Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics
`
`Mechanism of Action of Glatiramer Acetate in Treatment of
`Multiple Sclerosis
`
`Martin S. Weber,* Reinhard Hohlfeld,†‡ and Scott S. Zamvil*
`
`*Department of Neurology and Program in Immunology, University of California, San Francisco, CA 94143; †Institute for
`Clinical Neuroimmunology, Klinikum Grosshadern, Ludwig Maximilians University, Munich, 81377, Germany; and ‡Department
`of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried, 82152, Germany
`
`Summary: Glatiramer acetate (GA) (Copolymer-1, Copaxone,
`Teva, Israel, YEAK) is a polypeptide-based therapy approved for
`the treatment of relapsing-remitting multiple sclerosis. Most in-
`vestigations have attributed the immunomodulatory effect of GAs
`to its capability to alter T-cell differentiation. Specifically, GA
`treatment is believed to promote development of Th2-polarized
`GA-reactive CD4⫹ T-cells, which may dampen neighboring in-
`flammation within the central nervous system. Recent reports in-
`dicate that the deficiency in CD4⫹CD25⫹FoxP3⫹ regulatory T-
`
`cells in multiple sclerosis is restored by GA treatment. GA also
`exerts immunomodulatory activity on antigen presenting cells,
`which participate in innate immune responses. These new findings
`represent a plausible explanation for GA-mediated T-cell immune
`modulation and may provide useful insight for the development of
`new and more effective treatment options for multiple sclerosis.
`Key Words: Multiple sclerosis, glatiramer acetate, immuno-
`modulatory agents, mechanism of action, antigen presenting
`cells.
`
`INTRODUCTION
`
`Glatiramer acetate (GA) (Copolymer-1, Copaxone,
`Teva, Israel, YEAK) is a pool of synthetic peptides ran-
`domly composed of L-tyrosine (Y), L-glutamic acid (E),
`L-alanine (A), and L-lysine (K) with an average length of
`40 to 100 residues. GA was synthesized in this manner
`more than 30 years ago to most closely resemble the
`encephalitogenic properties of myelin basic protein
`(MBP), one suspected auto-antigen in multiple sclerosis
`(MS). Surprisingly, instead of inducing experimental au-
`toimmune encephalomyelitis (EAE), the murine model
`of MS, immunizations with GA protected mice from
`subsequent attempts to induce EAE.1 This seminal ob-
`servation was
`followed by various clinical
`trials.
`Whereas early open-label studies already suggested clin-
`ical benefit in the 1980s,2,3 these findings had to be
`interpreted with caution as drug production was not yet
`standardized. In 1991, a phase III multicenter, double-
`blind, placebo-controlled trial with standardized GA
`preparation was initiated in 11 medical centers in the
`
`Address correspondence and reprint requests to: Scott S. Zamvil,
`M.D., Ph.D., Department of Neurology, Program in Immunology, Uni-
`versity of California, San Francisco, 513 Parnassus Avenue, S-268, San
`Francisco, CA 94143. E-mail: zamvil@ucsf.neuroimmnunol.org.
`
`United States, with 251 relapsing–remitting MS pa-
`tients.4 Within two years of treatment, the relapse rate
`decreased approximately 30% in GA-treated patients
`leading to approval of GA treatment of MS in many
`countries worldwide in 1995. A later double-blind, pla-
`cebo-controlled study demonstrated a reduction in the
`number of gadolinium-enhancing lesions in patients re-
`ceiving GA compared to a placebo during a nine-month
`study period.5 Additional data showed that GA may also
`have a favorable effect in preventing tissue loss at a later
`diseased stage.6,7 Based on these favorable clinical and
`imaging data, subcutaneously administered GA is one of
`the most widely prescribed drugs used today for the
`treatment of relapsing–remitting MS.
