Size and targeting to PECAM vs ICAM control endothelial delivery, internalization and protective effect of multimolecular SOD conjugates.

Controlled endothelial delivery of SOD may alleviate abnormal local surplus of superoxide involved in ischemia-reperfusion, inflammation and other disease conditions. Targeting SOD to endothelial surface vs. intracellular compartments is desirable to prevent pathological effects of external vs. endogenous superoxide, respectively. Thus, SOD conjugated with antibodies to cell adhesion molecule PECAM (Ab/SOD) inhibits pro-inflammatory signaling mediated by endogenous superoxide produced in the endothelial endosomes in response to cytokines. Here we defined control of surface vs. endosomal delivery and effect of Ab/SOD, focusing on conjugate size and targeting to PECAM vs. ICAM. Ab/SOD enlargement from about 100 to 300nm enhanced amount of cell-bound SOD and protection against extracellular superoxide. In contrast, enlargement inhibited endocytosis of Ab/SOD and diminished mitigation of inflammatory signaling of endothelial superoxide. In addition to size, shape is important: endocytosis of antibody-coated spheres was more effective than that of polymorphous antibody conjugates. Further, targeting to ICAM provides higher endocytic efficacy than targeting to PECAM. ICAM-targeted Ab/SOD more effectively mitigated inflammatory signaling by intracellular superoxide in vitro and in animal models, although total uptake was inferior to that of PECAM-targeted Ab/SOD. Therefore, both geometry and targeting features of Ab/SOD conjugates control delivery to cell surface vs. endosomes for optimal protection against extracellular vs. endosomal oxidative stress, respectively.

Anti-inflammatory effect of targeted delivery of SOD to endothelium: mechanism, synergism with NO donors and protective effects in vitro and in vivo.

Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect. Ab/SOD, but not non-targeted SOD injected in mice alleviated endotoxin-induced leukocyte adhesion in the cerebral vasculature and protected brain from ischemia-reperfusion injury. Transfection of endothelial cells with SOD, but not catalase inhibited NFκB signaling and expression of Vascular Cell Adhesion Molecule-1 induced by both cytokines and TLR agonists. These results affirmed that Ab/SOD-quenched superoxide anion produced by endothelial cells in response to proinflammatory agents mediates NFκB activation. Furthermore, Ab/SOD potentiates anti-inflammatory effect of NO donors in endothelial cells in vitro, as well as in the endotoxin-challenged mice. These results demonstrate the central role of intracellular superoxide as a mediator of pro-inflammatory activation of endothelium and support the notion of utility of targeted interception of this signaling pathway for management of acute vascular inflammation.

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 thrombin's 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.

Modulation of endothelial targeting by size of antibody-antioxidant enzyme conjugates.

Endothelial targeting of antioxidant enzymes attenuates acute vascular oxidative stress in animal studies. Superoxide dismutase (SOD) and catalase conjugated with antibodies to Platelet-Endothelial Cell Adhesion Molecule-1 (anti-PECAM/SOD and anti-PECAM/catalase) bind to endothelium, accumulate in the pulmonary vasculature, and detoxify reactive oxygen species. In order to define the role of conjugate size in the efficacy and specificity of endothelial targeting, we synthesized anti-PECAM/enzyme conjugates of controlled size (40nm-10,000nm). Binding of anti-PECAM/enzymes to endothelial cells increased with conjugate size from 300nm to 2µm (from 2.5 to 8.5% of bound fraction), and was specific, as conjugates did not bind to PECAM-negative cells. Pulmonary uptake of anti-PECAM/enzyme conjugates injected intravenously in mice also increased from 4.5 to 16% of injected dose for particles from 200 to 800nm. However, control conjugates larger than 300nm showed elevated non-specific pulmonary uptake, indicating that the targeting specificity of anti-PECAM/enzyme conjugates in vivo has a bell-shaped curve with a maximum close to 300-nm diameter. These results show that: i) the size of an antibody/enzyme conjugate modulates efficacy and specificity of targeting, and ii) a size optimum should be defined in vivo to account for parameters that are difficult to model in cell culture.

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.

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 FeCl(3) 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.

Platelet-endothelial cell adhesion molecule-1-directed endothelial targeting of superoxide dismutase alleviates oxidative stress caused by either extracellular or intracellular superoxide.

Targeting of the antioxidant enzyme catalase to endothelial cells protects against vascular oxidative stress induced by hydrogen peroxide (H(2)O(2))(Am J Physiol 285:L283-L292, 2003; Nat Biotechnol 21:392-398, 2003; Am J Physiol 293:L162-L169, 2007). However, another reactive oxygen species, superoxide anion, is also involved in many forms of vascular oxidative stress, including ischemia/reperfusion, hypertension, and inflammation. To protect endothelium against superoxide attack, we designed and tested antibody-directed targeting of superoxide dismutase (SOD) to the endothelial surface determinant, platelet-endothelial cell adhesion molecule (PECAM)-1. We synthesized anti-PECAM/SOD conjugates that retained 70% of enzymatic activity (superoxide anion dismutation) and specifically bound to endothelial cells, but not PECAM-negative cells. The effect of anti-PECAM/SOD delivery to cells was tested in two distinct models of oxidative stress induced by either extracellular or intracellular generation of superoxide anion. In the first model, anti-PECAM/SOD, but not unconjugated SOD, protected endothelial cells against injury caused by superoxide produced in the medium by hypoxanthine-xanthine oxidase. At the optimal dose, anti-PECAM/SOD provided up to 40 to 50% protection against cell death in this model. In the second model, anti-PECAM/SOD at the optimal dose provided complete protection against necrosis caused by paraquat-induced intracellular superoxide generation. Endothelial targeting of SOD represents a new molecular antioxidant approach that could be used for the management of vascular oxidative stress.

