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Originally published online as doi:10.1189/jlb.0804460 on November 17, 2004

Published online before print November 17, 2004
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(Journal of Leukocyte Biology. 2005;77:129-140.)
© 2005 by Society for Leukocyte Biology

Targeting leukocyte integrins in human diseases

Karyn Yonekawa* and John M. Harlan{dagger},1

* Division of Nephrology, Department of Pediatrics, and
{dagger} Division of Hematology, Department of Medicine, University of Washington, Seattle

1 Correspondence: Division of Hematology, Harborview Medical Center, Mailstop 359756, 325 Ninth Avenue, Seattle, WA 98115. E-mail: jharlan{at}u.washington.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 INTEGRINS
 LEUKOCYTE INTEGRINS
 APPROACHES TO TARGETING...
 TARGETING LEUKOCYTE INTEGRINS...
 CONCLUSION
 REFERENCES
 
As our understanding of integrins as multifunctional adhesion and signaling molecules has grown, so has their recognition as potential therapeutic targets in human diseases. Leukocyte integrins are of particular interest in this regard, as they are key molecules in immune-mediated and inflammatory processes and are thus critically involved in diverse clinical disorders, ranging from asthma to atherosclerosis. Antagonists that interfere with integrin-dependent leukocyte trafficking and/or post-trafficking events have shown efficacy in multiple preclinical models, but these have not always predicted success in subsequent clinical trials (e.g., ischemia-reperfusion disorders and transplantation). However, recent successes of integrin antagonists in psoriasis, inflammatory bowel disease, and multiple sclerosis demonstrate the tremendous potential of antiadhesion therapy directed at leukocyte integrins. This article will review the role of the leukocyte integrins in the inflammatory process, approaches to targeting leukocyte integrins and their ligands, and the results of completed clinical trials.

Key Words: adhesion • clinical • trials • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
INTEGRINS
 LEUKOCYTE INTEGRINS
 APPROACHES TO TARGETING...
 TARGETING LEUKOCYTE INTEGRINS...
 CONCLUSION
 REFERENCES
 
Inflammatory and immune diseases affect millions of people in the United States alone [1 , 2 ], providing an impetus to develop new anti-inflammatory and immunomodulatory therapies. Although critical for host defense and repair, leukocytes are also the primary effectors of inflammation and immune responses. During the process of emigration from bloodstream to extravascular tissue, leukocytes may injure the vessel wall, provoking thrombosis or promoting edema. Emigrated leukocytes may initiate and sustain tissue damage by release of diverse mediators, such as oxidants, proteases, bioactive lipids, and cytokines.

Over the past two decades, there has been dramatic progress in elucidating the molecular basis of leukocyte trafficking from bloodstream to extravascular tissue (reviewed in refs. [3 4 5 6 7 ]), thus identifying potential new targets for therapeutic intervention. The integrin receptors involved in leukocyte trafficking have been recognized as particularly attractive targets for several reasons. First, integrins are critically involved in several steps of the adhesion cascade, and preclinical studies have shown that leukocyte integrin blockade is efficacious in diverse disease models [8 ]. Second, elegant studies have provided important details of integrin structure and function, thereby facilitating drug development (reviewed in refs. [9 10 11 12 ]). Third, platelet integrin antagonists have already been shown to be effective drugs in acute coronary syndromes (reviewed in refs. [13 , 14 ]), thus validating integrins as "drugable" targets. Consequently, many pharmaceutical and biotechnology companies have worked to develop leukocyte integrin antagonists (reviewed in refs. [12 , 15 16 17 18 19 ]). In this article, we will review therapeutic approaches directed at leukocyte integrins and their ligands, emphasizing those therapies that have completed clinical trials.


   INTEGRINS
TOP
 ABSTRACT
 INTRODUCTION
 INTEGRINS
LEUKOCYTE INTEGRINS
 APPROACHES TO TARGETING...
 TARGETING LEUKOCYTE INTEGRINS...
 CONCLUSION
 REFERENCES
 
The term "integrin" was first proposed in 1986 to describe a protein complex that was felt to be integral to the transmembrane connection between the extracellular matrix (ECM) and the cytoskeleton [20 ]. Soon after, homology with other transmembrane proteins was discovered, and integrin came to describe a family of structurally related cell-surface receptors. These molecules are heterodimeric receptors comprised of noncovalently associated {alpha} and ß subunits. Integrins are expressed by all multicellular animals, although the repertoire varies amongst phyla [21 ]. The human integrin family now includes at least 18 known {alpha}-subunits and eight known ß-subunits. Some {alpha}-subunits contain an inserted I domain, which is a major ligand-binding site [22 ]. A given {alpha}-subunit may interact with more than one ß-subunit, resulting in 24 different heterodimers identified to date. Integrin expression varies from one cell type to another. However, certain integrins are limited to one cell lineage or to a specific cell type. ß2 integrins, for example, are expressed only on hematopoietic cells, and {alpha}Eß7 is expressed primarily by T lymphocytes in mucosal tissue [23 ].