`Many investigators have attempted to address the im-
`munologic basis for the clinical effects of GA in MS and
`MS models.8,9 Although different potential mechanisms
`have been considered, most investigations have attrib-
`uted the immunomodulatory activity of GA to alterations
`in T-cell antigen reactivity, focusing on its influence on
`the adaptive immune response. Early in vitro studies
`established that GA can bind to major histocompatibility
`complex (MHC) class II molecules and suggested that
`GA might preferentially alter presentation of myelin an-
`tigens to auto-reactive T-cells.10,11 Studies in EAE and
`MS have extensively demonstrated that GA treatment
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`promotes development of Th2-polarized GA-reactive
`CD4⫹ T cells.1,12–14 These Th2 cells can potentially
`accumulate in the CNS15 where they might release anti-
`inflammatory cytokines12,15–17 and neurotrophic fac-
`tors,18 dampening the activity of nearby auto-aggressive
`T cells, a process known as “bystander suppression.”15
`Recent reports indicate that GA treatment also exerts
`immunomodulatory activity on antigen presenting cells
`(APCs),19 –23 a part of the innate immune system. These
`newer findings may provide a plausible explanation for
`the observed Th2 deviation under GA treatment and raise
`the question as to whether GA is solely an antigen-
`specific T cell-directed immunotherapeutic agent as cur-
`rently assumed. In this article, we review the various
`effects of GA on the adaptive and innate immune system
`and describe how these two arms of the immune system
`interact with one another during GA therapy.
`
`Effects on the adaptive immune system
`As with conventional peptide antigens, GA can bind to
`MHC class II molecules on the surface of APCs.10,24 In
`association with MHC class II molecules, GA is recog-
`nized by T cells via their antigen-specific T-cell receptor.
`Early in vitro studies indicated that GA may compete
`with myelin antigens for the binding to MHC class II.
`Specifically, it was observed that GA binding to MHC
`class II could inhibit the activation of T cell lines specific
`for MBP.25 However, a later study demonstrated that the
`stereoisomer of GA, D-GA, which contains solely D-
`amino acids, could effectively bind to MHC class II,26
`but failed to suppress EAE.27 These findings indicate that
`GA may not primarily act as an MHC class II antagonist.
`It is well established that in most MS patients, GA
`treatment induces a population of CD4⫹ GA-reactive
`Th2 cells,12–14,28 which is associated with clinical bene-
`fit.29 It seems very unlikely that sufficient amounts of
`GA can reach the CNS to locally activate GA-reactive T
`cells. It is believed that GA-reactive Th2 cells are gen-
`erated in the periphery, accumulate (along with patho-
`genic non-GA-specific elements) in the CNS of patients
`with MS and release anti-inflammatory cytokines in a
`process termed “bystander suppression” (see FIG. 1).
`Many studies in EAE and MS have generated the con-
`cept
`that GA-reactive Th2 cells may be reactivated
`within the CNS through cross-recognition of myelin an-
`tigen.12,16 This assumption was supported by two obser-
`vations. First, GA-reactive Th2 cells could be identified
`in the CNS of GA-treated mice protected from EAE.15
`Second, in some but not all studies,30 several GA-spe-
`cific Th2 cell lines generated from MS patients or mice
`could cross react with MBP at the level of cytokine
`secretion.12,15–17,31
`CD4⫹CD25⫹ regulatory T cells (Treg) are an important
`subclass of regulatory cells that engage in the maintenance
`of immunologic tolerance by actively suppressing self-
`
`Neurotherapeutics, Vol. 4, No. 4, 2007
`
`reactive lymphocytes.32,33 Forkhead transcription factor
`Foxp3 is the key transcription factor in the physiological
`development of Treg.34 Its genetic defect results in im-
`paired function of Treg, which is associated with in an
`autoimmune and inflammatory syndrome in humans as
`well as in mice.35 Similarly, the experimental deletion of
`Treg in mice causes various spontaneous organ-specific
`autoimmune diseases.36 Viglietta et al.37 reported that in
`patients with MS, similar to other autoimmune condi-
`tions,38 effector function and frequency of Treg is sig-
`nificantly decreased in the peripheral blood. Several
`studies provided evidence for a role and mechanism of
`action of GA in the induction of CD4⫹CD25⫹ Treg. In
`vitro exposure to GA resulted in an elevated production
`of interleukin-10 (IL)-10 by Treg.39 In another study, GA
`promoted the conversion of CD4⫹CD25- to CD4⫹CD25⫹
`Treg through the activation of Foxp3.40 GA treatment led to
`a significant increase in Foxp3 expression in CD4⫹ T cells
`in MS patients whose Foxp3 expression was reduced at
`baseline. GA-reactive CD4⫹CD25⫹ T-cell lines generated
`from GA-treated MS patients expressed high levels of
`Foxp3 that correlated with increased T-cell regulation.40
`Thus, besides the well-known preferential Th2 differentia-
`tion of T cells, GA appears to normalize frequency and
`function of Treg in MS, which represents an additional
`immunomodulatory effect of GA.
