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的风险，而不需要 伴随出血的风险。调查小组最近发现了单链抗体 其选择性地阻断活化的血小板并在血栓形成的部位富集治疗剂。在 在这一过程中，我们发现，选择性靶向活化的血小板， 抗血栓药物是有效的，远低于引起出血的全身浓度。在这 我们假设，选择性靶向活化的血小板提供了一种局部集中的手段， 抗血小板、抗凝剂和纤维蛋白溶解剂，用于静脉血栓生长部位， 基本止血过程。我们相信，这一策略将使设计高效能的药物， 静脉血栓预防，但不会增加大出血的风险。在本提案中，我们打算： (1)明确血栓靶向“双途径”抗血栓药物预防静脉血栓形成的能力 抑制血栓后静脉壁纤维化而不影响止血。“双重”的功效 SCE 5-TAP是一种具有抗血小板和抗凝剂双重作用的单链抗体， 使用静脉血栓形成的鼠模型来定义活性。这些研究将评估 SCE 5-TAP和相关的新变体，以防止静脉血栓形成，限制早期和晚期炎症 反应，并抑制血栓诱导的静脉壁重塑，而不会出现止血障碍。 (2)确定显示抗血小板和纤维蛋白溶解的凝块靶向抗血栓药物的能力 抑制静脉血栓形成和血栓形成后静脉壁重塑的活性。的有效性 SCE 5-scuPA是一种具有抗血小板和纤溶活性的单链抗体， 使用静脉血栓形成的鼠模型单独或与SCE 5-TAP或相关新的 变体。我们还将设计重组凝块靶向杂交构建体（SCE 5-TAP-scuPA）， 抗血小板、抗凝血和纤维蛋白溶解活性。这些药剂抑制血栓形成、促进血栓形成的能力是显著的。 血栓消退和限制静脉壁纤维化的发展。 (3)通过设计功能性抗血栓药物减轻对凝块靶向抗血栓药物的免疫应答 通过缺失免疫原性T细胞表位来去除去免疫化TAP变体。蜂窝 TAP（蜱抗凝肽）的免疫原性将被评估，T细胞表位，其驱动 在幼稚人外周血单核细胞中绘制的应答。计算工具将用于 设计功能性去免疫化TAP变体，其将在体外和离体评价其维持 的FXa抑制活性和它们的细胞免疫原性的降低。野生型和由此产生的铅 将在人源化HLA转基因小鼠中评估候选人的体内细胞和体液免疫原性。 TAP的成功去免疫化将使我们能够产生去免疫化的凝块靶向抗血栓药物。

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.