Increasing efficiency of sdFv production in a tobacco-based system for synthetic platelet design

项目总结 失控出血是平民和军事创伤中的一个重大临床问题； 在这两种情况下，住院前失血都是导致男性和女性死亡的主要原因。 此外，创伤后的愈合可能会因感染、瘢痕疙瘩形成、血流不足、 或糖尿病和肥胖症等疾病。血栓形成是创伤后止血的关键 包括激活循环中的血小板，使其磨练到损伤部位并聚集形成血小板 堵塞，止血。活化的血小板还结合在损伤部位形成的纤维蛋白纤维形成血小板- 纤维蛋白网。然后，血小板利用肌动蛋白-肌球蛋白机械向凝块网络施加力，收缩和 稳定血栓并促进其作为临时基质的作用，以支持随后的细胞渗透 伤口环境。在创伤的情况下，失血会导致血小板耗尽， 削弱了他们止血和促进愈合的能力。输注血小板是目前的护理标准； 然而，孤立的血小板的保质期很短，导致了重大的供应链问题。另外， 与输血产品相关的潜在免疫学问题突出了关键的未得到满足的需求 开发治疗创伤后出血的血小板替代品。我们最近开发出了合成的类血小板 由高度变形的微粒与纤维蛋白靶向抗体片段偶联而成的微粒(PLP) 能够通过与损伤部位形成的纤维蛋白高亲和力结合来磨练损伤。我们最初的 研究表明，PLP能够概括天然血小板的几种功能，包括凝血 体外增强，减少体内创伤啮齿动物模型的出血次数和总失血量，以及 改善了体内损伤后的愈合反应；然而，提供 到目前为止，这些对PLP的能力是在细菌表达系统中小批量生产的，限制了放大 和翻译潜力。该项目的长期目标是开发大批量生产技术 这些纤维蛋白特异性抗体片段有助于翻译止血PLP以用于紧急情况 用于增强凝血和减少因失血而导致的失血和相关死亡的药物应用。 这项应用的目的是优化大批量生产纤维蛋白特异性抗体片段的方法 烟草植物表达系统的构建及扩增效果的验证 凝血。我们的中心假设是利用本氏新城疫原虫产生的抗体片段产生PLP 表达系统的稳定性和有效性将与之前设计的使用 在大肠杆菌中产生的抗体片段，以及N.benthamiana表达系统将提供更大的 抗体片段产量具有与在大肠杆菌表达系统中实现的类似的稳定性和均一性， 从而支持将这项技术向前推进到大型动物模型的进一步临床前开发。 本项目的具体目标是：1)优化和评价抗体的产量、稳定性和均质性。 在N.benthamiana植物表达系统中产生的片段和2)确定纤维蛋白结合和凝块 所得到的PLP的增强效果。

PROJECT SUMMARY Uncontrolled bleeding is a significant clinical problem in both civilian and military traumatic injuries; in both cases, exsanguination prior to hospitalization is the primary cause of death for both men and women. Furthermore, healing following trauma can be complicated by infection, keloid formation, insufficient blood flow, or conditions such as diabetes and obesity. Clot formation is critical to the cessation of bleeding after trauma and involves the activation of circulating platelets that hone to the site of injury and aggregate to form a platelet plug, stemming bleeding. Activated platelets also bind fibrin fibers forming at a site of injury to form a platelet- fibrin mesh. Platelets then utilize actin-myosin machinery to apply forces to the clot network, contracting and stabilizing the clot and facilitating its role as a provisional matrix to support subsequent cellular infiltration of the wound environment. In cases of traumatic injury, exsanguination can cause platelets to become depleted, impairing their ability to stop bleeding and promote healing. Platelet transfusion is the current standard of care; however, isolated platelets have a short shelf-life, contributing to major supply chain issues. Additionally, potential immunologic concerns associated with transfusion of blood products highlights the critical unmet need to develop platelet alternatives to treat bleeding after trauma. We have recently developed synthetic platelet-like particles (PLPs) created from highly deformable microparticles coupled to fibrin-targeting antibody fragments that are capable of honing to injuries through high affinity binding to fibrin forming at the sites of injury. Our initial studies demonstrate that PLPs are able to recapitulate several functions of native platelets, including clot augmentation in vitro, decreasing bleeding times and overall blood loss in in vivo rodent models of trauma, and improved healing responses in vivo following injury; however, the fibrin-targeting antibody fragments that confer these abilities to PLPs are thus far produced in bacterial expression systems in small batches, limiting scale up and translational potential. The long-term goal of this project is to develop large batch production techniques for these fibrin-specific antibody fragments to facilitate the translation of hemostatic PLPs for use in emergency medicine applications to augment clotting and decrease blood loss and associated deaths due to exsanguination. The objective of this application is to optimize large batch production of fibrin-specific antibody fragments in a Nicotiana benthamiana plant expression system and validate the efficacy of the resultant PLPs for augmenting clotting. Our central hypothesis is that PLPs created using antibody fragments produced in the N. benthamiana expression system will have comparable stability and efficacy as previously designed PLPs created using antibody fragments produced in E. coli, and that the N. benthamiana expression system will provide larger antibody fragment yield at comparable stability and homogeneity as those achieve in E. coli expression systems, thereby supporting moving this technology forward into further preclinical development in large animal models. The specific aims of this project are: 1) Optimize and evaluate yield, stability, and homogeneity for antibody fragments produced in an N. benthamiana plant expression system and 2) Determine fibrin-binding and clot augmentation efficacy of the resultant PLPs.