Targeting Antioxidant and Antithrombotic Biotherapeutics to Endothelium

Abstract

The endothelium is one of the key targets for pharmacological interventions in oxidative stress and thrombosis, two conditions that are notoriously difficult to treat due to limited efficacy and precision of action of current drugs. Design of molecular and nano-devices that deliver potent antioxidant and antithrombotic therapeutic enzymes to the endothelium holds promise to improve the potency, localization, timing, specificity, safety, and mechanistic precision of these interventions. In particular, cell adhesion molecules expressed on the surface of resting and pathologically altered endothelial cells can be used for drug delivery to the endothelial surface (preferable for thrombolytics) and into intracellular compartments (preferable for antioxidants). Drug delivery platforms including protein conjugates, recombinant fusion constructs, and stealth polymer carriers designed to target these drugs to endothelium are reviewed in this article.

The endothelium represents an important therapeutic target in thrombosis and vascular oxidative stress. Advances in molecular biology and biotechnology paved the way for the design of potent and specific recombinant therapeutic proteins including antithrombotic and antioxidant enzymes. Thus plasminogen activators (PAs) catalyze dissolution of fibrin clots providing re-perfusion, whereas superoxide dismutase (SOD) and catalase detoxify reactive oxygen species (ROS) that may inflict tissue damage and inflammation. These enzymes represent labile, yet potentially advantageous biotherapeutic agents that require precise delivery to desired sites of action in the target cells (i.e., endothelial luminal surface for fibrinolytics and intracellular compartments for antioxidants).

Unfortunately, these biotherapeutics have no endothelial affinity and their delivery to the desired site of their action is inadequate; hence adverse effects limit clinical utility of fibrinolytic PAs and inadequate effectiveness precludes the use of antioxidant enzymes. In theory, medical application of these biotherapeutics may benefit from targeted delivery to selected compartments in endothelium. Endowing these agents or their carriers with an affinity to endothelial cells that provides targeted delivery to either the endothelial lumen or its intracellular compartments holds promise to localize therapeutic interventions in desired endothelial compartments in vascular areas of interest and optimize treatment of these vascular disorders. This article briefly discusses the current status of the experimental design and development of targeted delivery of antioxidants and fibrinolytics to the endothelium.

MOLECULAR TARGETS FOR ENDOTHELIAL DRUG DELIVERY

Ideally, binding of targeted drugs to the target should cause therapeutically beneficial side effects and provide the proper subcellular localization of a drug. Depending on the therapeutic goal, a drug should either be retained on the cell surface or traffic to a proper subcellular compartment. Binding of targeted drugs may activate endothelial cells or induce shedding and/or internalization of target determinants, change their functionality, or otherwise disturb the endothelium. Obviously, targeting of antioxidant or antithrombotic agents should not cause harmful side effects. In this aspect, targeting protective drugs is different versus targeting toxic agents to tumor endothelium. In the latter scenario, toxic effect to the target cells is a bonus. Therefore, the specificity of tumor targeting must be maximal to avoid collateral damage. In contrast, endothelial disturbance by targeted antioxidant and antithrombotic drugs must be minimized to avoid aggravation of oxidative stress, inflammation, and thrombosis. However, the criteria of specificity of targeting are less stringent in this case because drugs alleviating these conditions (often associated with systemic pathologies) are less likely to cause systemic harmful effects; thus pan-endothelial delivery of antioxidants or antithrombotic therapeutics throughout the vasculature is a suitable option. Of note, the pulmonary vasculature is the major capillary network, containing ~30% of the endothelial surface in the body and receiving the entire cardiac output. As a result, agents with an endothelial affinity accumulate in the lungs after intravenous (IV) injection, even if their target determinants are relatively evenly distributed throughout all types of endothelial cells in the body.¹ This vascular bed is an important target for treatment of acute lung injury, oxidative stress, thrombosis, and inflammation, among other conditions.