Although originally identified as adhesion molecules, integrins are now known to mediate a wide variety of signaling functions, and consequently, integrins influence many biologic systems. They are involved in hematopoiesis, hemostasis, immune regulation, and the inflammatory response. Additionally, they are crucial for embryonic development, influencing cellular survival, proliferation, and differentiation [24 ]. Genetic deletion models highlight the impact that the integrin has on organism viability as well as the unique role of each integrin subunit [21 ].

Several human genetic diseases provide tangible evidence of the clinical importance of this family of cell adhesion receptors (reviewed in refs. [25 , 26 ]). Originally described in 1918, Glanzmann’s thrombasthenia is an autosomal recessive disease characterized by serious mucocutaneous bleeding. Almost 60 years later, it was found to result from mutations in the platelet integrin {alpha}IIbß3 [integrin nomenclature: {alpha}IIbß3, glycoprotein (GP)IIb/IIIa, CD41/CD61; {alpha}Mß2, macrophage adhesion molecule-1, CD11b/CD18; {alpha}Lß2, lymphocyte function-associated antigen-1, CD11a/CD18; {alpha}Xß2, p150, 95, CD11c/CD18; {alpha}Dß2, CD11d/CD18; {alpha}4ß7, lymphocyte Peyer’s patch adhesion molecule-1; {alpha}4ß1, very late antigen-4 (VLA-4), CD49d/CD29; {alpha}1ß1, VLA-1, CD49a/CD29; {alpha}Vß3, CD51/CD61; {alpha}5ß1, CD49e/CD29; refs. 27 , 28 ]. Leukocyte adhesion deficiency syndrome (LAD I) was also described before the molecular defect was discovered (reviewed in refs. [29 , 30 ]). LAD I patients exhibit recurrent bacterial infections and persistent, marked leukocytosis between infectious episodes. They were found to be quantitatively deficient in the ß2 integrins {alpha}Mß2, {alpha}Lß2, {alpha}Xß2 [31 ], and {alpha}Dß2 as a result of heterogeneous mutations in the common ß2 subunit. More recently, LAD I "variants" have been reported [32 33 34 35 ] and proposed to constitute a distinct syndrome [36 ]. These patients have dysfunctional but structurally intact ß1, ß2, and ß3 integrins, suggesting a defect in a common signaling molecule for integrin activation [36 ]. Other integrin-dependent diseases include epidermis bullosa variants caused by mutations in the {alpha}4ß6 complex [37 38 39 ] and congenital myopathy resulting from mutations in the {alpha}7 gene [40 , 41 ].


   LEUKOCYTE INTEGRINS
TOP
 ABSTRACT
 INTRODUCTION
 INTEGRINS
 LEUKOCYTE INTEGRINS
APPROACHES TO TARGETING...
 TARGETING LEUKOCYTE INTEGRINS...
 CONCLUSION
 REFERENCES
 
Trafficking
As evidenced by the diverse human diseases resulting from these genetic defects, integrins are crucial for physiologic homeostasis. Integrins are critically involved in the trafficking of leukocytes from the bloodstream to extravascular tissue at sites of active inflammation as well as during routine immune surveillance. Studies by intravital microscopy have established a sequence of adhesive events involved in leukocyte emigration. Under conditions of flow, leukocytes are first observed to roll along the endothelium of postcapillary venules at sites of inflammation. Subsequently, some of the rolling leukocytes stick firmly, migrate along the endothelial surface, diapedese between endothelial junctions, and then migrate through a subendothelial matrix. These early steps in emigration—rolling, firm adhesion, and transendothelial migration—result from the interaction of distinct leukocyte and endothelial receptors in an adhesion cascade (reviewed in refs. [3 4 5 6 7 ]; Fig. 1 ). Rolling is observed only under flow conditions and is the consequence of shear forces acting on the leukocyte and adhesive interactions between the leukocyte and endothelium. This primary phase of adhesion is mediated predominantly by leukocyte L-selectin and endothelial P- and E-selectins and their glycoprotein counter-receptors, particularly P-selectin glycoprotein-1 (reviewed in ref. [42 ]). P-selectin is rapidly translocated from Weibel-Palade bodies to the luminal surface upon endothelial cell activation, whereas E-selectin is expressed after several hours following de novo synthesis.



