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.

Public Health Relevance

Rapid elimination from bloodstream limits effects of many drugs, especially labile biotherapeutics. This dictates use of large doses and repetitive injection reducing the safety of therapy. Drug delivery systems including diverse carriers prolonging drug circulation 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, by fusing these cargoes with a polypeptide that binds to RBC. In this project, we will apply this approach for RBC-mediated vascular delivery of a novel targeted biotherapeutic agent, thrombomodulin, to optimize management of thrombosis and inflammation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Kindzelski, Andrei L
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University of Pennsylvania
Schools of Medicine
United States
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Villa, Carlos H; Pan, Daniel C; Johnston, Ian H et al. (2018) Biocompatible coupling of therapeutic fusion proteins to human erythrocytes. Blood Adv 2:165-176
Pan, Daniel C; Myerson, Jacob W; Brenner, Jacob S et al. (2018) Nanoparticle Properties Modulate Their Attachment and Effect on Carrier Red Blood Cells. Sci Rep 8:1615
Carnemolla, Ronald; Villa, Carlos H; Greineder, Colin F et al. (2017) Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury. FASEB J 31:761-770
Villa, Carlos H; Cines, Douglas B; Siegel, Don L et al. (2017) Erythrocytes as Carriers for Drug Delivery in Blood Transfusion and Beyond. Transfus Med Rev 31:26-35
Villa, Carlos H; Seghatchian, Jerard; Muzykantov, Vladimir (2016) Drug delivery by erythrocytes: ""Primum non nocere"". Transfus Apher Sci 55:275-280
Villa, Carlos H; Anselmo, Aaron C; Mitragotri, Samir et al. (2016) Red blood cells: Supercarriers for drugs, biologicals, and nanoparticles and inspiration for advanced delivery systems. Adv Drug Deliv Rev 106:88-103
Pan, Daniel; Vargas-Morales, Omayra; Zern, Blaine et al. (2016) The Effect of Polymeric Nanoparticles on Biocompatibility of Carrier Red Blood Cells. PLoS One 11:e0152074
Villa, Carlos H; Pan, Daniel C; Zaitsev, Sergei et al. (2015) Delivery of drugs bound to erythrocytes: new avenues for an old intravascular carrier. Ther Deliv 6:795-826
Rahmani, Sahar; Villa, Carlos H; Dishman, Acacia F et al. (2015) Long-circulating Janus nanoparticles made by electrohydrodynamic co-jetting for systemic drug delivery applications. J Drug Target 23:750-8
Muzykantov, Vladimir R (2013) Drug delivery carriers on the fringes: natural red blood cells versus synthetic multilayered capsules. Expert Opin Drug Deliv 10:1-4

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