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April 26, 2005; 64 (8) View & Review

Anti-α4 integrin therapy for multiple sclerosis

Mechanisms and rationale

George P.A. Rice, Hans-Peter Hartung, Peter A. Calabresi
First published April 7, 2005, DOI: https://doi.org/10.1212/01.WNL.0000158329.30470.D0
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Abstract

The symptoms, severity, and course of multiple sclerosis (MS) vary among patients, leading to complex treatment issues. In recent years, research has focused on specific adhesion molecules that participate in the activation and function of lymphocytes, especially the migration of these cells to sites of inflammation. In particular, the integrin, very late activation antigen (VLA)-4, has been implicated in mediating adhesion and migration of immune cells through interaction with its ligand, vascular cell adhesion molecule (VCAM)-1. VLA-4 is comprised of α4/β1 and is critical in mediating Th-1 cell migration in the animal model of MS, experimental autoimmune encephalomyelitis, and has been the target of several recent clinical trials in MS. The humanized monoclonal antibody to α4 integrin, natalizumab (Tysabri, Biogen Idec/Elan), was recently approved in the United States for the treatment of relapsing MS. The authors discuss the mechanisms by which α4 integrins alter lymphocyte function as a rationale for anti-α4 integrin use in MS.

The causes of multiple sclerosis (MS) are unknown, but the immune system plays a role in the creation and amplification of the brain lesions characteristic of the disease.1 MS is a dynamic lifelong illness mediated by chronic lymphocyte infiltration into the brain and an ensuing inflammatory cascade. The resulting demyelination and subsequent degeneration of axons account for the disability experienced by patients with MS. Strategies aimed at limiting the instigating inflammation seem to have a positive effect on the disease course. In this effort, an antibody to α4-integrin, natalizumab, was developed to inhibit transmigration of T-cells from the circulation into the brain parenchyma. In this review we describe the importance of α4-integrin in lymphocyte migration in MS and the rationale for anti-α4 integrin therapy in MS.

Inflammation and leukocyte migration.

Although they are concentrated in lymphatic tissues, low numbers of leukocytes can be found in all tissues that receive a blood supply including the brain, even in healthy individuals.2–4 To achieve this distribution, leukocytes must adhere to and traverse the vascular endothelium, migrate through tissue beds, and exit the tissue through the lymphatic system.5

Inflammation is associated with increased numbers of activated leukocytes at the site of the lesion. In MS, sites of new or active brain lesions are enriched in activated lymphocytes. Accumulation of these cells involves enhanced transmigration across the blood–brain barrier, proliferation within the brain, and possibly enhanced survival of lymphocytes within the brain. These processes also involve the activity of soluble mediators including proinflammatory cytokines, chemoattractant cytokines called chemokines, matrix metalloproteinases, and adhesion molecules.

Under the inflammatory conditions of MS, brain, CSF, and blood lymphocyte levels increase by clonal expansion of T-cell subsets.6,7 The subset of T cells involved may depend on the disease stage.6 Endothelial cells undergo changes during inflammation as well. Cytokines expressed at sites of inflammation activate the endothelial cells lining postcapillary venules and induce the expression of several cell adhesion molecules (CAMs), such as P- and E-selectins, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1).8–10 The expression of these CAMs on the surface of the endothelial cells facilitates tethering, rolling, and firm adhesion of circulating leukocytes to the endothelium,11,12 in preparation for transmigration of the leukocytes across the endothelium and into the inflamed tissue (figure). Initial contact between leukocytes and endothelial cells is mediated by the P-, E-, and l-selectins,11,12 and by α4 integrins.13,14 It has been demonstrated14 in vivo that experimental autoimmune encephalomyelitis (EAE)-inducing T cells in the bloodstream are captured on the endothelial vessel surface via α4 integrin/VCAM-1 interaction. α4 Integrins are also responsible for firm adhesion of leukocytes to the endothelial surface, and for initiating the process by which the leukocytes migrate across the endothelium. In addition, monocyte-chemoattractant protein-1, secreted from astrocytes, may play a role in leukocyte transmigration as demonstrated in a cell culture model of the blood–brain barrier.15 These adhesion molecules and cytokines are key players in a cellular cascade of events controlling leukocyte activation, expression of other CAMs, and even cell phenotype and survival. In MS, P-selectin and α4 integrin may mediate pioneering lymphocyte migration at disease onset,14,16 and α4 integrin-mediated leukocyte attachment may be more important once inflammation is established.