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  		Figure 1. Leukocyte trafficking and post-trafficking events. The trafficking of leukocytes from the bloodstream to extravascular tissue is a multistep process. Under conditions of flow, leukocytes are first captured and tethered to endothelium by predominantly selectin-mediated adhesive interactions that allow the leukocyte to roll along the vessel wall. The rolling cells are stimulated by chemokines or chemoattractants, triggering inside-out signaling that increases the affinity and/or clustering of integrins. The activated integrins bind avidly to endothelial ligands, firmly attaching the leukocyte to the endothelium. The subsequent migration of leukocyte across the endothelial surface, diapedesis between endothelial cells, and migration in extravascular tissue is also integrin-dependent. Furthermore, outside-in signaling via integrin receptors modulates a number of important post-trafficking events.

 
The initial contact of the leukocyte with the endothelial surface "tethers" the leukocyte, allowing endothelial membrane-bound chemokines, membrane-expressed platelet-activating factor, or locally secreted chemoattractants to activate leukocyte integrins. This "inside-out" activation results in changes in integrin affinity [10 ] or affinity-independent integrin clustering [43 ]. The activated leukocyte integrins initiate the secondary phase of adhesion with firm binding to endothelial ligands, which are members of the immunoglobulin gene superfamily (IgSF; IgSF nomenclature: intercellular adhesion molecule-1 (ICAM-1), CD54; ICAM-2, CD102; vascular cell adhesion molecule-1 (VCAM-1), CD106; platelet-endothelial cell adhesion molecule-1 (PECAM-1), CD31; mucosal addressin cell adhesion molecule-1 (MAdCAM-1); junctional adhesion molecule (JAM); receptor for advance glycation end products (RAGE); Fig. 2 ). These IgSF ligands are constitutively expressed (ICAM-1; ICAM-2) or further up-regulated (ICAM-1) or are induced (VCAM-1). Recently, the pattern recognition receptor RAGE, which is up-regulated by diabetic conditions [44 ], has been identified as an endothelial IgSF ligand for leukocyte ß2 integrins [45 ].



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  		Figure 2. Leukocyte integrins and their ligands. Leukocyte integrins bind to IgSF ligands on the vascular endothelium in nonlymphoid tissue and to high endothelial venules (HEV) in lymphoid tissue as well as to components of the ECM, such as the connecting segment-1 (CS-1) of fibronectin (FN), osteopontin (OPN), thrombospondin (TSP), collagen (COL), vitronectin (VN), and laminin (LN). Antagonists of the leukocyte integrins depicted have shown efficacy in preclinical models ({alpha}Mß2, {alpha}1ß1, {alpha}vß3) and in clinical trials ({alpha}Lß2, {alpha}4ß1, {alpha}4ß7).

 
Once firmly adherent, the leukocyte migrates over the endothelial cell surface, using {alpha}Lß2 and {alpha}Mß2 integrins and their endothelial IgSF ligands [46 ], and then emigrates through an intercellular junction. The process of transendothelial migration involves several junctional proteins, including the IgSF proteins PECAM-1 [47 ] and JAMs [48 , 49 ] as well as CD99 [50 ]. In some vascular beds, leukocytes may enter the extravascular tissue by traversing through, rather than between, endothelial cells [51 ].

Immune surveillance of tissue is mediated by similar adhesion mechanisms. Lymphocytes recirculate between blood and lymphatics, gaining entrance to the latter at specialized HEV of postcapillary venules in lymphoid tissue [7 ]. The HEV express vascular addressins recognized by specific lymphocyte-homing receptors that support the primary phase of adhesion. L-selectin recognizes sialyl LewisX-like sugars on the peripheral node-addressin, and the integrin {alpha}4ß7 binds to MAdCAM-1 [52 ]. In the secondary phase of firm adhesion, the binding of locally expressed chemokines to cognate lymphocyte receptors activates {alpha}Lß2, attaching the lymphocyte firmly to the endothelial cell. When similarly activated, {alpha}4ß1 and {alpha}4ß7 also promote firm adhesion. Once attached, the lymphocyte can then diapedese between endothelial cells and enter lymphoid tissue.

