Brain Advance Access originally published online on August 22, 2003
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Brain, Vol. 126, No. 12, 2638-2647,
December 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg269
Blocking effects of serum reactive antibodies induced by glatiramer acetate treatment in multiple sclerosis
1 Multiple Sclerosis Research Unit, Department of Neurology, Baylor-Methodist Multiple Sclerosis Center, 2 Department of Immunology, Baylor College of Medicine, Houston, Texas, USA, and 3 Department of Neurology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
Correspondence to: Hassan H. Salama MD, Department of Neurology, Mansoura University, Mansoura, EgyptE-mail: hassansalama{at}yahoo.com
Received December 2, 2002. Revised May 28, 2003. Second revision June 20, 2003. Accepted June 23, 2003.
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Glatiramer acetate (GA) is a treatment option for multiple sclerosis. Although its mechanism of action remains unclear, evidence has emerged supporting the role of GA as an immunomodulatory drug that regulates T-cell function. It has been demonstrated that long-term GA treatment induces a serum antibody response; however, the functional properties of these ‘reactive antibodies’ are unknown. It has been speculated that GA-induced antibodies may have a blocking effect that can inhibit the immunologic activity of GA. This study was conducted to determine whether serum antibodies induced by GA treatment can block the in vitro immunoregulatory effects of GA on T-cell proliferation and cytokine production. Forty-two patients with relapsing-remitting multiple sclerosis who were treated with GA for 1–5 years were examined for GA antibody titres using enzyme-linked immunoabsorbent assay (ELISA). Thirty-three percent of patients developed high antibody titres [antibody binding index (ABI) = 16–64] and 14% had low antibody titres (ABI = 4) after 1 year on treatment. Results showed that purified GA antibodies blocked the stimulatory effects of GA on GA-specific T-cell lines of Th0 cytokine profile. The increase in interleukin-10 (IL-10) and IL-4 levels and the decrease in IL-12 and tumour necrosis factor-
levels, normally seen with GA stimulation, were reversed in the presence of GA antibodies. The study has important implications in our understanding of the potential role of high-titre GA antibodies in the treatment of multiple sclerosis. Keywords: glatiramer acetate; Copaxone; cytokines; serum reactive antibodies; multiple sclerosis
Abbreviations: ABI= antibody binding index; CPM = counts per minute; EDSS = Expanded Disability Status Scale; ELISA = enzyme-linked immunoabsorbent assay; GA = glatiramer acetate; HEL = hen egg lysozyme; Ig = immunoglobulin; IL = interleukin; OD = optical density; PBMCs = peripheral blood mononuclear cells; TNF- = tumour necrosis factor-
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Introduction |
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The development of immunomodulatory agents, including glatiramer acetate (GA) and interferon ß, represents a major advance in the treatment of multiple sclerosis. As with interferon ß, it has been shown that GA favourably alters the natural history of the disease by reducing relapse rate and decreasing brain inflammation as measured by MRI (Johnson et al., 1995
; Comi et al., 2001a, b). Although the mechanism of action of GA remains unclear, there is evidence that the clinical effects of the agent are related to its immunomodulatory properties. In particular, GA has been found to increase the ratio of anti-inflammatory (Th2) to pro-inflammatory (Th1) cytokines (Aharoni et al., 1997, 1998, 2000, 2002; Chen et al., 2001, 2002; Farina et al., 2001; Hussien et al., 2001; Neuhaus et al., 2001; Maron et al., 2002). Other mechanisms that may account for the treatment effects of GA include: (i) interaction/competition with major histocompatibility complex class II molecules (specifically DR2 molecules), thereby interfering with presentation of self-myelin antigens to autoreactive T-cells (Racke et al., 1992; Teitelbaum et al., 1992, 1996; Fridkis-Hareli et al., 1994; Ben-Nun et al., 1996; Fridkis-Hareli and Stominger, 1998); (ii) induction of anergy that renders T-cells unresponsive to myelin antigens (Gran et al., 2000); (iii) upregulation of CD8+ regulatory T-cells (Karandikar et al., 2002); (iv) modification of dendritic cell costimulation processes (Hussien et al., 2001) or activity as a weak/partial T-cell receptor agonist to activate naïve T-cells (Wiesemann et al., 2001); and (v) impairment of activated T-cells to interact with microglia that produce cytokines in the CNS (Chabot et al., 2002).