Numerous molecules localized on the surface of endothelial cells of diverse phenotypes have been identified by high-throughput approaches² including selective proteomics of the endothelial plasmalemma^3,4 and in vivo phage display⁵ and low throughput approaches including immunological techniques.¹ Specific domains in the endothelial plasmalemma are enriched in certain molecules. For example, rat glycoprotein GP85 is predominantly localized on the luminal surface of the plasmalemma domain and belongs to a thin part of the endothelial cell body that lacks major organelles and separates alveolar and vascular compartments.⁶ Antibodies to GP85 accumulate in rat pulmonary vasculature without internalization and deliver conjugated tissue plasminogen activator (tPA) into the pulmonary vasculature.⁷ A human counterpart of this antigen could be an interesting candidate for drug delivery to the surface of alveolar capillaries. Further, determinants in the caveoli provide effective intracellular and transendothelial delivery of antibodies and small protein conjugates.⁸ However, utility of the caveolar determinants for sustained anchoring of drugs on the cell surface as well as for targeting carriers larger than 50 to 80 nm is questionable. Surface density of some endothelial molecules is reduced upon disease conditions associated with oxidative stress and ischemia, hindering targeting to these determinants.⁹

This section provides a brief overview of endothelial targets suitable for delivery of antioxidant and antithrombotic agents (Table 1). For example, angiotensin-converting enzyme (ACE) is a transmembrane constitutive glycoprotein preferentially expressed at the endothelial luminal surface in pulmonary capillaries.^9–11 ACE converts Ang I into Ang II, a vasoactive peptide that exerts vasoconstricting, prooxidant, prothrombotic, and proinflammatory activities.¹² Endothelial cells internalize ACE antibodies (anti-ACE) and anti-ACE conjugates.¹³ Labeled anti-ACE selectively accumulates in the lungs after IV injection in rats, mice, cats, primates, and humans.^11,14–16 Pilot tests did not reveal harmful effects of anti-ACE in animals^10,17 and humans.¹⁷ Anti-ACE formulations are being used for targeting drugs including antioxidants and fibrinolytics to the pulmonary endothelium in animals.^18–20 Ischemia, oxidants, cytokines, and other pathological agents suppress ACE expression and thus inhibit targeting to ACE.^21,22

Table 1.

Candidate Targets for Endothelial Drug Delivery

Target	Function and Localization	Targeting Advantages	Potential Problems
ACE	Peptidase, converts Ang I into Ang II and cleaves bradykinin, ACE enriched in the lung capillaries	Selective targeting to lung EC, intracellular delivery, vasodilating and anti-inflammatory effects of ACE inhibition	Inflammation suppresses targeting; ACE inhibition may be dangerous
PECAM-1	Facilitates transmigration of leukocytes, stably expressed in EC borders	Intracellular delivery of anti-PECAM conjugate may also suppress inflammation	PECAM signaling and side effects are not understood
ICAM-1	Mediates leukocyte adhesion to EC, stably expressed and upregulated by pathologies	Similar to PECAM, but inflammation enhances targeting	Similar to PECAM
E-selectin	Supports leukocyte adhesion, expressed only on altered EC	Intracellular targeting to EC in inflammation	Targeting is transient and not robust; surface targeting is not effective due to endocytosis
P-selectin	Similar to E-selectin	Similar to E-selectin	Similar to above, platelets also bind P-selectin ligands
gp90	Function unknown; localized in EC caveoli	Transendothelial targeting in rat lungs	Human analog and side effects are not known
Gp85	Function unknown; EC avesicular zone in alveolar capillaries	Targeting to the EC surface in rat lungs	Similar to above
gp60	Albumin-binding protein in EC caveoli	Transendothelial targeting	Unclear side effects and specificity of targeting
APN	Digestion of peptides by proteolysis	Upregulated in tumor vasculature	Expression in other cell types
APP	Cleavage of bradykinin and substance P	Localized in the caveoli, enriched in lungs, and offers very effective and fast endothelial targeting	Inhibition of APP may cause side effects including edema

ACE, angiotensin-converting enzyme; EC, endothelial cells; PECAM, platelet-endothelial adhesion molecule; ICAM, intercellular adhesion molecule; gp, glycoproteins; APN, aminopeptidase N; APP, aminopeptidase P.

