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 不受控制的出血在平民和军事创伤中都是一个重要的临床问题; 在这两种情况下，住院前放血是男女死亡的主要原因。 此外，创伤后的愈合可能会因感染、瘢痕疙瘩形成、血流不足， 或诸如糖尿病和肥胖症的病症。血凝块的形成对创伤后出血的停止至关重要 并涉及循环血小板的活化，这些血小板聚集到损伤部位并聚集形成血小板 栓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以形成血小板- 纤维蛋白网。然后，血小板利用肌动蛋白-肌球蛋白机制向凝块网络施加力，使其收缩， 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，放血会导致血小板耗尽， 削弱了他们止血和促进愈合的能力。血小板输注是目前的标准治疗; 然而，分离的血小板具有较短的保存期限，从而导致主要的供应链问题。此外，本发明还 与血液制品输注相关的潜在免疫学问题突出了关键的未满足需求 开发血小板替代品来治疗创伤后出血。我们最近开发出了合成的血小板样物质 由与纤维蛋白靶向抗体片段偶联的高度可变形微粒产生的颗粒（PLP） 其能够通过与在损伤部位形成的纤维蛋白的高亲和力结合而对损伤进行珩磨。我们最初 研究表明，PLPs能够重现天然血小板的几种功能，包括凝血功能， 体外增强，减少体内啮齿动物创伤模型中的出血时间和总失血量，以及 损伤后体内愈合反应的改善;然而， 到目前为止，PLPs的这些能力在细菌表达系统中以小批量产生，限制了规模放大 和平移潜力。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促进止血PLPs的翻译， 用于增强凝血和减少失血以及由于失血而导致的相关死亡的医学应用。 本申请的目的是优化纤维蛋白特异性抗体片段的大批量生产， 本氏烟草（Nicotiana benthamiana）植物表达系统，并验证所得PLPs用于增强 凝血我们的中心假设是，PLPs是用N.本氏 表达系统将具有与先前设计的PLP相当的稳定性和功效，所述PLP使用 在E. coli，N. benthamiana表达系统将提供更大的 抗体片段产量与E.大肠杆菌表达系统， 从而支持将该技术向前推进到大型动物模型中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、稳定性和均一性 在N. benthamiana植物表达系统和2）测定纤维蛋白结合和凝块 增强所得到的PLP的功效。