There are a number of exceptions to the general model of selectin-mediated rolling followed by integrin-mediated firm adhesion and transmigration depicted in Figure 1 . Tethering and rolling can also occur via {alpha}4ß7 [52 ], {alpha}4ß1 [53 ], and under some circumstances, {alpha}Lß2 [54 ] integrins. CD44 [55 ] and vascular adhesion protein-1 [56 ] may also support selectin-independent rolling. Moreover, selectins do not appear to play a major role in leukocyte emigration in the pulmonary microcirculation, where emigration occurs predominantly in capillaries [57 ], or in the liver microvasculature, where leukocytes emigrate primarily in sinusoids [58 ]. Additionally, leukocyte interactions with platelets may contribute to recruitment to inflammatory sites. Following a vascular injury that produces endothelial denudation or retraction, platelets rapidly adhere to the exposed subendothelium. The platelets adherent to the damaged vessel wall may recruit leukocytes directly by initiating selectin- and integrin-dependent leukocyte adhesion to surface-bound platelets [59 60 61 ]. Finally, there are a number of other potentially important adhesion pathways that have yet to be fully characterized in vivo [62 ], including leukocyte integrin-dependent interactions such as binding of {alpha}Mß2 to ICAM-1 via bridging by fibrinogen [63 ], {alpha}Mß2 to GPIb [64 ], {alpha}Lß2 to JAM-A [48 ], {alpha}Mß2 to JAM-C [49 , 65 ], {alpha}4ß1 to JAM-B [66 ], and {alpha}9ß1 to VCAM-1 [67 ].

Post-trafficking events
Leukocyte integrins also play a critical role in multiple "post-trafficking" events that follow transendothelial migration. Neutrophil {alpha}Mß2 binds to plasma-derived proteins, such as iC3b, factor X, and fibrinogen, which may be present tissue at sites of vascular leakage. These interactions may contribute significantly to the pathogenesis of the local inflammatory response (e.g., ref. [68 ]). A variety of stimuli drives the continued movement of the leukocyte through the subendothelial matrix and the extravascular tissue to the site of inflammation or immune reaction. The interaction of leukocyte integrins with ECM components is critical for migration in tissue (reviewed in refs. [69 70 71 72 ]). In addition to regulating migration, "outside-in" signaling via leukocyte integrins modulates the activation, proliferation, and survival of leukocytes in extravascular tissue (reviewed in refs. [21 , 73 74 75 76 ]; Figs. 1 and 2 ). The importance of these integrin-dependent post-trafficking events in inflammatory and immune responses of leukocytes is illustrated by several in vivo studies of blockade of integrins on tissue leukocytes. In sheep [77 ] and in mice [78 ], blockade of {alpha}4 on intrapulmonary leukocytes (in contrast to circulating leukocytes) decreased allergen-induced lung inflammation. Integrin {alpha}1ß1 is expressed on activated lymphocytes and monocytes in tissue but not on circulating leukocytes in the bloodstream. Nevertheless, blockade of {alpha}1ß1 significantly ameliorates inflammatory responses in animal models of arthritis [79 ], colitis [80 ], influenza infection [81 ], and asthma [82 ], demonstrating that post-trafficking signaling via {alpha}1ß1 regulates leukocyte function. Finally, {alpha}vß3 is expressed on monocytes in culture [83 ] and on monocyte-derived macrophages, including osteoclasts. A peptidomimetic antagonist of {alpha}vß3 inhibited bone resorption in vitro and osteoporosis in a rat model [84 ].

Leukocyte development and hematopoiesis
The ß1 integrins, particularly {alpha}4ß1, play a critical role in B lymphocyte development in bone marrow and germinal centers and T lymphocyte development in the thymus [74 , 85 86 87 ]. The ß1 integrins, {alpha}4ß1 and {alpha}5ß1, are essential for fetal hematopoiesis [88 , 89 ], and in the adult, {alpha}4ß1 is important in the distribution of progenitors and regeneration of hematopoiesis after stress [90 ]. The interaction of {alpha}4ß1 on hematopoietic stem cells (HSC) with VCAM-1 on marrow stromal cells is pivotal in the mobilization and homing of hematopoietic progenitors [91 , 92 ]. The ß2 integrins interact synergistically with {alpha}4ß1 in HSC mobilization [93 ]. Antagonists of {alpha}4ß1 have been proposed as adjunctive agents for mobilization of HSC for transplantation. It is important that chronic blockade of {alpha}4 in clinical trials in patients with multiple sclerosis (MS; see below) has not, to date, revealed a defect in hematopoiesis.

Outside-in signaling via {alpha}4ß1 interactions with stromal VCAM-1 or fibronectin is also important in the survival of leukemic cells in the marrow microenvironment [94 ]. Expression of {alpha}4ß1 has been correlated with poor prognosis in myeloid leukemia, and blockade of {alpha}4 sensitizes leukemic cells to chemotherapy-induced killing [94 ], raising the possibility that {alpha}4ß1 antagonists could synergize with chemotherapy in treatment of leukemia.


