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

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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.