`More recently, it was reported that GA treatment also
`induces a population of CD8⫹ GA-reactive T cells. In
`untreated MS patients, GA-reactive CD8⫹ T-cell re-
`sponses were found to be significantly lower compared
`with healthy individuals. Treatment with GA restored
`these CD8⫹ responses41 and enhanced release of IFN-␥
`by these cells,42 which appears to be associated with a
`positive clinical response.42 Although the in vivo func-
`tion of these cells is still not entirely understood, a recent
`report indicated that GA-reactive CD8⫹ T cells may
`suppress pro-inflammatory effector T-cell function in a
`manner similar to CD4⫹CD25⫹ Treg.43,44
`Besides activation and alteration of T cells, GA treat-
`ment also induces a humoral response to itself in most
`patients, which peaks approximately 3 months after treat-
`ment initiation.45 Just as individuals who are naive to GA
`treatment sometimes have pre-existing (naive) GA reac-
`tive T cells,28 some untreated MS patients reveal an
`unprimed humoral response against GA, mainly of an
`IgM, IgG1, and IgG2 isotype.46 GA-treated MS patients
`also produce IgG1 and IgG2 anti-GA antibodies, but in
`contrast to unexposed individuals, GA-treated MS pa-
`tients frequently develop high titers of IgG4 antibodies
`against GA.46 Preferential secretion of IgG4 antibodies
`might occur secondary to the induction of GA-reactive
`Th2 cells, as isotype switching to IgG4 is regulated by
`the Th2 cytokine IL-4. To date, it is considered contro-
`versial whether antibodies against GA are of clinical
`relevance. In general, IgG4 antibodies have strong neu-
`
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`GLATIRAMER ACETATE TO TREAT MS
`
`649
`
`Periphery
`
`CNS
`
`?
`
`CD4+CD25+
`FoxP3+ Treg
`
`Y
`GA/Ag
`
`MHC
`
`Type II
`APC
`
`GA/Ag
`
`Y
`
`CD4+
`Th-2
`
`CD4+
`Th-2
`
`YAg
`
`Type II
`APC
`
`local
`or
`recruited
`
`Neurotrophic
`factors
`
`Anti-inflammatory
`cytokines
`
`Type II
`microglia
`
`GA
`
`CD8+
`T cell
`
`?
`
`?