   APPROACHES TO TARGETING LEUKOCYTE INTEGRINS AND THEIR IgSF LIGANDS
TOP
 ABSTRACT
 INTRODUCTION
 INTEGRINS
 LEUKOCYTE INTEGRINS
 APPROACHES TO TARGETING...
TARGETING LEUKOCYTE INTEGRINS...
 CONCLUSION
 REFERENCES
 
There are a number of approaches to "antiadhesion" therapy directed to leukocyte integrins and their IgSF ligands (Fig. 3 ).



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  		Figure 3. Approaches to targeting leukocyte integrins and their IgSF ligands. In addition to direct blockade of receptor-ligand interactions by monoclonal antibody (mAb) or ligand mimetics, there are small molecule antagonists that inhibit the conformational changes necessary for increases in receptor affinity. Antisense technology has been used to target IgSF ligands. Signaling pathways regulating integrin function and those involved in IgSF ligand expression are potential targets for small molecule inhibitors.

 
Receptor-ligand blockade
This approach is the most obvious, and mAb that block integrin binding to ligand have been used extensively in preclinical and clinical trials. A murine mAb to ICAM-1 was tested in several clinical trials [95 , 96 ]; however, most other mAb (e.g., anti-{alpha}4, anti-{alpha}L, and anti-{alpha}4ß7) in clinical trials have been humanized by various techniques to minimize mouse sequences and thereby reduce host antibody response. Many peptidomimetic and small molecule ligand mimetic antagonists of leukocyte integrins have also been developed (reviewed in refs. [12 , 15 16 17 18 ]). Several {alpha}4ß1 ligand mimetic antagonists have been shown to be effective in animal models (e.g., refs. [97 , 98 ]).

Allosteric inhibitors
Conformational changes in the I domain of the {alpha}-subunit of ß2 integrins are necessary for an increase in the binding affinity of the metal ion-dependent adhesion site (MIDAS), which is the ligand-binding site in the I domain [22 ]. Two classes of allosteric I domain inhibitors have been identified [12 ]. Several small molecules inhibit ligand binding to the distal MIDAS by binding to and stabilizing the closed conformation of the I domain, preventing its conversion to the high-affinity, open conformation [99 100 101 ]. Another class of small molecule antagonists maintains the I domain in the low-affinity, closed conformation by affecting interactions between the I domain and the I-like domain in the ß-subunit [102 , 103 ].

Inside-out and outside-in signaling pathways
The signaling pathways modulating integrin-affinity modulation and clustering have been studied intensively. Several key proteins involved in inside-out signaling have been identified (reviewed in refs. [70 , 104 , 105 ]), including the GTPase Rap1 and its ligand RAPL [106 ], the GTPase RhoA [107 ], the tyrosine kinase Pyk2 [108 ], and the cytoskeletal protein talin [109 ]. Inside-out signaling is an important target for small molecule inhibitors of integrin function. For example, statin drugs have the potential to block integrin function by preventing the prenylation and thereby, inhibiting the function of Rap1 [110 ]. Similarly, the multiple outside-in signaling pathways initiated by integrin ligation (reviewed in refs. [21 , 73 74 75 76 ]), which regulate important post-trafficking events, are also potential candidates for disruption by small molecule antagonists. For example, in neutrophils, Vav guanine nucleotide exchange factors [111 ] and the Src homology 2 domain-containing adaptor leukocyte phosphoprotein of 76 kD [112 ] have been shown to regulate several key functions induced by ligation of ß2 integrin.

Intracellular pathways regulating expression of IgSF ligands
Antisense oligonucleotides or RNA interference [113 ] can be used to decrease synthesis of adhesion molecules. Antisense oligonucleotides that target ICAM-1 have undergone extensive clinical trials with only limited success (Table 1 ). RNA interference has been used in vitro to decrease the expression of integrin receptors (e.g., ref. [138 ]), but trials targeting integrins or IgSF ligands in vivo have not yet been reported. The signaling pathways that up-regulate expression of endothelial IgSF ligands by cytokines are also potential targets for small molecule inhibitors (e.g., refs. [139 , 140 ]).


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  		Table 1. Clinical Trials Targeting Leukocyte Integrins and Their IgSF Ligandsa

 

   TARGETING LEUKOCYTE INTEGRINS AND THEIR IgSF LIGANDS IN HUMAN DISEASE
TOP
 ABSTRACT
 INTRODUCTION
 INTEGRINS
 LEUKOCYTE INTEGRINS
 APPROACHES TO TARGETING...
 TARGETING LEUKOCYTE INTEGRINS...
CONCLUSION
 REFERENCES
 
In reviewing clinical trials of antiadhesion therapy directed at leukocyte integrins or their IgSF ligands, we will consider only completed Phase II and III studies (Table 1) .