Platelet-endothelial adhesion molecule (PE-CAM)-1, CD31 and intercellular adhesion molecule (ICAM)-1, CD54 are transmembrane glycoproteins constitutively expressed on endothelial cells throughout the vasculature. Their ligands (e.g., anti-ICAM and anti-PECAM) infused locally in a conduit artery accumulate in the downstream vascular areas including cardiac,²³ cerebral,²⁴ and mesentery^25,26 vasculature. Anti-PECAM and anti-ICAM tend to accumulate in the lungs after IV injection and may be used for drug targeting to either normal and/or pathologically altered pulmonary and systemic endothelium.²⁷ PECAM and ICAM are involved in mechanisms of cellular recognition, adhesion, and transendothelial migration of leukocytes.²⁸ Thus drug targeting to these molecules may inhibit leukocyte trafficking.²⁹ Leukocyte infiltration is generally viewed as an injurious factor,³⁰ and attenuation of leukocyte infiltration would be a bonus in the treatment of inflammation. Endothelial cells do not internalize anti-ICAM and anti-PECAM or their monovalent fragments, but they do internalize multimeric, multivalent conjugates causing excessive cross-linking of ICAM and PECAM.^31,32 Therefore, based on the molecular design of the conjugates or fusion proteins targeted to these cell adhesion molecules, either intracellular or surface addressing of the cargoes is possible.

PECAM is stably expressed on the endothelium at the level of a million copies per cell predominantly localized in interendothelial borders.³³ Multivalent binding of PECAM-targeted nanocarriers offers effective means for endothelial drug delivery.^11,32 Diverse reporter,³⁴ enzymatic,^23,35,36 and genetic materials³⁷ conjugated to anti-PECAM accumulate and display their functional activity in the endothelium as soon as 10 minutes after IV injection in mice, rats, and pigs.

In contrast to constitutively and stably expressed PECAM-1, quiescent confluent endothelial cells in culture do not express appreciable amounts of ICAM-1, but expression is markedly increased after cytokine treatment.³⁸ Endothelial cells in the vasculature express ICAM-1 at a surface density of 2 × 10⁴ to 2 × 10⁵ surface copies per cell, and this level doubles upon proinflammatory challenge.³⁹ Other cell types also express ICAM, yet the blood-accessible ICAM is located predominantly on endothelial cells. Anti-ICAM and anti-ICAM conjugates bind to the vascular endothelium after IV administration in animals.^40,41 Pathological stimuli including oxidants, cytokines, and abnormal shear stress stimulate de novo synthesis and surface expression of ICAM by endothelial cells⁴² and thereby facilitate anti-ICAM endothelial targeting.^40,41,43,44 Conjugation of anti-ICAM to therapeutics,^41,45 liposomes,⁴⁶ or polymer carriers²⁶ providing multivalent binding to the endothelium further enhances drug delivery.

Vascular cellular adhesion molecule (VCAM)-1) and selectins are normally absent on the vascular lumen but are exposed on pathological endothelium. For example, pathological mediators cause mobilization of intracellular P-selectin to the endothelial surface within 10 to 30 minutes³⁰ and induce de novo synthesis and surface expression of E-selectin⁴⁷ and VCAM-1³³ within several hours. Selectins and VCAM-1 facilitate adhesion of leukocytes to endothelial cells.⁴⁸ Selectin and VCAM-1 ligands represent attractive affinity moieties for delivery diagnostics and therapeutic agents to activated endothelium. Experiments in cell culture and animals show that anti-selectins permit drug targeting to cytokine-activated endothelium.^49,50 Endothelial cells constitutively internalize selectins via clathrin-coated pits,^51–53 permitting entry into endothelial cells of anti-E-selectin targeted liposomes,⁵⁴ anti-inflammatory drugs,^54,55 and genetic materials.⁵⁶ Yet, even at maximal activation, selectins and VCAM-1 are exposed at surface densities lower than PECAM-1 and ICAM-1; hence robustness may be suboptimal for therapies requiring delivery of large doses of drugs. P-selectin targeted compounds also bind to activated platelets.⁵⁷ E-selectin and VCAM-1 seem to be more readily expressed in activated endothelium outside the pulmonary vasculature (e.g., in arteries and skin microvasculature).⁵⁸ These determinants seem to be useful for diagnostic visualization of activated endothelium in inflammation foci by delivery of conjugated isotopes⁵⁹ or ultrasound contrasts.^57,60

TARGETING OF ANTIOXIDANT ENZYMES

Cells normally produce low levels of ROS such as superoxide anion and hydrogen peroxide, but excessive vascular ROS generation is postulated to underlie the pathogenesis of many diseases.⁶¹ Both acute (acute lung injury, ischemia/reperfusion injury, inflammation, and complications of organ transplantation) and chronic (e.g., hypertension, diabetes, and atherosclerosis) vascular disorders are associated with abnormally high level of ROS produced by activated leukocytes and endothelial cells.⁶² Therefore, the endothelium is an important target for antioxidant interventions.