Figure

Figure. Extravasation is a multistep process.

α4 Integrins.

α4 Integrin is a member of a large family of cell-surface adhesion molecules that mediate both cell-cell and cell-extracellular matrix (ECM) interactions.17 α4 Integrin is expressed predominantly on lymphocytes, monocytes, eosinophils, and basophils, and is usually undetectable on neutrophils.18–20 It heterodimerizes with two β-subunits, β1 and β7, to form a functional molecule.

As shown in the figure, one of the ligands for α4β1 and α4β7 integrins is VCAM-1, which is expressed on vascular endothelial cells, especially those activated by cytokine.21,22 α4 Integrin interaction with VCAM-1 promotes firm adhesion of lymphocytes to the endothelium in preparation for transmigration of the lymphocyte. Binding of lymphocyte cell lines to activated endothelial cells was dependent on the level of expression of α4β1 integrin in the lymphocyte-derived cells.21 α4 Integrin binding to endothelial cells via VCAM-1 also initiates a number of cellular processes, leading to activation of the lymphocyte and alteration of its phenotype to promote migratory function,23 activation,24 and proliferation.25 The close apposition of lymphocytes and endothelial cells allows locally produced cytokines and chemokines to activate the lymphocytes and further promote adhesion and transmigration into inflamed tissue.26,27

Although adhesion of α4-expressing leukocytes and their subsequent migration across the blood–brain barrier involve interactions between the α4 integrin and VCAM-1 on the blood–brain barrier endothelial cells and glial cells, interactions between α4-expressing leukocytes also occur with the extracellular matrix molecules, fibronectin,28 and osteopontin,29,30 and may perpetuate the inflammatory cascade within the brain parenchyma. Interactions with fibronectin have important functional implications for lymphocytes.31 Studies using immobilized ligands, or specific peptide ligands for the fibronectin binding site of α4β1, have suggested that the α4β1-fibronectin interaction is important for migration of lymphocytes through the extracellular matrix toward the site of inflammation.32,33 The α4β1-fibronectin interaction also acts as a costimulatory or second signal (in addition to T-cell-receptor signaling) for lymphocyte proliferation and activation.34–36 In addition, in vitro experiments demonstrate that transmigrated α4β1-expressing T cells downregulate α4 expression and display increased binding to fibronectin via upregulated α5β1.37

Osteopontin is a matrix protein that has both adhesive and Th-1-like cytokine activity. Its expression is increased in areas of remodeling,30 in the spinal cord of rats with EAE, and in lesions from patients with MS.38 Osteopontin also acts as a costimulatory signal for T-lymphocyte activation.39 Interestingly, mice deficient in osteopontin were relatively resistant to EAE.38

Activated T lymphocytes have also been shown to bind, via α4β1, to a glycoprotein component of the extracellular matrix, thrombospondin, that is upregulated in many tissues during damage or inflammation.40 The importance of this interaction is under investigation.

Preclinical studies of α4-integrin inhibitors.

In vitro and animal studies demonstrate the crucial role of α4 integrins in leukocyte trafficking, activation, differentiation, and proliferation. Because α4 integrin is not expressed by neutrophils, blockade of α4 function is unlikely to inhibit the response of these cells to bacterial infection. These properties make α4 integrin an attractive target for therapeutic intervention against diseases that involve chronic inflammation, such as MS.