Ischemia-reperfusion disorders
Ischemia-reperfusion injury has been postulated to contribute to many pathologic conditions, including MI, stroke, and shock. Preclinical studies in animal models of ischemia-reperfusion disorders showed striking protection against reperfusion injury with blockade of leukocyte or endothelial adhesion molecules (reviewed in ref. [141 ]). It is disappointing, however, the clinical trials that followed in these indications were not successful.

MI
Two multicenter, randomized, placebo-controlled trials tested the efficacy of ß2 blockade in the setting of acute MI. Rovelizumab, a recombinant, humanized anti-ß2 mAb, failed to reduce infarct size following angioplasty [124 ]. In the second study, erlizumab, another recombinant, humanized anti-ß2 mAb, did not modify cardiac endpoints when administered in conjunction with thrombolytic therapy [123 ].

Stroke
Rovelizumab was also tested in acute stroke, but Phase III studies were discontinued at approximately 15 months after initiation as a result of a lack of efficacy [132 ]. Likewise, Phase II trials in acute stroke of recombinant neutrophil inhibitory factor protein, which inhibits {alpha}Mß2, were discontinued secondary to early determination that treatment had no effect on outcome [131 ]. A Phase III trial of enlimomab, a murine anti-ICAM-1 mAb, showed no efficacy and in fact, worsened stroke outcome [95 ]. It has been postulated that the adverse outcome observed with enlimomab treatment in this trial was a result of complement-dependent activation of neutrophils by the antibody [142 ].

Traumatic shock
Two Phase II trials tested blockade of ß2 in the setting of hemorrhagic shock following trauma. Neither rovelizumab nor erlizumab demonstrated efficacy compared with control upon analysis of a number of clinical endpoints [136 , 137 ].

There are several potential explanations for the failure of adhesion blockade in these clinical disorders with putative ischemia-reperfusion injury. The design of the trials may not have been adequate to test the underlying hypothesis. For example, in the case of the two hemorrhagic shock trials [136 , 137 ], mortality in all groups was low compared with the reported rates in similar populations, making the studies too small to have adequate power to demonstrate efficacy in reducing mortality. Second, it is possible that selectins rather than ß2 integrins are a better target. However, in the preclinical models, ß2 integrin or selectin blockade appeared equally efficacious in preventing reperfusion injury [141 ], including comparisons in the same model (e.g., refs. [143 , 144 ]). Moreover, to date, there have been no reports of successful clinical trials of selectin antagonists in MI, stroke, or shock [18 ]. Third, it is possible that leukocytes are not a significant component of clinical reperfusion injury, particularly with longer ischemic periods [145 146 147 ]. Other mediators, such as complement or oxidants, may play larger roles in this setting. Finally, the general issue remains that animal models may not always accurately reflect human disease processes.

Atherosclerosis
The endothelial IgSF ligand VCAM-1 is thought to play a pivotal role in atherogenesis (e.g., ref. [148 ]). AGI-1067 is an oral antioxidant that inhibits the transcription of VCAM-1 as well as several other cytokine-induced, redox-sensitive genes [139 ]. AGI-1067 improved luminal dimensions at the intervention site and reduced restenosis in the Canadian antioxidant restenosis trial [149 ]. Whether the benefit of AGI-1067 was a result of an inhibition of VCAM-1 expression or to other mechanisms is uncertain.

Asthma
The asthmatic response to allergen is characterized by airway hyper-responsiveness and inflammation with the accumulation of lymphocytes and eosinophils. In animal models, the trafficking of these effector cells involves {alpha}Lß2 and {alpha}4ß1 (e.g., refs. [77 , 78 , 150 , 151 ]). {alpha}4ß1 [78 ] and {alpha}1ß1 [82 ] are also involved in post-trafficking events regulating the allergic response. Efalizumab, a humanized anti-{alpha}L mAb, was tested in patients with mild asthma. Although the number of activated eosinophils was significantly decreased after 4 weeks of treatment, the early and late percent fall in forced expiratory volume at 1 s after allergen challenge did not reach statistical significance [114 ]. Recently, an oral, dual {alpha}4ß1/{alpha}4ß7 antagonist was reported to show some efficacy in a Phase II trial in asthma [115 ].

