Drug delivery by carrier erythrocytes

描述（由申请人提供）：优化药物的药代动力学和生物利用度，特别是在血液中有限生命期的生物制剂，将改善许多临床环境的结果。包括聚乙二醇（PEG）隐身化学和脂质体和聚合物载体在内的药物输送系统被设计用于实现这一目标。在这些载体中，红细胞（RBC）似乎非常有吸引力，是持续递送作用于血管内靶点的药物的天然载体。我们设计了一个原始的方法，涉及生物相容性偶联治疗蛋白的红细胞表面。为了避免体外装载和重新输注修饰的红细胞，我们将这些货物与单克隆抗体（mAb）的单链片段融合到小鼠红细胞（scFv）中。这些融合物：i)注射后与血液中的红细胞结合；Ii)长时间安全循环；iii)被病理介质局部激活，并赋予人类疾病小鼠模型中未见的非靶向货物的保护作用。为了实现这种方法的临床翻译，我们利用了一个独特的噬菌体展示文库，该文库是由先前用人类红细胞免疫的猕猴创建的，分离出与人类和猪红细胞结合的灵长类动物scFv，并将其与治疗性片段融合，产生功能性的红细胞靶向融合物，可以在这些动物物种中进行测试、优化和使用。在这些成功的基础上，我们现在建议利用红细胞来运载本研究设计的一种新的靶向生物疗法，scfv与血栓调节素（TM）融合。我们最近的出版物和试点数据表明，scFv/TM在小鼠模型中具有独特的抗血栓和抗炎作用。该项目的目标是更好地了解这种新型靶向药物的抗血栓和抗炎作用，确定其作用机制，并将其转化为结合猪和人红细胞的灵长类scFv/TM嵌合蛋白，并将我们的研究推向临床应用。我们将追求三个目标：目标1：评估scFv/TM的收益/风险比。在本研究中，我们将验证红细胞靶向scFv/TM在小鼠血栓和炎症模型中比sTM或APC更有效和安全的假设。目的2：明确scFv/TM的作用机制。我们将区分凝血酶和apc介导的与scFv/TM的直接作用，测试红细胞糖萼在scFv/TM调节中的作用，并确定TM的细胞因子猝灭凝集素和凝血素猝灭EGF结构域在其抗血栓和抗炎活性中的作用。目标3：将scFv/TM转化为临床有用的格式。我们将建立灵长类动物scFv/TM融合，确定人类红细胞结合位点，测试scFv/TM与人类和猪红细胞的结合和功能，研究其对红细胞生物相容性、循环和猪的影响。

DESCRIPTION (provided by applicant): Optimizing pharmacokinetics and bioavailability of drugs, especially biological agents with limited life-time in the blood, will improve outcome in a number of clinical settings. Drug delivery systems including polyethylene glycol (PEG)-stealth chemistry and liposomal and polymeric carriers are devised to achieve this goal. Among these carriers, red blood cells (RBC) seem very attractive, natural carriers for sustained delivery of drugs acting upon intravascular targets. We devised an original approach for involving biocompatible coupling of therapeutic proteins to the surface of RBC. To avoid ex vivo loading and re-infusion of modified RBC, we fused these cargoes with a single chain fragment of a monoclonal antibody (mAb) to mouse RBC (scFv). These fusions: i) bind to RBC in the bloodstream after injection; ii) safely circulate for a prolonged time; and, iii) get activated loclly by pathological mediators and confer protective effects not seen with non-targeted cargoes in mouse models of human diseases. To enable clinical translation of this approach, we took an advantage of a unique phage-display library created from a macaque previously immunized with human RBC to isolate primate scFv binding to human and pig RBC and fused it with therapeutic moiety, producing functional RBC-targeted fusions that can be tested, optimized and used in these animal species. Capitalizing on these successes, we now propose to employ RBC for carriage of a novel targeted biotherapeutic designed in this study, scFv-fusion with thrombomodulin (TM). Our recent publication and pilot data show that scFv/TM exerts unique anti-thrombotic and anti-inflammatory benefits in mouse models. The goal of this project is to better understand the anti-thrombotic and anti-inflammatory effects of this novel targeted agent, define its mechanisms of action, and to convert it into a primate scFv/TM chimeric protein that binds both pig and human RBC and bring our studies towards clinical application. We will pursue three Aims: Aim 1: Assess scFv/TM benefit/risk ratio. In this Aim we will test the hypothesis that RBC-targeted scFv/TM is more effective and safe than either sTM or APC in mouse models of thrombosis and inflammation. Aim 2: Define mechanisms of action of scFv/TM. We will differentiate thrombin and APC-mediated vs. direct effects of scFv/TM, test the role of the RBC glycocalyx in scFv/TM regulation and define the role of the cytokine-quenching lectin vs. the thrombin-quenching EGF domains of TM in mediating its anti-thrombotic vs. anti-inflammatory activities. Aim 3: Translate scFv/TM into a clinically useful format. We will produce primate scFv/TM fusion, identify the human RBC binding site, test scFv/TM binding and functionality with human and pig RBC, examine its effects on RBC biocompatibility, circulation and effects in pigs.