Factors modulating the delivery and effect of enzymatic cargo conjugated with antibodies targeted to the pulmonary endothelium.

Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM(34) or anti-TM(201)) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H(2)O(2) leading to lung damage, a clear physiologic endpoint. Results of in vivo studies in mice showed that the efficiency of cargo delivery and its effect are influenced by a number of factors including: 1) The level of pulmonary uptake of the targeting antibody (anti-TM(201) was more efficient than anti-TM(34)); 2) The amount of an active drug delivered to the target; 3) The amount of target antigen on the endothelium (animals with suppressed TM levels showed less targeting); and, 4) The substrate availability for the enzyme cargo in the target tissue (hyperoxia augmented GOX-induced injury). Therefore, both activities of the conjugates and biological factors control targeting and effects of enzymatic cargo. Understanding the nature of such "modulating biological factors" will hopefully allow optimization and ultimately applications of drug targeting for "individualized" pharmacotherapy.

Tumor necrosis factor-alpha differentially regulates the expression of proinflammatory genes in human airway smooth muscle cells by activation of interferon-beta-dependent CD38 pathway.

Recent evidence suggests that CD38, an ectoenzyme that converts NAD(+) to cyclic ADP-ribose (cADPr), may play a role in cytokine-induced airway smooth muscle (ASM) cell hyper-responsiveness, a key feature associated with chronic asthma. In the present study, we investigated the major signaling pathways by which tumor necrosis factor-alpha (TNFalpha) induces CD38 expression and its role in regulating gene expression in human ASM cells. Using flow cytometry analyses, TNFalpha enhanced CD38 expression in a manner that was time-(0-24 h), concentration-(0.1-40 ng/ml), and protein synthesis-(cycloheximide blockade) dependent. A selective agonistic antibody against tumor necrosis factor receptor (TNFR) 1 also augmented CD38 expression, whereas anti-TNFR2 antagonistic antibody did not prevent the TNFalpha response. Inhibition of the Janus activated kinase/signal transducer and activator of transcription pathways using the soluble inhibitor 2-(1,1-dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz-[h]imidaz[4,5-f]isoquinolin-7-one (DBI) or with neutralizing antibody against interferon beta (IFNbeta) completely abrogated TNFalpha-induced CD38 expression at both protein and mRNA levels. Combining TNFalpha (0.1 and 1 ng/ml) and IFNbeta (100 IU/ml) at concentrations alone that had little effect on CD38 expression induced a robust synergistic induction of CD38 mRNA and protein levels. 8-Bromo-cADPr, a cADPr antagonist, significantly augmented TNFalpha-induced interleukin-6 secretion, whereas regulated on activation normal T cell expressed and secreted secretion was suppressed. 8-Bromo-cADPr, however, did not affect TNFalpha-induced cell surface expression of intercellular adhesion molecule-1. Our study is the first to demonstrate that IFNbeta-dependent activation of CD38 pathway is a novel component by which TNFalpha differentially regulates the expression of inflammatory genes in ASM cells.

Tumor necrosis factor alpha modulates airway smooth muscle function via the autocrine action of interferon beta.

Current evidence suggests that tumor necrosis factor alpha (TNFalpha) and the family of interferons (IFNs) synergistically regulate many cellular responses that are believed to be critical in chronic inflammatory diseases, although the underlying mechanisms of such interaction are complex, cell-specific, and not completely understood. In this study, TNFalpha in a time-dependent manner activated both janus tyrosine kinase 1 and Tyk2 tyrosine kinase and increased the nuclear translocation of interferon-regulatory factor-1, STAT1, and STAT2 in human airway smooth muscle cells. In cells transfected with a luciferase reporter, TNFalpha stimulated gamma-activated site-dependent gene transcription in a time- and concentration-dependent manner. Using neutralizing antibodies to IFNbeta and TNFalpha receptor 1, we show that TNFalpha-induced secretion of IFNbeta mediated gamma-activated site-dependent gene expression via activation of TNFalpha receptor 1. In addition, neutralizing antibody to IFNbeta also completely abrogated the activation of interferon stimulation response element-dependent gene transcription induced by TNFalpha. Secreted IFNbeta acted as a negative regulator of TNFalpha-induced interleukin-6 expression, while IFNbeta augmented TNFalpha-induced RANTES (regulated on activation normal T cell expressed and secreted) secretion but had little effect on TNFalpha-induced intercellular adhesion molecule-1 expression. Furthermore TNFalpha, a modest airway smooth muscle mitogen, markedly induced DNA synthesis when cells were treated with neutralizing anti-IFNbeta. Together these data show that TNFalpha, via the autocrine action of IFNbeta, differentially regulates the expression of proinflammatory genes and DNA synthesis.