Burns
Tissue injury following a burn can worsen as a result of progressive destruction from the acute inflammatory response [152 ]. The murine mAb to human ICAM-1, enlimomab, was tested in the treatment of partial thickness burns. There was improved healing at high-risk sites with enlimomab compared with placebo and trends toward significance for the primary endpoint of reduction in the total area of primary grafts [96 ]. However, the scar ratings at long-term follow-up were not statistically different from control [96 ].

Inflammatory bowel disease
Although distinct diseases, Crohn’s disease and ulcerative colitis are chronic inflammatory conditions, which are theorized to stem from repeated stimulation of the immune system by normal bowel flora [153 ]. Endothelial IgSF ligands VCAM-1 [154 , 155 ], ICAM-1 [154 ], and MAdCAM-1 [156] are expressed in normal and inflamed bowel. Consequently, there have been several trials of antiadhesion therapy directed at the IgSF ligands or leukocyte integrins.

Alicaforsen, an antisense oligonucleotide to ICAM-1, failed to show efficacy in Phase II trials for Crohn’s disease [118 , 119 ].

A humanized mAb against {alpha}4ß7 (MLN02) has a completed Phase II trial for ulcerative colitis. There were significant improvements in the clinical remission rates of patients treated with MLN02 compared with placebo [120 ]. A Phase II clinical trial in Crohn’s disease did not achieve superior clinical response compared with placebo, the primary endpoint of the trial. However, the clinically important, secondary endpoint of disease remission was achieved in the higher dose group [117 ].

Natalizumab is a recombinant, humanized mAb to the {alpha}4 subunit, which inhibits binding of {alpha}4ß7 to MAdCAM-1 and {alpha}4ß1 to VCAM-1. In Phase II trials in Crohn’s disease, patients who received two infusions of natalizumab had higher clinical response rates and improved remission rates when compared with placebo at predetermined time-points [116 ]. Additionally, the midpoint analysis of Phase III trials showed that those patients who had previously achieved a clinical response and who had received monthly infusions of natalizumab were better able to maintain clinical response and remission at 6 months [157 ]. With these positive results, there are plans to file for approval for the use of natalizumab in Crohn’s disease in Europe [158 ].

MS
MS is a presumed autoimmune disorder characterized pathologically by demyelinated plaques in the white matter of the central nervous system (CNS). The majority of patients has relapsing-remitting MS with the relapses secondary inflammation in the CNS white matter mediated by autoreactive T lymphocytes [159 ]. Blockade of {alpha}L, {alpha}M [160 ], or {alpha}4 [161 ] showed efficacy in rodents with experimental allergic encephalomyelitis (EAE), a model for MS. However, in a Phase II trial of patients with acute exacerbations of MS, the humanized anti-ß2 mAb, rovelizumab, did not demonstrate significant clinical benefit as determined by changes in the Scripps scale, a neurological instrument [122 ].

Over a decade after the seminal study by Yednock et al. [161 ], demonstrating the efficacy of {alpha}4 blockade in the EAE model, a Phase II trial of natalizumab showed short-term efficacy in relapsing-remitting MS [121 ]. At 6 months, patients who received monthly natalizumab had fewer new enhancing lesions, as assessed by monthly magnetic resonance imaging scans. Based on a 1-year analysis of ongoing Phase III trials, natalizumab has been submitted for approval for treatment of MS to the U.S. Food and Drug Administration (FDA; Rockville, MD) [162 ] and European Medicines Agency [163 ].

Psoriasis
Psoriasis is an inheritable, chronic, inflammatory condition, which primarily affects the skin with scaly papular or plaque-like lesions and extracutaneous manifestions including psoriatic arthritis and inflammatory bowel disease. The pathophysiology of psoriasis remains unknown, but one possibility is that it begins with stimulation of the keratinocytes by, for example, mild trauma. Dendritic cells and T cells are then activated, initiating the inflammatory cascade with the ensuing release of cytokines and growth factors causing keratinocyte proliferation. Eventually, epidermal thickening, an angiogenic tissue reaction, and plaque formation result [164 ].

The interaction of {alpha}Lß2 with ICAM-1 is important for lymphocyte trafficking to inflamed skin as well as for immunologic synapse formation between resident T lymphocytes and antigen-presenting cells [165 ]. In Phase III trials, patients that received weekly subcutaneous injections of the humanized anti-{alpha}L mAb efalizumab exhibited significant improvement in their psoriasis compared with controls for all endpoints [125 126 127 ]. Efalizumab was approved by the U.S. FDA in October 2003 for the treatment of chronic, moderate-to-severe plaque psoriasis [166 ]. For psoriatic arthritis, however, preliminary results from a Phase II trial using efalizumab failed to demonstrate any clinical benefit [128 ].