Superoxide anion O₂⁻ is not particularly toxic by itself, but at excessive levels can yield a highly reactive and damaging hydroxyl radical OH. Further, it consumes a protective agent nitric oxide (NO·) in a fast reaction producing a strong oxidant peroxynitrite that aggravates vascular oxidative stress, inflammation, vasoconstriction, and thrombosis.^61,63 Superoxide dismutases (SODs) are metal-containing enzymes that catalyze superoxide conversion into H₂O₂, thus alleviating these pathological mechanisms.⁶⁴ There are three types of human SOD: cytosolic CuZnSOD (SOD1), mitochondrial MnSOD (SOD2), and extracellular SOD (SOD3).^64,65 Catalase, a tetrameric enzyme localized in the cytosol and intracellular vesicles called peroxisomes, decomposes H₂O₂ into water and oxygen.

Use of SOD and catalase for antioxidant interventions was proposed almost four decades ago.⁶⁶ However, their medical utility is precluded by rapid elimination and inadequate delivery to target cells.⁶⁷ Diverse modifications of SOD and catalase including coupling with polyethylene glycol or other chemicals and encapsulation into liposomes have been designed to solve this problem (Table 2).^68–70 Some of these derivatives and SOD mimetics showed protective effects after intravascular or local administration in animal models of systemic or focal oxidative stress, respectively.^68,71–73 There were also attempts to optimize cellular binding of SOD and catalase. For example, lecithinized SOD (PC-SOD) binds to some cell types including endothelial cells in vitro and was protective in several animal models of human pathologies including myocardial infarction, colitis, and tumor growth.^74–76 SOD conjugation with folate enhances enzyme uptake by macrophages.⁷⁷ A recombinant fusion of mitochondrial MnSOD (SOD2) and heparin-binding domain of EC-SOD (SOD3) has been synthesized⁷⁸; this SOD2/3 chimera binds to cellular glycocalyx and alleviates vascular dysfunction in models of myocardial ischemia.^79,80 Other SOD fusion proteins have also been reported (e.g., SOD-hemoglobin fusion⁸¹ and bacterial Fe-SOD fused with a single-chain variable fragment (scFv) LC-1 antibody to lung cancer cells⁸²).

Table 2.

Antioxidant and Antithrombotic Interventions

Antioxidant Interventions
Cargo	Target	Disease	Effect
PC-SOD	EC membrane	Myocardial ischemia, colitis, burn trauma, etc.	Attenuation of tissue injury
Chimera SOD2/3	EC HSPGs	I/R	Attenuation of tissue injury
Antibody/SOD conjugate	PECAM-1	Hypertension	Vasoreactivity normalization, BH4 protection against oxidation
Antibody/catalase conjugate	PECAM-1	I/R	↓ Alveolar edema and attenuation of arterial blood oxygenation
Antithrombotic Interventions
tPA	ICAM-1	CVD	↑ Rate of fibrinolysis
uPA	PECAM-1	CVD	↑ Rate of fibrinolysis
uPA-T	PECAM-1	CVD	↑ ↑ Rate of fibrinolysis
TM	PECAM-1	ALI	↓ Lung thrombosis and inflammation

PC, phosphatidylcholine; SOD, superoxide dismutase; EC, endothelial cells; HSPG, heparan sulfate proteoglycan; I/R, ischemia/reperfusion; PECAM-1, pulmonary endothelial cell adhesion molecule-1; BH₄, tetrahydrobiopterin; tPA, tissue plasminogen activator; ICAM-1, intercellular adhesion molecule-1; CVD, cardiovascular disease; uPA, urokinase plasminogen activator; uPA-T, urokinase plasminogen activator–thrombin; TM, thrombomodulin; ALI, acute lung injury.

However, the pharmacokinetics and specificity of endothelial binding of these antioxidant formulations remain suboptimal and did not provide controlled delivery into these cells, the goal that seems necessary for interception of intracellular ROS.⁸³ Inadequate delivery precludes the therapeutic use of catalase and SOD. Alas, despite remarkable efforts to improve their pharmacokinetics,⁸⁴ effective and specific endothelial delivery remain elusive.