Monoclonal antibodies directed against α4 integrin have been used to study the function of this molecule in several models of inflammation including contact hypersensitivity reactions, allograft rejection, ulcerative colitis, lung antigen challenge, and EAE.19,41

Antibodies to α4β1 prevented the interaction of lymphocytes with endothelial cells in vitro,21 which suggests that antibodies to α4β1 integrin reduce the transmigration of lymphocytes to areas of inflammation. It has been reported that α4β1-expressing Th-1 lymphocytes translocate to the brain parenchyma, whereas those that lack expression of α4β1 do not.42 Other researchers have reported that memory T cells that circulate to areas of inflammation express α4β1.43,44 Furthermore, blockade of α4 integrin-VCAM-1 interaction increases apoptosis of T cells,45 including those involved in an inflammatory response.46

Under normal conditions, VCAM-1 cannot be detected in the brain parenchyma. However, there is evidence that it may be expressed by certain glial cells near sites of lesions in patients with MS.47 In vitro studies have also found that expression of VCAM-1 by glial cells is induced by cytokines.48 These early in vitro studies raise the intriguing possibility that lymphocytes in the brain parenchyma may interact with glial cells through α4β1-VCAM-1 binding,49,50 which may contribute to the mechanism of action of an α4-integrin inhibitor on lymphocytes already present in brain tissue.

α4-Integrin inhibitors in laboratory models of MS.

The most commonly used experimental model of MS is EAE. Although there are many variants of the model, the general concept involves injection of a myelin-derived protein (such as myelin basic protein [MBP]) in conjunction with complete Freund’s adjuvant (CFA) into a rodent. In most cases, the rodent will develop an immune response to the antigen, including antibodies and activated T cells specific for the antigen. These T cells then migrate to the brain where they initiate inflammation. The inflammation leads to secondary recruitment of additional lymphocytes and monocytes from the blood. Disability results from the inflammatory attack on the axonal myelin sheath. The course and characteristics of EAE have many similarities to MS.51–54

The first evidence that α4 integrin was involved in the EAE model of MS was obtained by Yednock et al.55 These investigators used a variant of EAE in which an activated T-cell clone specific for myelin basic protein was administered to rats by intraperitoneal injection. After 4 to 5 days, the rats experienced paralysis of their tails and hind limbs. This paralysis was accompanied by infiltration of lymphocytes and monocytes into the brain, and inflammation of blood vessels in the brainstem and spinal cord.

The ability of day-5 brain sections from animals with EAE to support leukocyte attachment was also examined in this study. Monocytic cell lines, including human peripheral blood monocytes and lymphocytes, and freshly isolated rat and mouse lymphocytes, selectively bound inflamed vessels in brain sections from animals with EAE, but not in sections taken from control animals. Binding of human peripheral neutrophils to EAE vessels was not observed. In this model system, binding of the human monocytic cell line U937 to EAE vessels was almost completely (>95%) inhibited by pretreatment with a monoclonal antibody to α4 integrin. In addition, intraperitoneal injection of this antibody 2 days after administration of the inflammation-inducing T-cell clone completely prevented the development of paralysis of the treated animals, and delayed and reduced the severity of paralysis in those animals that developed the disease. The protective effect of the antibody was dose-dependent, a finding also observed in a study by others.56 The therapeutic effect of the anti-α4 antibody used in this study was dependent on the total amount of antibody injected, as well as the mean disease score. When administered to mice with severe disease, the antibody significantly delayed the lethal outcome of EAE. Animals with mild disease were provided longer periods of protection from disease after the last antibody injection. Studies subsequent to that of Yednock et al.55 have confirmed that the ability of lymphocytes to migrate to inflamed regions of the brain is strongly correlated with their surface expression of α4β1.42,57,58 Additional studies in guinea pigs have corroborated that anti-α4 antibodies are capable of reversing the signs of EAE observed by brain MRI.59 A peptide inhibitor of α4 integrin has also recently been shown to be beneficial for treatment of EAE.60 In another EAE model for relapses triggered by viral infection, mice partially resistant to EAE were made susceptible by exposure to a virus.61 Antibodies to α4 integrin inhibited development of EAE and reduced migration of lymphocytes into the brain. Antibodies to other adhesion molecules were less effective. The authors concluded that relapses associated with viral infections resulted from facilitation of leukocyte entry into the brain. Antagonism of α4 inhibited this process.61

