有来自人蛋白的单个结构域， 鼠标背景通过检查免疫谱，将进一步研究明显的差异。 通过杂交瘤技术与先前用FVIII建立的抗体对FV的应答进行比较。第二 这一假设将研究对这两种蛋白质的不同反应是否与机制有关。 差异用于维持每个原辅因子的功能静止。目标2下的研究 解决FVIII与vWF的结合是否在调节对vWF的免疫应答中起重要作用， 使用具有不同受损的vWF结合的FVIII突变体的免疫策略。第二 该方法将测试正常情况下在其生命周期中产生的定义的FVIII蛋白水解衍生物是否发挥作用， 以前未考虑的作用，询问免疫系统，并确定抑制反应， 第VIII节。目标3利用我们在FV（a）衍生物方面的结构性进展，来探讨结构性框架是否 考虑抑制性抗体与FVIII结合的影响受到几何约束的影响 在由晶体堆积施加的C域内。我们提出了小角度散射和高分辨率的低温- 在溶液中的FVIII的EM，并结合到膜纳米盘，以解决是否构象灵活性内 C结构域是其溶液构象的基础，并且当与膜结合时。我们将使用prototype C1和C2的抗体抑制剂以及vWF的重组D 'D3片段，以测试 这些配体稳定FVIII内的C结构域，使其高亲和力相互作用处于次优状态， 用膜。最后，从FV（a）和FXa的结构进展中，我们将使用x射线 晶体学与其他结构方法相结合，以测试抑制性的假设， A2结构域的抗体分离成影响辅因子与FIXa、FX或两者结合的抗体 在某种程度上，中间。我们提出的多管齐下的方法，在这个U 54的更大背景下，将 揭示了FVIII输注抑制剂反应的生物化学基础的基本细节， 血友病A患者。

Project 4-Abstract We approach the investigation of the biochemical and structural basis for the inhibitory immune response to FVIII, with a fresh perspective drawn from our extensive background in the biochemistry of the homologous and related coagulation protein, FV. We will, for the first time, systematically address the implication from human patient data that the antibody response to human FVIII differs fundamentally, in character and intensity, to that of human FV. Using comparable B domain deleted variants of both proteins injected into wild type mice, we propose to compare the total inhibitory antibody response. Further mapping of the differential response will be performed using chimeric variants of FVIII or FV containing individual domains from the human protein on the mouse background. Obvious differences will be further studied by examining the spectrum of the immune response to FV by hybridoma technology in comparison to that previously established with FVIII. A second hypothesis will investigate whether the differential response to the two proteins relates to mechanistic differences employed to maintain functional quiescence in each procofactor. Studies under Aim 2 further address whether the binding of FVIII to vWF plays a fundamental role in regulating the immune response to the cofactor using immunization strategies with FVIII mutants with variously impaired vWF binding. A second approach will test whether defined proteolytic derivatives of FVIII, normally produced during its life cycle, play a previously unconsidered role in interrogating the immune system and determining the inhibitory response to FVIII. Aim 3 draws on our structural advances with FV(a) derivatives to ask whether the structural framework for considering the effects of inhibitory antibody binding to FVIII is compromised by geometric constraints within the C domains imposed by crystal packing. We propose small angle scattering and high resolution Cryo- EM of FVIII in solution and bound to membrane nanodiscs to address whether conformational flexibility within the C domains underlies its solution conformation and when bound to membranes. We will use prototypic antibody inhibitors to C1 and C2 as well as the recombinant D’D3 fragment of vWF to test for the commonality of the idea that these ligands stabilize the C domains within FVIII in a sub-optimal for its high affinity interaction with membranes. Finally drawing from structural advances with FV(a) and FXa, we will use x-ray crystallography in combination with the other structural approaches to test the hypothesis that inhibitory antibodies to the A2 domain segregate into those that impact the binding of the cofactor to FIXa, to FX or both to some intermediate extent. Our proposed multi-pronged approach, within the larger context of this U54, will shed new light on basic details of the biochemistry underlying the inhibitor response to FVIII infusions in hemophilia A patients.