Targeting catalase and SOD to specific endothelial targets may help to solve this problem.^31,32 Conjugates with antibodies to ACE,¹⁸ PECAM,⁸⁵ and ICAM¹⁹ specifically bind to endothelium in vitro and in vivo and accumulate in the pulmonary and systemic endothelium after IV injection. Multivalent catalase conjugates with anti-ICAM or anti-PECAM,^18,19,86,87 as well as catalase loaded to polymer nanocarriers coated by these antibodies enter endothelial cells.³⁸ Catalase delivered to endocytic vesicles degrades H₂O₂ and protects endothelial cells from oxidative stress.³⁸ IV injected anti-PECAM/catalase conjugates protect against acute oxidative stress in the pulmonary vasculature in mice,⁸⁶ reduce lung edema and improve blood oxygenation in a mouse model of pulmonary ischemia/reperfusion,⁸⁸ and improve the outcome of lung transplantation in rats.⁸⁹ IV injection of anti-ACE/catalase conjugate in a donor animal also protects lungs in a model of transplantation in rats.²⁰ Similarly, multivalent anti-PECAM/SOD conjugates bind to endothelial cells with high affinity and specificity and protect cells against oxidative stress caused by superoxide anion.⁹⁰ Further, IV-injected anti-PECAM/SOD binds to the endothelium and alleviates superoxide-mediated oxidative stress in blood vessels and vascular contractility in animals.⁸⁸

Of note, targeting SOD and catalase to the same endothelial determinant, PECAM-1, provides different and specific protective effects in several animal models of acute vascular oxidative stress, emphasizing importance of interception of specific ROS in given pathologies.⁸⁸ Of note, lysosomal degradation of antioxidant enzymes⁹¹ terminates the protective effect within a few hours after internalization.⁹² Drugs affecting lysosomal trafficking and degradation prolongs the protective effects of anti-ICAM/catalase formulations.^91,92 Further, using catalase encapsulated into PECAM-targeted bio-compatible polymer nanocarriers selectively permeable to H₂O₂, but not to proteases, permits a prolonged antioxidant effect. A freeze-thaw modified double emulsion allows encapsulation of active catalase into such protective nanocarriers with controlled size and shape, appropriate for vascular delivery into endothelial cells.^93,94 Typical carrier morphologies, controlled by chemistry of the polymers utilized, are spheres with diameters <500 nm⁹³ or flexible filaments that are a few microns in length and ~50 nm in cross section.⁹⁴ Catalase-loaded polymer spherical nanocarriers targeted to PECAM-1 deliver the enzyme to the pulmonary vasculature after IV injection in animals and protect endothelial cells against H₂O₂-induced injury for an extended period.⁹⁵

ENDOTHELIAL TARGETING OF ANTITHROMBOTIC DRUGS

Thrombosis is caused by intravascular blood clots. Pathologically altered vasculature (e.g., due to oxidative stress, inflammation, or ischemia) is predisposed for thrombosis, in part due to suppression of natural antithrombotic mechanisms in endothelium.⁹⁶ It is tempting to postulate that anchoring of exogenous antithrombotic proteins on the endothelial lumen may help to compensate for this dysfunction and thereby improve management of thrombosis.

Fibrinolytic PAs convert plasminogen to plasmin that in turn cleaves fibrin facilitating clot degradation and restoration of blood perfusion. These drugs are used in clinics for urgent fibrinolytic therapy but are marred by hemorrhagic and other side effects, due to inadequate delivery to the therapeutic site. Attempts to improve delivery by conjugating PAs with antibodies to clot components have not yielded decisive improvement in therapy, likely due to impermeability of clots.^97,98 None of the current PA formulations are useful for short-term thromboprophylaxis despite the urgent and critical un-met medical need for such intervention in identified patient cohorts.