One preclinical study of EAE in mice raised some concern that anti-α4 integrin therapy may worsen disease in some patients with MS.58 The authors of this study found that treatment with an anti-α4 antibody before induction or onset of disease inhibited the onset and reduced the severity of disease. However, when given during the peak of an acute exacerbation, or in the period between exacerbations, the anti-α4 antibody worsened signs of disease and increased the number of activated T cells in the CNS. The reason for this reaction in EAE mice is unclear. Similarly, because an α4-integrin antibody essentially traps cells in the periphery and does not fix complement or kill very late activation antigen (VLA)-4-expressing T cells, it has been postulated that an increased migration may occur upon cessation of treatment. Alternatively, if regulatory cells expressed VLA-4, the monoclonal antibody could antagonize the beneficial effect of this population of cells. However, these findings have not been observed to date in human studies of the humanized monoclonal antibody to α4 integrin, natalizumab, in patients with MS.

Antibodies to α4 integrin will block the function of the α4β7 heterodimer, as well as the function of α4β1. The major role of α4β7 is in mediating migration of gut-homing T cells through ligation with the mucosal addressin cell adhesion molecule (MAdCAM), and it appears to contribute to the efficacy of α4 antagonists in Crohn’s disease. In preclinical in vivo studies of EAE mice, α4β1 appears to play a more dominant role in EAE development,62 whereas α4β7 appears to be important in the chronic phase of a non-remitting form of the disease.63,64 In one study, antibodies directed against α4 or VCAM-1 significantly delayed the onset of EAE, whereas antibodies against α4β7 or β7 had no influence on EAE development. However, in a study of mice that already developed a chronic non-remitting form of EAE, an antibody against β7 significantly attenuated clinical and histopathologic symptoms of the disease.63 In addition, a MAdCAM-1 antibody (the preferred ligand for α4β7) prevented progression of chronic progressive EAE.64

Models of EAE have also provided evidence that the effects of α4 inhibitors on the lymphocyte activation and proliferation functions of α4 integrins may have therapeutic implications.65 Thus, inhibition of leukocyte adhesion is not likely to explain the entire mechanism of action of these agents. It is not yet clear whether these results extend to lesion development in patients with MS.

Natalizumab (Tysabri, Biogen Idec/Elan).

Natalizumab is the first α4-integrin antagonist in the class of therapeutics referred to as selective adhesion molecule (SAM) inhibitors to be used for the treatment of MS and Crohn’s disease. Natalizumab is a humanized monoclonal antibody raised against human α4 integrin, therefore it blocks both α4β1 and α4β7 integrins. Studies with the murine form of the antibody (AN100226m) prior to humanization had demonstrated that it was a potent inhibitor of in vitro interactions between α4β1 and VCAM-1, and that it suppressed and reversed EAE.63

Humanization of AN100226m was undertaken to reduce potential immunogenicity, increase the in vivo half-life, and allow repeated administration for increased therapeutic benefit.66 Grafting of the complementarity-determining regions (CDRs) of AN100226m onto a human immunoglobulin (Ig) G4 framework resulted in an antibody, natalizumab, that was approximately 99% human-derived. The IgG4 isotype of IgG was chosen as the framework for natalizumab, because this subclass does not activate complement and appears to persist longer in circulation than other forms of human IgG.65 In vitro functional comparisons between natalizumab and AN100226m demonstrated nearly identical binding affinities to an α4β1-expressing cell line. Binding saturation was achieved at the same concentration, and natalizumab was as efficacious as AN100226m in inhibiting binding of α4β1-expressing cells to human VCAM-1. Importantly, and consistent with results observed with AN100226m in the guinea pig model, administration of natalizumab after the onset of EAE resulted in a reversal of both symptoms and T cell accumulation within the CNS, and decreased levels of monocyte infiltration into the CNS.63

Clinical results of natalizumab for MS.

