Factor VIII Immunogenicity-Biology and Structure: Project 4

项目4-摘要 我们探讨了抑制免疫反应的生化和结构基础。 FVIII，从我们广泛的同源生物化学背景中提取了一个新的视角 以及相关的凝血蛋白Fv。我们将首次系统地解决以下问题 人类患者数据表明，对人类FVIII的抗体反应在性质和强度上有根本的不同， 对人类生殖器的影响。将这两种蛋白的B结构域缺失的类似变体注射到野生型小鼠中， 我们建议比较总抑制性抗体反应。差异响应的进一步映射将 使用FVIII或Fv的嵌合变体来执行，该嵌合变体包含从人类蛋白质到 鼠标背景。通过检查免疫的光谱，将进一步研究明显的差异。 杂交瘤技术对FV的反应与以前与FVIII建立的技术相比。一秒钟 假说将调查对这两种蛋白质的不同反应是否与机制有关。 用来维持每个原癌因子功能静止的差异。目标2下的进一步研究 FVIII与vWF的结合是否在调节免疫应答中发挥重要作用 辅因子使用免疫策略与不同受损的vWF结合的FVIII突变体。一秒钟 方法将测试FVIII的特定蛋白水解衍生物是否在其生命周期中发挥作用 在询问免疫系统和确定抑制反应方面以前没有考虑过的作用 Fviii.目标3利用我们的Fv(A)衍生品的结构进步来询问结构框架是否 考虑到与FVIII结合的抑制性抗体的影响，几何约束是折衷的 在晶体堆积施加的C域内。我们提出了小角散射和高分辨率的低温系统。 EM的FVIII在溶液中，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，当与膜结合时。我们将使用原型 抗C_1、C_2抗体抑制剂及重组vWF D‘D3片段的共性 认为这些配体以一种次优的方式稳定FVIII内的C结构域，因为它的高亲和力相互作用 有膜的。最后，借鉴Fv(A)和FxA的结构进展，我们将使用x射线 结晶学与其他结构方法相结合来检验抑制作用的假说 抗A2区的抗体分离为影响辅因子与FixA、Fx或两者结合的抗体 在某种程度上是中等程度的。我们建议的多管齐下的方法，在这一U54的更大背景下，将 揭示了FVIII输注后抑制物反应的基本生物化学细节 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.