`
`Plasma cell
`
`Y Y
`Y
`GA-reactive IgG4
`Y
`Y
`FIG. 1. Cross-talk between type II antigen presenting cells (APCs) and regulatory T-cell populations in glatiramer acetate (GA)-mediated
`immune modulation. GA treatment exerts effects on APC and T cells that result in the induction of a specific population of Th2 cells and
`CD4⫹CD25⫹FoxP3⫹ regulatory T cells (Treg) in the periphery. Type II APC and Th2 cells may facilitate the development of each in a
`positive feedback mechanism, as type-2 monocytes tend to induce Th2 cells, and Th2 cell-derived anti-inflammatory cytokines may
`promote development of type II APC. GA-reactive Th2 cells are believed to cross the blood-brain barrier and to be locally reactivated
`within the CNS through cross recognition of myelin antigen. In response, these cells may secrete anti-inflammatory cytokines and
`neurothrophic factors dampening neighboring inflammation (“bystander suppression”). Another feedback loop between APC and T cells
`may develop within the CNS, as Th2-cytokines might promote type II differentiation of resident APC, such as microglia. GA treatment
`is also associated with induction of GA-reactive CD8⫹ T cells, although their in vivo function remains to be determined. Finally,
`consistent with the Th2 shift, GA-reactive plasma cells secrete anti-GA antibodies ( Ɱ), preferentially of an IgG4 isotype. Whether these
`antibodies enter the CNS or may neutralize some of the immunomodulatory effects of GA is not yet known. (GA-Ag ⫽ glatiramer acetate
`antigen; ? ⫽ Presumed transmigration of immune cells across the blood-brain barrier.)
`
`tralizing activity, although they do not bind to fragment
`crystallizable (Fc) receptors or activate the complement
`system. However, serum from GA-treated patients con-
`taining antibodies against GA did not inhibit the ability
`of GA to stimulate GA-reactive T cells, indicating that in
`vitro anti-GA Ig had no neutralizing effect.45 Interest-
`ingly, Brenner et al.47 reported that relapse-free patients
`displayed higher titers against GA than patients with an
`active disease course under GA treatment, indicating a
`beneficial rather than effect-neutralizing role of antibod-
`ies against GA. In fact, in an animal model of CNS
`demyelinating disease, GA-specific antibodies were
`shown to promote myelin repair,48 an effect which might
`contribute to the proposed neuroprotective properties of
`GA in MS.
`
`Effects on the innate immune system
`Although past investigations of GA primarily focused
`on its effects on the adaptive immune system, especially
`on T cells, emerging evidence supports the concept that
`GA may also act on APCs. The interaction between
`APCs and T cells is fundamental for any adaptive T-cell
`immune response. Several groups have reported that in
`vitro GA treatment leads to a broad antigen-nonspecific
`alteration of APC function.19-21,49 –52 Possibly the first
`report regarding the effect of GA on the innate immune
`system was derived from an in vitro study in which GA
`altered the activation of a human monocytic cell line.49
`Specifically, GA inhibited the induction of HLA proteins
`as well as the release of tumor necrosis factor (TNF) and
`cathepsin B by THP-1 cells. In vitro GA treatment was
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`also shown to alter the activation status of freshly iso-
`lated human monocytes.19,20 Weber et al.19 reported that
`GA inhibited lipopolysaccharide (LPS)-mediated expres-
`sion of APC activation markers,
`including CD150/
`SLAM, CD25, and CD69. Furthermore, GA-treated
`monocytes released significantly lower levels of TNF-
`and IL-12, two inflammatory Th1-polarizing cytokines.
`Another study demonstrated that GA treatment not only
`reduced the release of inflammatory cytokines, but also
`enhanced production of Th2 polarizing IL-10 by mono-
`cytes.20 A similar cytokine shift was observed in micro-
`glial cells, an APC population that is believed to have a
`key role in the reactivation of T cells within the CNS. In
`vitro-generated human dendritic cells also released less
`TNF21 and IL-1250 on in vitro GA exposure. Most no-
`tably, GA-treatment of dendritic cells promoted Th2 dif-
`ferentiation of naive T cells without affecting APC ca-
`pability for inducing T-cell proliferation.21
`Two independent studies investigated how GA af-
`fects monocytes in MS patients.
`In both studies,
`monocytes were freshly isolated from GA-treated pa-
`tients without any additional in vitro exposure to GA.
`Compared to untreated MS patients and healthy sub-
`jects, monocytes from GA-treated MS patients ex-
`pressed significantly lower levels of the activation
`marker CD150/SLAM and released less TNF on stim-
`ulation with low concentration of LPS.19 In the second
`study, Kim and colleagues20 reported that the basal
`and induced release of IL-10 was significantly en-
`hanced in monocytes
`from GA-treated patients,
`whereas the production of IL-12 was reduced, defining
`an anti-inflammatory “type II” monocyte phenotype.