The effect of natalizumab on development of new brain lesions has been studied in three published, randomized, controlled phase II trials.67–69 In a randomized, double-blind trial of 72 patients with relapsing-remitting MS (RRMS) or secondary progressive MS (SPMS), patients were randomized to receive two IV infusions of natalizumab 3 mg/kg or placebo administered 4 weeks apart.67 They were monitored for 24 weeks by gadolinium-enhanced (Gd+) T1-weighted MRI. During the first 12 weeks, patients receiving natalizumab developed fewer new active (mean of 1.8 vs mean of 3.6 for the placebo group, p = 0.042) or new Gd+ (mean of 1.6 vs 3.3 for the placebo group, p = 0.017) lesions. There was no significant difference in the number of new active or new Gd+ lesions in the second 12 weeks of the study, after study drug was discontinued.

The study was not designed to assess the effects of natalizumab on relapse rate or disability, and no effects on relapse rate were observed during the first 12 weeks. However, during the second 12-week period, more relapses were observed in the active treatment group compared with the placebo group (p = 0.005 in favor of the placebo group), although the interpretation of this observation is unclear. These findings raised the question of an increase in relapses after cessation of treatment. However, quiescent MRIs during this period were reassuring. Overall, the MRI results support the hypothesis that natalizumab may alter the process of lesion formation in patients with MS.

Treatment with natalizumab was well tolerated in this trial, and adverse events were not significantly different between the treatment groups. Lymphocyte counts increased between weeks 1 and 12 in natalizumab-treated patients (mean increase of 56% to 60% compared with placebo); however, the mean count remained within the normal range. Lymphocyte counts remained slightly elevated at 16 weeks, and returned to baseline values at weeks 20 and 24. Approximately 11% of patients receiving natalizumab developed low-titer (1.3 to 5 μg/mL) anti-natalizumab antibodies.

It was hypothesized that the administration of natalizumab to patients in the midst of an acute clinical relapse might accelerate clinical recovery, by diminishing the egress of inflammatory cells into the CNS. This question has been addressed in a randomized, multicenter, placebo-controlled, double-blind trial.68 Patients with RRMS or SPMS (n = 180) and acute symptoms lasting longer than 24 hours but less than 96 hours were included in the study to capture the earliest phase of the clinical relapse. Patients were randomized to receive a single IV infusion of natalizumab (1 mg/kg or 3 mg/kg) or placebo. Patients were followed for 14 weeks.

The primary outcome measure was the average change in disability score, defined by the Expanded Disability Status Scale (EDSS), from baseline examination to 1 week. Other secondary outcome measures included the proportion of patients requiring corticosteroid rescue and the evolution of Gd enhancement. There was no change in EDSS score over time between the treatment groups; however, 50% of all patients had improved EDSS scores at weeks 2 and 4. Treatment with natalizumab was associated with a significant decrease in the volume of Gd+ lesions in the combined treatment groups compared with placebo at weeks 1 and 3. The treatment was well tolerated and no unexpected toxicities were identified.

In a randomized, double-blind, placebo-controlled trial designed to test the safety and efficacy of natalizumab in patients (n = 213) with RRMS or SPMS, patients were randomized to receive natalizumab (3 mg/kg or 6 mg/kg) or placebo by IV infusion monthly for 6 months.69 The primary outcome measure was the number of new brain lesions on monthly Gd+ T1-weighted MRI during the 6-month treatment period. Patients were also monitored by T2-weighted MRI and clinical measures including the EDSS,70 Multiple Sclerosis Functional Composite (MSFC),71 and number of clinical relapses.72

Patients receiving natalizumab exhibited significantly fewer new brain lesions on Gd+ MRI. The placebo group had a mean of 9.6 new Gd+ lesions per patient, whereas patients receiving 3 mg/kg had a mean of 0.7 (p < 0.001), and those receiving 6 mg/kg had a mean of 1.1 (p < 0.001). In the subgroup of patients with RRMS, natalizumab-treated patients showed significantly fewer new Gd+ lesions compared with placebo; the mean number of new Gd+ lesions per patient was 12.1 in the placebo group compared with means of 0.6 in the 3-mg/kg group (p < 0.001) and 0.6 in the 6-mg/kg group (p < 0.001). In the subgroup of patients with SPMS, patients who received natalizumab had fewer new Gd-enhanced lesions compared with placebo; this difference was significant for the 3-mg/kg group (p = 0.005), but not for the 6-mg/kg group (p = 0.08). Treatment with natalizumab was also associated with a marked reduction in the number of persistent Gd+ lesions, the total volume of Gd+ lesions, and the number of new active lesions.

