Novel Therapeutics for Acquired Thrombotic Thrombocytopenic Purpura

描述(申请人提供)：血栓性血小板减少性紫癜(TTP)是一种致命的综合征。获得性TTP主要是由抑制ADAMTS13酶的自身抗体引起的。血浆置换是迄今为止唯一有效的治疗方法。在本提案的目的1中，我们将对一系列新的重组ADAMTS13变异体进行重新设计和鉴定，这些变异体具有更高的比活性，但能抵抗获得性TTP患者的自身抗体的抑制。这一目标的完成将为ADAMTS13的结构与功能关系提供新的见解，并改变我们今天治疗TTP的方式。在目标2中，我们建议使用我们新颖的突破性的氢交换与质谱分析相结合的方法来确定氨基酸分辨率下的抗原结合表位。此外，我们还将确定一组抑制性单链抗体(S)在实验室建立的小鼠动脉血栓形成和TTP模型中的致病性。本研究获得的信息可能有助于我们理解获得性TTP的分子机制，并为未来的治疗合理设计具有所需特性(如抵抗自身抗体抑制)的新型重组ADAMTS13突变体。最后，在目标3中，我们将验证这样一个假设，即在血小板中异位表达野生型ADAMTS13和功能获得/抗体抵抗型ADAMTS13变体将直接将治疗酶靶向损伤部位，而不会被循环中的抗ADAMTS13抗体抑制。总之，这项研究的完成将为ADAMTS13的结构-功能关系提供新的见解，有助于我们理解获得性TTP的机制，并为这种致命的TTP综合征的潜在治疗提供新的工具。

DESCRIPTION (provided by applicant): Thrombotic thrombocytopenic purpura (TTP) is a fatal syndrome. Acquired TTP is mainly caused by autoantibodies that inhibit ADAMTS13 enzyme. Plasma exchange is the only effective therapy available to date. In Aim1 of this proposal, we will reengineer and characterize a series of novel recombinant ADAMTS13 variants that exhibit increased specific activity, but are resistant to inhibition by autoantibodies from patients with acquired TTP. The completion of this aim will provide novel insights into the structure-function relationship of ADAMTS13 and change how we treat TTP today. In Aim 2, we propose to determine the antigenic binding epitopes at the amino acid resolution using our novel and groundbreaking deuterium exchange coupled with mass spectrometric analysis approaches. In addition, we will determine the pathogenicity of a panel of inhibitory scFV(s) in murine models of arterial thrombosis and TTP established in the laboratory. The information gained from this study may help our understanding of the molecular mechanisms of acquired TTP and the rational designing of novel recombinant ADAMTS13 variants with desired properties (such as resistance to autoantibody inhibition) for future therapy. Finally, in Aim 3, we will test the hypothesis that ectopic expression of wild-type ADAMTS13 and gain-of-function/antibody-resistant ADAMTS13 variants in platelets would target the therapeutic enzyme directly to sites of injury without being inhibited by circulating anti-ADAMTS13 antibodies. Overall, the information obtained from the completion of the proposed study will provide novel insight into the structure-function relationship of ADAMTS13, help our understandings of the mechanisms of acquired TTP, and provide novel tools for potential therapy of such a fatal TTP syndrome.