FcRn-enabling strategies for improved thrombolytic therapy

项目总结/摘要 虽然近几十年来治疗性蛋白质的临床使用呈指数级增长，但效用通常受到以下因素的限制： 不利的药代动力学（PK），由快速消除介导。克服这种限制的一种方法是 半衰期延长（HLE），通过生物治疗剂与聚合物（PEG）、血浆蛋白 （白蛋白，IgG）和血细胞。有一个缺乏的数据描述货物蛋白质的性质的影响 和HLE赋予配体对使用这些策略测试的蛋白质的PK的影响。HLE拥有的一个领域是， 推进临床治疗的潜力是急性的、危及生命的、血栓形成（肺栓塞，缺血性中风， 等）。唯一可用的药物治疗是输注纤溶酶原 激活剂，具有不利的药理学特征，由几分钟的半衰期介导，快速失活 血浆中，以及严重的不良反应（如出血性转化）。试点数据表明， 在富含凝血酶的病理性血栓中选择性活化的尿激酶衍生物（scuPA-T）与 白蛋白结合纳米抗体（Nb）导致PK与RBC结合scuPA-T相同， Muzykantov和Cines博士的研究，尽管分布在血浆中，而不是血液的细胞部分。 该提议的中心假设是scuPA-T与白蛋白结合配体的偶联将 提供由新生儿Fc受体（FcRn）介导的延长循环和选择性溶栓 病理性血栓，使止血塞免于溶解。在辅导阶段， 将使用FcRn敲除小鼠直接鉴定FcRn激活的HLE上的货物药物的性质， 阐明由白蛋白结合赋予的HLE的FcRn介导的组分（Aim 1; K99）。基于导频 将在肺栓塞的小鼠模型中研究白蛋白结合scuPA-T的数据、安全性和功效。 这些结果将用于开发预测建模平台，该平台将用于进一步的工程设计。 HLE-scuPA-T构建体的PK优化（Aim 2; K99）。着眼于机械和翻译 为了推进这一策略，将使用以下方法定义白蛋白结合亲和力对scuPA-T PK的作用： 新开发的纳米抗体库（Aim 3; R 00）。此外，凝血酶可裂解的HLE配体将被 设计，允许血栓中scuPA-T的选择性释放，改善向凝块中的扩散和溶解（Aim 3; R 00）。 该提案的主要主题包括识别白蛋白介导的HLE的关键特征， 基于机制的建模，以指导蛋白质治疗的优化和再造。指导团队 已确定与专业知识跨越研究领域，在这个赠款，即血栓形成和 止血，抗体工程和生物系统的数学建模。指导研究将是 在宾夕法尼亚大学进行，它有一个高度合议和协作的教师和广泛的 现有资源进行拟议的研究。这项建议旨在获得专业知识， 作为一名终身教职人员，进行高质量的生物医学研究是成功的必要条件。

PROJECT SUMMARY/ABSTRACT While clinical use of therapeutic proteins has grown exponentially in recent decades, utility is often limited by unfavorable pharmacokinetics (PK), mediated by rapid elimination. One approach to overcome this limitation is half-life extension (HLE), achieved by attachment of biotherapeutics to polymers (PEG), plasma proteins (albumin, IgG), and blood cells. There is a paucity of data describing the impact of properties of cargo proteins and HLE-conferring ligands on PK of proteins tested using these strategies. One area where HLE has the potential to advance clinical therapy is acute, life-threatening, thrombosis (pulmonary embolism, ischemic stroke, etc.). The only pharmacologic treatment available for treatment of this condition is infusion of plasminogen activators, which have an unfavorable pharmacologic profile, mediated by a half-life of minutes, rapid inactivation in plasma, and severe adverse effects (e.g. hemorrhagic transformation). Pilot data demonstrates that conjugation of a derivative of urokinase selectively activated in thrombin-rich pathological thrombi (scuPA-T) to an albumin-binding nanobody (Nb) leads to PK that is identical to RBC-binding scuPA-T, an approach pioneered by Drs. Muzykantov and Cines, albeit with distribution in the plasma rather than the cellular fraction of blood. The central hypothesis of this proposal is that coupling of scuPA-T to albumin-binding ligands will provide prolonged circulation, mediated by the neonatal Fc receptor (FcRn), and selective thrombolysis of pathological thrombi, sparing hemostatic plugs from lysis. In the mentored stage, the impact of properties of the cargo drug on FcRn-enabled HLE will be identified, using FcRn knockout mice to directly elucidate the FcRn-mediated component of HLE conferred by albumin binding (Aim 1; K99). Based on pilot data, safety and efficacy of albumin-binding scuPA-T will be studied in a mouse model of pulmonary embolism. These results will be used to develop a predictive modeling platform that will be used to for further engineering and PK optimization of HLE-scuPA-T constructs (Aim 2; K99). With an eye on mechanistic and translational advancement of this strategy, the role of albumin-binding affinity on the PK of scuPA-T will be defined, using a newly developed library of nanobodies (Aim 3; R00). Additionally, thrombin-cleavable HLE ligands will be devised, permitting selective release of scuPA-T in thrombi, improving diffusion into clots and lysis (Aim 3; R00). Overarching themes of this proposal include identification of critical features of albumin-mediated HLE and mechanism-based modeling to guide optimization and reengineering of protein therapeutics. A mentoring team has been identified with expertise spanning the areas of research in this grant, namely, thrombosis and hemostasis, antibody engineering, and mathematical modeling of biological systems. Mentored research will be conducted at the University of Pennsylvania, which has a highly collegial and collaborative faculty and extensive resources available to conduct the proposed research. This proposal is geared towards gaining the expertise necessary to be successful as a tenured faculty member conducting high quality biomedical research.