Significantly fewer patients in the natalizumab groups experienced a relapse during the 6-month study period. Thirty-eight percent of the patients (27/71) in the placebo group experienced relapses, compared with 19% (13/68, p = 0.02) in the 3-mg/kg group and 19% (14/74, p = 0.02) in the 6-mg/kg group. More patients in the placebo group required corticosteroid treatment for relapse.

Natalizumab was well tolerated. Similar numbers of patients in each group experienced adverse events. One patient in each group developed a serum sickness-like illness, and one patient in the 3-mg/kg group developed urticaria and bronchospasm that rapidly responded to antihistamines and corticosteroids. There were substantial increases in lymphocyte, monocyte, and eosinophil counts that persisted only during the study period, although the mean values were not beyond the normal range. No increase in neutrophil counts was observed. The incidence of anti-natalizumab antibodies was 11%; whether these were binding or neutralizing antibodies was not determined.

A subset analysis of this trial was conducted to evaluate the effect of natalizumab on the conversion of Gd+ lesions to T1-hypointense lesions in patients with relapsing MS.73 As new Gd+ lesions were identified during the trial, their development into T1 lesions by month 12 was investigated. The two natalizumab treatment groups were combined for all analyses because the frequency of new Gd+ lesions was similar. In the combined natalizumab treatment group, significantly fewer patients developed T1 lesions from new Gd+ lesions compared with the placebo group (26% vs 68%; p < 0.01). In addition, the OR demonstrated a reduced probability of conversion of new Gd+ lesions to T1 lesions for patients who received natalizumab (0.48; 95% CI: 0.24, 0.94, p = 0.031). These results suggest that natalizumab reduces inflammatory activity within the lesions already present in patients with MS.

Discussion.

Mechanistic and early clinical studies suggest that inhibition of α4 integrins is a promising new approach for disease-modifying therapy of MS. Inhibition of α4 integrin reduces leukocyte migration to areas of inflammation, and reduces activation and proliferation of leukocytes in response to inflammation. Because α4 integrin is not involved in neutrophil migration or activity, inhibition of this molecule is unlikely to hinder response to bacterial infection. Therefore, it is likely that natalizumab suppresses inflammatory activity present in the brain of patients with MS and inhibits further recruitment of immune cells into inflamed tissue, thus reducing the formation of MS lesions. Furthermore, there is potential for combining α4-integrin inhibitors with existing therapies for MS.

Based on the encouraging results from earlier clinical trials of natalizumab, two large (>900 patients), 2-year, multicenter phase III trials are ongoing. The first trial will assess relapse rates at 1 year, and disability at 2 years in patients receiving either natalizumab (300 mg/month) or placebo. The second study assesses relapse rate at 1 year, and disability at 2 years in patients who have either natalizumab (300 mg/month) or placebo added to an established regimen of IM interferon beta-1a; this combination was shown to be safe and well tolerated in an open-label pilot study involving patients with RRMS.74 Parallel trials are also being done in patients with an incomplete response to glatiramer acetate and in patients with Crohn’s disease. Dosing for these studies was based on the finding that a monthly fixed dose of 300 mg produces exposures similar to that observed with 3-mg/kg and 6-mg/kg dosing.75 The outcomes are eagerly awaited.

Footnotes

  • G.P.A.R. has received honoraria from Biogen Idec. H.-P.H. has received honoraria for speaking and consulting from Biogen Idec. A.C. has received honoraria in excess of $10,000 for speaking and consulting from Biogen Idec.

    Received May 10, 2004. Accepted in final form January 10, 2005.

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