Clot-Targeted Antithrombotics for Venous Thromboprophylaxis

项目摘要 我们目前缺乏有效的治疗方法来降低静脉血栓栓塞症的风险 随之而来的是出血的风险。集结的调查小组最近发现了单链抗体 选择性地阻断活化的血小板，并在血栓形成部位丰富治疗药物。在 在这个过程中，我们发现选择性靶向激活的血小板允许各种各样的 抗血栓药物是有效的，远低于导致出血的全身浓度。在这 提议，我们假设选择性靶向活化的血小板提供了一种局部浓缩的方法 静脉血栓生长部位的抗血小板、抗凝剂和纤溶剂，而不破坏 基本的止血过程。我们相信，这一战略将使高效制剂的设计成为可能 静脉血栓预防，但不会增加大出血的风险。在这项建议中，我们打算： (1)明确凝血靶向抗血栓药物预防静脉血栓形成的能力 并抑制血栓形成后静脉壁纤维化，不影响止血。“双重”的功效 基于抗血小板和抗凝血剂的单链抗体SCE5-TAP的抗血栓药物 活性将使用静脉血栓形成的小鼠模型来定义。这些研究将评估 SCE5-TAP和相关新变种可预防静脉血栓形成，限制早期和晚期炎症 可抑制血栓引起的静脉壁重塑，而不影响止血。 (2)测定具有抗血小板和纤溶作用的凝血靶向抗血栓药的能力 抑制静脉血栓形成和血栓后静脉壁重塑的活性。的有效性。 抗血栓药物基于SCE5-scuPA，一种兼具抗血小板和纤溶活性的单链抗体将 单独使用小鼠静脉血栓模型或结合SCE5-TAP或相关新技术进行定义 变种。我们还将设计以血栓为靶点的重组杂交构建物(SCE5-TAP-scuPA)，以展示 抗血小板、抗凝血剂和纤溶活性。这些药物抑制血栓形成的能力，促进 血栓的消退，以及限制静脉壁纤维化的发展将被定义。 (3)通过功能性设计减轻血栓靶向抗血栓药物的免疫应答。 通过删除免疫原性T细胞表位来灭活TAP变异体。蜂窝手机 将评估TAP(扁虱抗凝肽)的免疫原性和驱动T细胞表位的T细胞表位 在幼稚的人外周血单核细胞中绘制了反应图谱。将使用计算工具来 设计功能失免疫的TAP变体，将在体外和体外评估其维护情况 抑制FXA活性和降低细胞免疫原性。野生型和由此产生的铅 候选者将在人源化的人类白细胞抗原转基因小鼠体内进行细胞和体液免疫原性评估。 TAP的成功灭活将使我们能够产生去免疫的凝血靶向抗血栓药物。

Project Summary We currently lack effective therapies that reduce the risk of venous thromboembolism without an attendant risk of bleeding. The assembled investigative team has recently identified single chain antibodies that selectively block activated platelets and enrich therapeutics at the site of a developing thrombus. In the process, we have discovered that selective targeting of activated platelets allows a wide variety of antithrombotic agents to be efficacious, well below systemic concentrations that cause bleeding. In this proposal, we postulate that selective targeting to activated platelets provides a means to locally concentrate antiplatelet, anticoagulant, and fibrinolytic agents at the site of a growing venous thrombus without disrupting essential hemostatic processes. We believe that this strategy will enable the design of high potency agents for venous thromboprophylaxis, but without an increased risk of major bleeding. In this proposal, we intend to: (1) Define the ability of clot-targeted “dual pathway” antithrombotics to prevent venous thrombosis and inhibit postthrombotic vein wall fibrosis without hemostatic disturbance. The efficacy of “dual pathway” antithrombotics based on SCE5-TAP, a single chain antibody with both antiplatelet and anticoagulant activity will be defined using murine models of venous thrombosis. These studies will evaluate the capacity of SCE5-TAP and related new variants to prevent venous thrombosis, limit early and late inflammatory responses, and inhibit thrombus-induced remodeling of the vein wall without hemostatic disturbance. (2) Determine the capacity of clot-targeted antithrombotics that display anti-platelet and fibrinolytic activity to inhibit venous thrombosis and postthrombotic vein wall remodeling. The effectiveness of antithrombotics based on SCE5-scuPA, a single chain antibody with both antiplatelet and fibrinolytic activity will be defined using murine models of venous thrombosis alone or in combination with SCE5-TAP or related new variants. We will also design recombinant clot-targeted hybrid constructs (SCE5-TAP-scuPA) that display antiplatelet, anticoagulant, and fibrinolytic activity. The ability of these agents to inhibit thrombosis, promote thrombus resolution, and limit the development of vein wall fibrosis will be defined. (3) Mitigate the immune response to clot-targeted antithrombotics with the design of functionally deimmunized TAP variants through deletion of immunogenic T cell epitopes. The cellular immunogenicity of TAP (tick anticoagulant peptide) will be assessed and T cell epitopes, which drive the response mapped in naïve human peripheral blood mononuclear cells. Computational tools will be used to design functionally deimmunized TAP variants that will be evaluated in vitro and ex vivo for their maintenance of FXa inhibitory activity and their reduction of cellular immunogenicity. The wild type and resulting lead candidate will be assessed for cellular and humoral immunogenicity in vivo in humanized HLA transgenic mice. The successful deimmunization of TAP will allow us to generate deimmunized clot-targeted antithrombotics.