Bi-Sen Ding

Targeted delivery of therapeutics to endothelium

The endothelium is a target for therapeutic and diagnostic interventions in a plethora of human disease conditions including ischemia, inflammation, edema, oxidative stress, thrombosis and hemorrhage, and metabolic and oncological diseases. Unfortunately, drugs have no affinity to the endothelium, thereby limiting the localization, timing, specificity, safety, and effectiveness of therapeutic interventions. Molecular determinants on the surface of resting and pathologically altered endothelial cells, including cell adhesion molecules, peptidases, and receptors involved in endocytosis, can be used for drug delivery to the endothelial surface and into intracellular compartments. Drug delivery platforms such as protein conjugates, recombinant fusion constructs, targeted liposomes, and stealth polymer carriers have been designed to target drugs and imaging agents to these determinants. We review endothelial target determinants and drug delivery systems, describe parameters that control the binding of drug carriers to the endothelium, and provide examples of the endothelial targeting of therapeutic enzymes designed for the treatment of acute vascular disorders including ischemia, oxidative stress, inflammation, and thrombosis.

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.

Cerebrovascular Thromboprophylaxis in Mice by Erythrocyte-Coupled Tissue-Type Plasminogen Activator

Background— Cerebrovascular thrombosis is a major source of morbidity and mortality after surgery, but thromboprophylaxis in this setting is limited because of the formidable risk of perioperative bleeding. Thrombolytics (eg, tissue-type plasminogen activator [tPA]) cannot be used prophylactically in this high-risk setting because of their short duration of action and risk of causing hemorrhage and central nervous system damage. We found that coupling tPA to carrier red blood cells (RBCs) prolongs and localizes tPA activity within the bloodstream and converts it into a thromboprophylactic agent, RBC/tPA. To evaluate the utility of this new approach for preventing cerebrovascular thrombosis, we examined the effect of RBC/tPA in animal models of cerebrovascular thromboembolism and ischemia. Methods and Results— Preformed fibrin microemboli were injected into the middle carotid artery of mice, occluding downstream perfusion and causing severe infarction and 50% mortality within 48 hours. Preinjected RBC/tPA rapidly lysed nascent cerebral thromboemboli, providing rapid, durable reperfusion and reducing morbidity and mortality. These beneficial effects were not achieved by preinjection of tPA, even at a 10-fold higher dose, which increased mortality from 50% to 90% by 10 hours after embolization. RBC/tPA injected 10 minutes after tail amputation to simulate postsurgical hemostasis did not cause bleeding from the wound, whereas soluble tPA caused profuse bleeding. RBC/tPA neither aggravated brain damage caused by focal ischemia in a filament model of middle carotid artery occlusion nor caused postthrombotic hemorrhage in hypertensive rats. Conclusions— These results suggest a potential RBC/tPA utility as thromboprophylaxis in patients who are at risk for acute cerebrovascular thromboembolism.

Targeting of a mutant plasminogen activator to circulating red blood cells for prophylactic fibrinolysis.

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl3 models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

Anchoring Fusion Thrombomodulin to the Endothelial Lumen Protects against Injury-induced Lung Thrombosis and Inflammation

RATIONALE Endothelial thrombomodulin (TM) regulates thrombosis and inflammation. Diverse forms of pulmonary and vascular injury are accompanied by down-regulation of TM, which aggravates tissue injury. We postulated that anchoring TM to the endothelial surface would restore its protective functions. OBJECTIVES To design an effective and safe strategy to treat pulmonary thrombotic and inflammatory injury. METHODS We synthesized a fusion protein, designated scFv/TM, by linking the extracellular domain of mouse TM to a single-chain variable fragment of an antibody to platelet endothelial cell adhesion molecule-1 (PECAM-1). The targeting and protective functions of scFv/TM were tested in mouse models of lung ischemia-reperfusion and acute lung injury (ALI) caused by intratracheal endotoxin and hyperoxia, both of which caused approximately 50% reduction in the endogenous expression of TM. MEASUREMENTS AND MAIN RESULTS Biochemical assays showed that scFv/TM accelerated protein C activation by thrombin and bound mouse PECAM-1 and cytokine high mobility group-B1. After intravenous injection, scFv/TM preferentially accumulated in the mouse pulmonary vasculature. In a lung model of ischemia-reperfusion injury, scFv/TM attenuated elevation of early growth response-1, inhibited pulmonary deposition of fibrin and leukocyte infiltration, and preserved blood oxygenation more effectively than soluble TM. In an ALI model, scFv/TM, but not soluble TM, suppressed activation of nuclear factor-kappaB, inflammation and edema in the lung and reduced mortality without causing hemorrhage. CONCLUSIONS Targeting TM to the endothelium using an scFv anchor enhances its antithrombotic and antiinflammatory effectiveness in models of ALI.

Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis

Thrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombins substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance. To overcome this problem, we fused TM with a single chain fragment (scFv) of an antibody targeted to red blood cells. scFv/TM catalyzes thrombin-mediated generation of activated protein C and binds to circulating RBCs without apparent damage, thereby prolonging its circulation time and bioavailability orders of magnitude compared with soluble TM. In animal models, a single dose of scFv/TM, but not soluble TM, prevents platelet activation and vascular occlusion by clots. Thus, scFv/TM serves as a prodrug and provides thromboprophylaxis at low doses (0.15 mg/kg) via multifaceted mechanisms inhibiting platelets and coagulation.

Delivery of Anti-Platelet-Endothelial Cell Adhesion Molecule Single-Chain Variable Fragment-Urokinase Fusion Protein to the Cerebral Vasculature Lyses Arterial Clots and Attenuates Postischemic Brain Edema

Efficacy and safety of current means to prevent cerebrovascular thrombosis in patients at high risk of stroke are suboptimal. In theory, anchoring fibrinolytic plasminogen activators to the luminal surface of the cerebral endothelium might arrest formation of occlusive clots in this setting. We tested this approach using the recombinant construct antiplatelet-endothelial cell adhesion molecule (PECAM) single-chain variable fragment (scFv)-urokinase-type plasminogen activator (uPA), fusing low-molecular-weight single-chain urokinase-type plasminogen activator with a scFv of an antibody directed to the stably expressed endothelial surface determinant PECAM-1, implicated in inflammation and thrombosis. Studies in mice showed that scFv-uPA, but not unconjugated uPA 1) accumulates in the brain after intravascular injection, 2) lyses clots lodged in the cerebral arterial vasculature without hemorrhagic complications, 3) provides rapid and stable cerebral reperfusion, and 4) alleviates post-thrombotic brain edema. Effective and safe thromboprophylaxis in the cerebral arterial circulation by anti-PECAM scFv-uPA represents a prototype of a new paradigm to prevent recurrent cerebrovascular thrombosis.

Vascular Immunotargeting to Endothelial Determinant ICAM-1 Enables Optimal Partnering of Recombinant scFv-Thrombomodulin Fusion with Endogenous Cofactor

The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC) and thrombomodulin (TM), have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1) exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR), the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1) favors scFv/TM collaboration with EPCR. Indeed: i) endothelial targeting scFv/TM to ICAM-1 provides ∼15-fold greater activation of protein C than its PECAM-targeted counterpart; ii) blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii) anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.

Dual targeting of therapeutics to endothelial cells: collaborative enhancement of delivery and effect

Anchoring pharmacologic agents to the vascular lumen has the potential to modulate critical processes at the blood‐tissue interface, avoiding many of the off‐target effects of systemically circulating agents. We report a novel strategy for endothelial dual targeting of therapeutics, which both enhances drug delivery and enables targeted agents to partner enzymatically to generate enhanced biologic effect. Based on the recent discovery that paired antibodies directed to adjacent epitopes of platelet endothelial cell adhesion molecule (PECAM)‐1 stimulate each others binding, we fused single‐chain fragments (scFv) of paired anti‐mouse PECAM‐1 antibodies to recombinant murine thrombomodulin (TM) and endothelial protein C receptor (EPCR), endothelial membrane proteins that partner in activation of protein C (PC). scFv/TM and scFv/EPCR bound to mouse endothelial PECAM‐1 with high affinity (EC50 1.5 and 3.8 nM, respectively), and codelivery induced a 5‐fold increase in PC activation not seen when TM and EPCR are anchored to distinct cell adhesion molecules. In a mouse model of acute lung injury, dual targeting reduces both the expression of lung inflammatory markers and trans‐endothelial protein leak by as much as 40%, as compared to either agent alone. These findings provide proof of principle for endothelial dual targeting, an approach with numerous potential biomedical applications.—Greineder, C. F., Brenza, J. B., Carnemolla, R., Zaitsev, S., Hood, E.D., Pan, D.C., Ding, B.‐S., Esmon, C. T., Chacko, A. M., Muzykantov, V. R. Dual targeting of therapeutics to endothelial cells: collaborative enhancement of delivery and effect. FASEB J. 29, 3483‐3492 (2015). www.fasebj.org