Theoretically, enhancing antithrombotic activity on the vascular luminal surface could be used for thromboprophylaxis. Gene transduction of antithrombotic proteins in the vasculature of laboratory animals supports this notion,⁹⁹ but this approach is impractical in acute settings. It is conceivable that immunotargeting of antithrombotic proteins to the endothelial surface would rapidly restrict and sustain their activity to the vascular lumen. This approach can potentially provide thromboprophylaxis in patients with a high propensity for thrombosis, particularly in settings where the risk of bleeding prohibits use of systemic anticoagulation.¹⁰⁰

Several endothelial determinants have been employed for this function. For example, tPA chemically conjugated with anti-ACE retained fibrinolytic and antigen-binding activities and exhibited sustained preferential accumulation in rat pulmonary vasculature.¹⁰¹ Other endothelial determinants have also been explored for this goal; both tPA and urokinase-type PA (uPA) chemically conjugated with antibodies against antigens enriched in the pulmonary endothelium accumulated in the pulmonary vasculature.^102,103

ICAM-1 and PECAM-1, stably expressed by endothelium and not supporting endocytosis of single antibodies, represent an attractive target for anchoring antithrombotic drugs on the endothelial luminal surface (Table 2). After IV injection in rats, pulmonary uptake of anti-ICAM/tPA conjugates is almost two orders of magnitude higher than that of control IgG/tPA, which resulted in enhanced fibrinolysis of subsequent pulmonary emboli.¹⁰⁴ Anti-ICAM carriers provide robust targeting to the pulmonary endothelium, on par with the best candidate carriers tested to date including antibodies to ACE and caveoli-associated antigens.^101,103 However, in contrast to these endothelial determinants, ICAM-1 offers enhanced pulmonary targeting in the context of inflammation that is frequently intertwined with thrombosis.^105,106 In addition, thrombin upregulates the expression of ICAM-1, which provides an additional rationale for its use as a target for delivering antithrombotic agents.¹⁰⁷

Chemical conjugation of proteins with antibodies yields multivalent conjugates that cause endocytosis.⁸⁵ This is undesirable in the context of sustained retention of fibrinolytics on the endothelial lumen. In contrast, recombinant fusion of enzymes with genetically engineered antibody fragments, single-chain Fv (scFv, comprising variable domains of heavy chain V_H and light chain V_L) yields a monovalent (thus no internalization via ICAM or PECAM), homogeneous, and relatively small (50 to 70 kDa, hence low immunogenicity and lack of Fc-fragment mediated side effects) bifunctional recombinant drug. As a proof of principle for prophylactic thrombolysis by endothelium-targeted thrombolytic fusions, using a linker of three Gly₄Ser repeats, an anti-PECAM scFv was fused with uPA, which exists as an inactive single-chain zymogen of 411 amino acid residues (single-chain uPA, scuPA¹⁰⁸). In the presence of fibrin, endogenous plasminogen activators convert plasminogen to plasmin, which in turn cleaves the Lys158-Ile159 peptide bond in scuPA and generates fully active two-chain uPA (tcuPA) consisting of two polypeptide chains linked by a Cys148-Cys279 disulfide bond.¹⁰⁹

The natural scuPA consists of three domains: the N-terminal domain homologous to the epidermal growth factor, the kringle domain, and the C-terminal catalytic domain. Urokinase binding to a cellular receptor via the “growth factor-like domain” (GFD) activates vascular cells,¹¹⁰ but deletion of the GFD provides a low molecular weight form of scuPA (MW 32 kDa) that has enzymatic features similar to those of full-length scuPA. After IV injection, the protein composed of an anti-PECAM scFv fused with lmw-scuPA (scFv/uPA) preferentially accumulated in the lungs of wild-type but not PECAM-deficient mice, persisted in the lungs for at least 3 hours and remained on the endothelial surface. Compared with nontargeted uPA, scFv/uPA augmented local lysis of pulmonary emboli in a mouse pulmonary thrombotic model.¹⁰⁸ Further, scFv/uPA accumulated in the cerebral vasculature after intra-arterial and IV injection, dissolved cerebral clots, and improved blood reperfusion without hemorrhagic complications, thereby mitigating postthrombotic brain edema in a mouse model of cerebral embolism.²⁴

