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，特别是在血液中寿命有限的生物制剂，将改善许多临床环境中的结果。药物递送系统包括聚乙二醇（PEG）-隐形化学和脂质体和聚合物载体被设计来实现这一目标。在这些载体中，红细胞（RBC）似乎非常有吸引力，是作用于血管内靶点的药物持续递送的天然载体。我们设计了一种新颖的方法，用于将治疗性蛋白质与RBC表面进行生物相容性偶联。为了避免修饰的RBC的离体加载和再输注，我们将这些货物与针对小鼠RBC的单克隆抗体（mAb）的单链片段（scFv）融合。这些融合：i）在注射后结合血流中的RBC; ii）安全地循环延长的时间;和iii）被病理介质局部激活并赋予在人类疾病的小鼠模型中非靶向货物所未见的保护作用。为了使这种方法能够临床转化，我们利用了从先前用人RBC免疫的猕猴产生的独特噬菌体展示文库来分离与人和猪RBC结合的灵长类scFv，并将其与治疗部分融合，产生可以在这些动物物种中测试、优化和使用的功能性RBC靶向融合物。利用这些成功，我们现在建议使用RBC来携带本研究中设计的新型靶向生物素，scFv融合血栓调节蛋白（TM）。我们最近的出版物和试验数据显示，scFv/TM在小鼠模型中发挥独特的抗血栓形成和抗炎益处。本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，并将其转化为灵长类scFv/TM嵌合蛋白，结合猪和人RBC，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的效益/风险比。在这个目标中，我们将测试RBC靶向scFv/TM在血栓形成和炎症的小鼠模型中比sTM或APC更有效和安全的假设。目的2：确定scFv/TM的作用机制。我们将区分凝血酶和APC介导与scFv/TM的直接作用，测试RBC糖萼在scFv/TM调节中的作用，并确定TM的精氨酸淬灭凝集素与凝血酶淬灭EGF结构域在介导其抗血栓形成与抗炎活性中的作用。目标3：将scFv/TM翻译成临床上有用的形式。我们将制备灵长类scFv/TM融合体，鉴定人RBC结合位点，测试scFv/TM与人和猪RBC的结合和功能，检查其对RBC生物相容性、循环的影响以及在猪中的作用。

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.