`These studies clearly indicate a systemic effect of GA
`treatment on monocytes that may promote Th2 differ-
`entiation of T cells in vivo. Theoretically, these find-
`ings raise the possibility that GA treatment may com-
`promise innate immune responses in GA-treated MS
`patients. However, GA-treatment does not appear to
`be predisposed to infections. In this regard, one in
`vitro finding might be of relevance (i.e., GA only
`inhibited activation of monocytes that were challenged
`with suboptimal concentrations of toll-like receptor
`ligands, such as LPS).19 Higher concentrations of LPS
`could override the inhibitory effect of GA, which
`could explain why the capability of monocytes to ef-
`ficiently clear infections is not diminished in GA-
`treated MS patients. Future longitudinal studies are
`necessary to define whether initiation of GA treatment
`truly leads to a reduction of APC reactivity in the
`individual MS patient. This type of study will also
`allow correlation between altered APC reactivity and a
`drug-related benefit
`to determine the extent of the
`clinical relevance of these GA-mediated effects on the
`APCs.
`
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`
`Cross-talk between type II APC and regulatory T-
`cell populations
`It has become established that the phenotype of APC
`influences differentiation of T cells and that reciprocally
`differentiated T cells modify APC function. In this re-
`gard, monocytes cultured with Th2 supernatants devel-
`oped a phenotype similar to GA-treated monocytes. This
`finding indicates that GA-reactive Th2 cells can exert a
`positive feedback on the development of type II mono-
`cytes. In fact, type II monocyte development may even
`occur secondary to the induction of GA-reactive Th2
`cells. However, other evidence suggests the opposite
`scenario (i.e., that APCs may be the primary target of GA
`and that GA-induced type II APCs mediate T-cell devi-
`ation). First, in vitro, GA exerted a direct effect on var-
`ious APC populations resembling its effect in vivo, in the
`absence of T cells.19,20 These GA-treated APCs were
`capable of promoting development of Th2 cells when
`co-cultured with naive (untreated) Th0 cells in the ab-
`sence of GA.21 Second, in vivo GA treatment exerted a
`systemic effect on monocytes and possibly on monocyte-
`derived APCs. However, the frequency of GA reactive
`Th2 cells in the peripheral blood of GA-treated MS
`patients is only approximately 1 in 20,000, raising the
`question as to whether Th2 cytokines derived from these
`cells could be sufficient to mediate type II APC devel-
`opment. Most strikingly, studies in genetically altered
`mice indicate that in vivo GA treatment can induce type
`II monocytes in the absence of T cells.53 Further studies
`are necessary to determine the pathway by which GA
`treatment may alter APC and T-cell function in MS
`patients.