The scuPA zymogen (prourokinase) can be activated by trace amounts of plasmin over time, and formed tcuPA may cause adverse effects that would limit prophylactic use.¹¹¹ Because tcuPA is rapidly inactivated by plasminogen activator inhibitor (PAI-1), the duration and effectiveness of prophylaxis would also be limited. Further, thrombin inactivates uPA by cleaving Arg156-Phe157, negating its effect at sites of active thrombosis.¹¹² These problems might be solved by deleting Phe157 and Lys158, which yields a plasmin-resistant mutant activated by thrombin (uPA-T).¹¹³ This pro-enzyme will not be activated by plasmin in vivo (thus avoiding systemic effects and premature PAI-1 inactivation), and thrombin will activate it locally at sites of nascent thrombosis within seconds of clotting. Replacing the native plasmin activation site in the uPA moiety of scFv/uPA with a thrombin activation site provided thrombin-activated anti-PECAM scFv/uPA-T.¹¹⁴ This construct was also found to contain an intrinsic thrombin-sensitive cleavage site in the anti-PECAM scFv moiety, providing a built-in mechanism for local drug release. The scFv/uPA-T is latent and resists the PA inhibitor PAI-1 until activated by thrombin. After IV injection in mice, scFv/uPA-T did not consume plasma fibrinogen, in contrast with scFv/uPA that has this liability. However, scFv/uPA-T bound to the endothelium and accumulated in the vascularized organs, particularly the lungs. In a mouse model of thrombin-induced pulmonary thrombosis, scFv/uPA-T provided more potent and durable thromboprophylaxis than both plasmin-sensitive scFv/uPA and lmw-scuPA. Further, injection of mice with scFv/uPA-T prior to unilateral lung ischemia/reperfusion attenuated pulmonary fibrin deposition, to a significantly greater extent than plasmin-sensitive scFv/uPA, and restored arterial oxygen tension; PAI-1 susceptible plasmin-activated scFv/uPA did not improve blood oxygenation.¹⁰²

The suppression of endothelial thrombomodulin activity (TM, a transmembrane glycoprotein that switches procoagulant and proinflammatory effects of thrombin to opposing effects via activation of protein C) is characteristic of acute lung injury, and some success has been achieved with a replacement therapy using soluble recombinant TM and activated protein C (APC).¹¹⁵ Further, TM fused with a tissue factor antibody has potent antithrombotic activity in a rat model.¹¹⁶ Yet these biotherapeutics seem unsuitable for prophylaxis due to fast disappearance from the vascular lumen. To solve this problem, a new anti-PECAM scFv/TM fusion has been produced and shown to bind and reside on endothelial surface, accumulate in the pulmonary vasculature, and attenuate thrombosis and tissue damage in mouse models of lung ischemia-reperfusion and endotoxin-induced acute inflammatory lung injury to a greater extent than nontargeted soluble TM, without causing bleeding (a known liability of APC treatment).¹¹⁵

In summary, endothelium-targeted thromboprophylaxis triggered by a prothrombotic enzyme illustrates a novel approach to time- and site-specific regulation of “on demand” reactions that can be modulated for therapeutic benefit. In clinical settings, this strategy of targeting antithrombotic drugs to the endothelial surface may provide local thromboprophylaxis in patients with an acute risk of developing new or recurrent thrombi and prevent clot extension.

CONCLUSION

Endothelial targeting of antioxidant and antithrombotic enzymes has been achieved in intact animals and animal models of human pathologies. Recent animal studies showed that ACE and especially ICAM and PECAM are good potential anchors for targeting these biotherapeutics to endothelium. Their functions are fairly well understood, which helps avoid unintentional side effects in a given pathological context. Careful selection of targets and modulation of features of the antibody-drug conjugates, such as valency and size, prodrug activation features, and encapsulation into protective polymeric carriers, provide powerful tools for the control of subcellular addressing, activation, and duration of the effects of the enzymes.

Scaling up synthesis and quality control of targeted drug delivery systems with a standard, Food and Drug Administration acceptable level of homogeneity is a challenge for their industrial development and clinical utility. Recombinant mutant thrombolytic prodrugs fused with affinity peptides such as anti-PECAM or anti-ICAM scFv provide more industrially and clinically suitable biotherapeutics. Molecular design of these constructs permits deletion of unnecessary parts of molecules or insertion of point mutations endowing products with novel favorable pharmacokinetics and/or functional features. Clinical testing of antioxidant and antithrombotic biotherapeutics targeted to endothelial cells has been recently conducted and demonstrated promising levels of safety and efficacy. It is tempting to hope that in the next decade targeted interventions into endothelial cells will be translated from initial successes reported in laboratory animals to medical practice.