RA
RA is a chronic, inflammatory condition, primarily affecting the joints, which can lead to debilitating destruction of cartilage and bone. As with other immune disorders, leukocytes are believed to be critical effectors of tissue injury in arthritis, and blockade of {alpha}4ß1/VCAM-1 and {alpha}Lß2/ICAM-1 interactions has demonstrated efficacy in animal models (e.g., ref. [167 ] and reviewed in ref. [168 ]).

Phase I/II trials were conducted in early and refractory RA using a murine anti-ICAM-1 mAb. Although the studies were not placebo-controlled, the majority of patients responded to treatment with greater apparent efficacy in early versus refractory disease [169 , 170 ]. A Phase II trial of the humanized anti-{alpha}Lß2 mAb efalizumab in moderate-to-severe RA was terminated, as there was no overall benefit compared with control [129 ]. A randomized, placebo-controlled trial of an antisense oligodeoxynucleotide to ICAM-1 in the treatment of severe RA did not show significant efficacy at the primary endpoint [130 ].

Transplant
Graft survival in solid organ transplant has improved as immunosuppressive therapy has evolved. However, rejection remains an important post-transplant complication. Transplanted organs and organs undergoing rejection show leukocyte accumulation and increased expression of IgSF ligands [171 , 172 ], and leukocyte integrin antagonists have shown efficacy in animal models of graft rejection (e.g., refs. [19 , 173 174 175 ]). There have been several clinical trials of adhesion blockade in clinical transplantation. There was a reduction of graft failure by a murine mAb to {alpha}Lß2 after human leukocyte antigen nonidentical bone marrow transplantation in children [176 ]. In patients receiving partially incompatible bone marrow transplants, a high rate of engraftment was achieved by the use of anti-{alpha}Lß2 and anti-CD2 antibodies, but there was a high incidence of lethal infections and relapses as a result of long-lasting immunodeficiency [177 ]. However, a Phase II trial of a murine anti-{alpha}Lß2 mAb odulimomab in renal transplant showed no difference in the incidence and severity of acute rejection in the first 3 months compared with treatment with rabbit antithymocyte globulin [133 ]. A Phase III trial of this drug also showed little efficacy [134 ]. Short-term induction therapy with the murine anti-ICAM-1 mAb enlimomab after cadaveric renal transplantation did not reduce the rate of acute rejection or delayed onset of graft function [135 ]. In a Phase I/II dose-finding study of the humanized anti-{alpha}Lß2 mAb efalizumab in patients receiving cadaveric or living donor kidneys, there was a worrisome occurrence of post-transplant lymphoproliferative disease in several patients receiving the high dose, presumably reflecting potent immunosuppresion [178 ].

Emigrated leukocytes are also the effectors of graft-versus-host disease (GVHD) [179 , 180 ]. Blockade of leukocyte integrins was shown to ameliorate experimental GVHD (e.g., refs. [181 , 182 ]). In a small clinical trial, an anti-{alpha}Lß2 antibody was effective in treatment of steroid-resistant, acute GVHD [183 ], and the murine anti-{alpha}Lß2 mAb odulimomab has been used in France for prevention of GVHD [18 ].


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 INTEGRINS
 LEUKOCYTE INTEGRINS
 APPROACHES TO TARGETING...
 TARGETING LEUKOCYTE INTEGRINS...
 CONCLUSION
REFERENCES
 
Antiadhesion therapy directed at leukocyte integrins and their IgSF ligands has demonstrated efficacy in multiple preclinical models of inflammatory and immune diseases. Although positive results in clinical trials have been somewhat elusive, the field has been buoyed by recent successes with recombinant, humanized mAb to {alpha}L in psoriasis and to {alpha}4 in MS and inflammatory bowel disease. Humanized, anti-integrin antibodies are being tested in other indications, and multiple companies are pursuing development of small molecule ligand mimetic antagonists. Insights into integrin structure and inside-out and outside-in signaling will also likely lead new therapies. The future of leukocyte integrin antagonist therapy is exciting, promising a new class of therapeutics with the potential to impact a broad spectrum of human diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by U.S. Public Health Service Grants HL18645 (J. M. H.) and DK007662 (K. Y.).

Received August 17, 2004; revised September 13, 2004; accepted October 4, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 INTEGRINS
 LEUKOCYTE INTEGRINS
 APPROACHES TO TARGETING...
 TARGETING LEUKOCYTE INTEGRINS...
 CONCLUSION
 REFERENCES
 

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