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

提高基于烟草的合成血小板设计系统中 sdFv 的生产效率

• 批准号: 10384333
• 负责人: Seema Nandi
• 金额: $ 25.85万
• 依托单位: SELSYM BIOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-10 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384333
• 关键词: Actins; Affinity; Animal Model; Area; Binding; Biological Products; Bleeding time procedure; Blood Coagulation Disorders; Blood Platelets; Blood Transfusion; Blood flow; Cause of Death; Cell Surface Receptors; Cellular Infiltration; Cessation of life; Clinical; Coagulation Process; Complex; Contracts; Coupled; Diabetes Mellitus; Emergency Medicine; Escherichia coli; Fiber; Fibrin; Goals; Hemorrhage; Hemostatic Agents; Hospitalization; Immunoglobulin Fragments; Immunologics; Impairment; In Vitro; Infection; Injury; Keloid; Life; Mediating; Military Personnel; Mission; Morphology; Myosin ATPase; Nicotiana; Obesity; Particle Size; Pharmacologic Substance; Plants; Platelet Transfusion; Post-Translational Protein Processing; Production; Property; Proteins; Public Health; Risk; Rodent Model; Role; Safety; Shapes; Site; System; Techniques; Technology; Therapeutic; Tobacco; Translations; Trauma; Traumatic injury; United States National Institutes of Health; Woman; base; blood product; clinical development; cost; cost effectiveness; design; efficacy validation; fibrin receptor; healing; improved; in vivo; innovation; large scale production; men; novel; particle; plant growth/development; preclinical development; response; scale up; standard of care; stem; translational potential; vascular injury; wound environment; wound healing

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.

项目摘要 在平民和军事创伤性伤害中，不受控制的出血是一个重大的临床问题。在 两种情况下，住院前的解血都是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，乳杆状形成，血液流量不足而变得复杂 或糖尿病和肥胖等疾病。凝块形成对于创伤后出血至关重要 并涉及循环血小板的激活，这些血小板磨损受伤部位并骨料形成血小板 插头，茎出血。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 - 肌球蛋白机械将力施加到凝块网络，收缩和 稳定凝块并促进其作为临时基质的作用，以支持随后的细胞浸润 伤口环境。在创伤性损伤的情况下，解血会导致血小板耗尽， 损害他们停止流血和促进康复的能力。血小板输血是当前的护理标准； 但是，孤立的血小板的保质期短，导致了主要的供应链问题。此外， 与输血有关的潜在免疫问题突出了至关重要的需求 开发血小板替代方案以治疗创伤后出血。我们最近开发了类似合成的血小板样 颗粒（PLP）是由高度可变形的微粒与触觉靶向抗体片段相连的 能够通过在受伤部位与纤维蛋白形成的高亲和力结合来磨害伤害。我们的最初 研究表明，PLP能够概括天然血小板的几个功能，包括凝块 体外增强，减少出血时间和体内创伤模型的总体失血，并 受伤后体内改善愈合反应；但是，赋予纤维蛋白靶向抗体片段 到目前 和翻译潜力。该项目的长期目标是开发大型批处理生产技术 这些纤维蛋白特异性的抗体片段，可促进止血PLP的翻译用于紧急情况 医学应用，以增加凝结并减少血液流失以及由于解血而导致的相关死亡。 该应用的目的是优化A中的大量纤维蛋白特异性抗体片段的产生 尼古蒂亚纳本尼亚娜植物表达系统，并验证所得PLP的功效增强 凝结。我们的中心假设是使用N. benthamiana产生的抗体片段产生的PLP 表达系统将具有与先前设计的PLP相当的稳定性和功效 大肠杆菌中产生的抗体片段，而本奈米亚乳杆菌的表达系统将提供更大的 在大肠杆菌表达系统中，抗体碎片的产量在可比的稳定性和均匀性下， 从而支持将这项技术推向大型动物模型的进一步临床前发展。 该项目的具体目的是：1）优化和评估抗体的产量，稳定性和均匀性 本氏菌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PLP的增强功效。