`Assuming that APCs are the primary target through
`which GA mediates T-cell immune deviation, one would
`anticipate that Th2 deviation and/or induction of Treg
`should not be restricted to GA-reactive T cells. A recent
`study by Allie et al.54 investigated the phenotype of
`T-cell lines specific for GA, MBP, or tetanus toxoid
`generated from MS patients before and after GA treat-
`ment. T-cell differentiation was assessed by the ratio
`between IFN-␥ and IL-5 release. In this longitudinal
`study, in vivo GA treatment biased differentiation of all
`T cell lines toward a Th2 phenotype, indicating that Th2
`differentiation occurred independent of T-cell antigen
`specificity.54 However, another study did not describe an
`antigen-independent Th2 deviation of established T-cell
`responses on GA treatment, and supported the concept
`that Th2 deviation may primarily occur in GA-reactive T
`cells.42 Although apparently conflicting, both findings
`might be valid. First, a cross-sectional study comparing
`untreated patients to GA-treated patients may be less
`sensitive to detect minor changes in T-cell differentiation
`compared to a longitudinal study investigating the same
`patients before and after treatment. Second, it is plausible
`that an APC-driven Th2 deviation may be pronounced in
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`GLATIRAMER ACETATE TO TREAT MS
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`GA-reactive T cells, as every APC that presents GA
`should have been in contact with GA and undergone type
`II differentiation prior to T-cell activation. The concept
`of an antigen-nonspecific effect of GA is further sup-
`ported by the fact that GA treatment has been shown to
`be clinically beneficial in other models of autoimmune or
`inflammatory conditions, such as arthritis, uveoretini-
`tis,55 inflammatory bowel disease,56 and graft rejec-
`tion.57
`Although T cells might not be the primary target of
`GA, they are most likely the effector cells of GA-medi-
`ated immune modulation. Deficiencies in regulatory T
`cells have been associated with MS pathogenesis38 and
`GA-mediated restoration of T-cell regulation correlates
`with clinical benefit.40 In EAE, adoptive transfer of GA-
`reactive T cells alone can inhibit EAE induction by var-
`ious encephalitogens,16,31,58 similar to GA treatment it-
`self, and GA-reactive T cells accumulate in the CNS of
`protected animals. Thus, whereas GA may mediate a
`primary effect on APC independent of T cells, the type II
`APC-induced regulatory T cells may be the effector cells
`of GA-mediated immune modulation.
`
`Possible neurotrophic effects
`Some experimental data indicate that GA may have
`direct neuroprotective properties. In vitro, GA-reactive T
`cells can produce neurotrophic factors such as brain-
`derived neurotrophic factor (BDNF).17,18 BDNF is an
`important factor for differentiation and survival of neu-
`rons and is required for maintenance of various glial cell
`functions.59 Although the ability to produce BDNF is
`unlikely to be restricted to GA-reactive T cells, it may
`relate to the activation status of immune cells (e.g., T
`cells).60 In this regard, the continuous activation by daily
`GA application may promote BDNF production of GA-
`reactive T cells in vivo. As activation of immune cells
`also facilitates their transmigration across the blood-
`brain barrier,61 accumulation of BDNF-producing GA-
`reactive T cells within the CNS of patients with MS may
`occur, in proportion to the population frequency of these
`cells. This concept is supported by findings derived from
`EAE studies. Adoptively transferred GA-reactive T cells
`are detected within the CNS of mice with EAE15 and
`produce BDNF in situ.62 These putative neurotrophic
`effects of GA may not be restricted to CNS autoimmune
`disease. In an optic-nerve injury model, GA-specific T
`cells prevented the secondary degeneration of axons and
`similarly accumulated at the site of injury producing
`neurotrophic factors.63 In an animal model of glaucoma,
`GA reduced loss of retinal ganglion cells without affect-
`ing intraocular pressure.64 GA administration protected
`motor neurons from acute and chronic degeneration65
`and adoptive transfer of GA-reactive T cells enhanced
`survival of dopaminergic neurons in a mouse model of
`Parkinson’s disease.66,67 Thus, GA may exert neurotro-
`
`phic and/or protective properties in addition to immuno-
`modulatory effects. Their relevance in human neurode-
`generative diseases,
`including MS,
`remains
`to be
`determined.
`
`CONCLUSIONS
`
`Although GA is one of the most widely prescribed
`drugs for treatment of relapsing–remitting MS, its mech-
`anism of action is still not entirely understood. GA treat-
`ment induces a preferential Th2 deviation of T cells and
`promotes restoration of frequency and function of Treg
`in MS. Recent reports demonstrated that GA also exerts
`immunomodulatory effects on APCs, such as monocytes.
`These new findings may provide a plausible explanation
`for GA-mediated T-cell immune modulation. Whereas it
`remains to be determined whether APCs, T cells, or both
`are the primary pharmacological target for GA, immune
`modulation of APC and T cells appears to engage a
`positive feedback mechanism. These novel observations
`should contribute to a better understanding of the mech-
`anism of action of GA and may provide useful insight for
`the development of new and more efficient agents.
`
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