Standard treatment with daily subcutaneous injections of GA is known to induce the development of ‘reactive antibodies’ by the host immune system (Brenner et al., 2001; Farina et al., 2002). It has been speculated that GA antibodies may interfere with the regulatory properties of GA by blocking the active groups on the molecule or by forming complexes that are rapidly cleared by the immune system. The issue is particularly relevant in the clinical management of multiple sclerosis because there is a large body of evidence that neutralizing antibodies develop to varying degrees to interferon ß formulations; the incidence of neutralizing antibodies has been reported in 2–6% of patients receiving intramuscular injections of interferon ß-1a (Herndon et al., 1999; Jacobs et al., 2000; Clanet et al., 2002), 12–25% of patients receiving subcutaneous injections of interferon ß-1a (Antonelli et al., 1998; PRISMS Study Group, 1998; Panitch et al., 2002), and 28–45% of patients receiving interferon ß-1b (IFNB Multiple Sclerosis Study Group, 1993, 1996; European Study Group on Interferon Beta-1b in Secondary Progressive Multiple Sclerosis, 1998). Data from two Phase III trials of interferon ß in multiple sclerosis patients have shown that neutralizing antibodies are associated with diminished efficacy starting after 18–24 months of treatment (IFNB Multiple Sclerosis Study Group, 1996; PRISMS Study Group, 2001).
To date, no study has been reported that addresses the biological effects or therapeutic implications of serum antibodies induced by GA treatment in patients with multiple sclerosis. This paper reports the results of a study conducted to determine whether GA antibodies influence the stimulatory and regulatory effects of GA on T-cells in functional assays. Because both the activation of Th2 cells and the regulation of cytokine production by T-cells are considered to be important regulatory mechanisms of GA, the functional assays were designed to analyse the effects of GA antibodies on these parameters. In addition, although the number of patients in the study is limited and insufficient for meaningful statistical analysis, preliminary attempts were made to evaluate whether the occurrence and titres of GA antibodies are associated with a decrease in the therapeutic effects of GA.
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Methods |
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Study design and patients
Multiple sclerosis patients who had started GA treatment (20 mg subcutaneously once daily) at the Baylor Methodist Multiple Sclerosis Center between 1996 and 2000, and who had been tested for serum reactive antibodies to GA before the treatment (as pre-treatment specimens) and periodically after the treatment (as post-treatment specimens), were identified for inclusion in the study by investigators via retrospective chart review. Serum specimens from all patients were stored at –80°C within 2 h of blood draw at various time points. Serum specimens were included in the study if patients met the following criteria: (i) a diagnosis of clinically definite multiple sclerosis and evidence of gadolinium-enhanced T1-weighted lesions of the brain and the spinal cord on MRI scans; (ii) a relapsing-remitting course of multiple sclerosis at the start of GA treatment; and (iii) complete clinical records documenting concurrent use of medications, Expand Disability Status Scale (EDSS) scores, and annual relapse rate 1 year before GA treatment and during the course of uninterrupted treatment over a period of 1–5 years. Serum specimens were excluded from the study if patients received treatment with an interferon ß product or prolonged immunosuppressive therapy during the study. Patients were treated for acute exacerbations with methylprednisolone (1 g administered intravenously for 3 days with an oral prednisone taper) when episodes occurred.
Table 1 shows the demographic and clinical characteristics of 42 patients who met inclusion/exclusion criteria and were included in the study. Sixty-four percent of patients were female, the mean age was 44 ± 11.4 years, and patients had multiple sclerosis for a mean of 11.3 ± 7.1 years. Patients were treated with GA for a mean of 3.3 ± 1.6 years, ranging from 1 to 5 years.
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Reagents
Culture medium used in the study was RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum and L-glutamine, sodium pyruvate, non-essential amino acids and 10 mM HEPES buffer (Hyclone, Logan, UT, USA). GA (Copaxone) used in this study was obtained from Teva Pharmaceuticals, Inc. (St Louis, MO, USA). Control antigens, hen egg lysozyme (HEL) and phytohemagglutinin, were purchased from Sigma (St Louis, MO, USA).
Antibody reactivity by enzyme-linked immunoabsorbent assay (ELISA)
Stored serum specimens (at –80°C) were examined for antibody reactivity to GA. Briefly, microtitre plates were coated overnight at 4°C with either GA or HEL at a concentration of 1 µg/well. Wells were then blocked at 37°C for 2 h with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (BSA) (Sigma) and were subsequently washed four times with 0.02% Tween 20 in a 0.9% NaCl solution. Each diluted serum sample and its control were added to the adjacent wells and incubated for 2 h. Plates were washed four times and incubated for 30 min with a goat anti-human immunoglobulin (Ig) (IgG) IgG/IgM antibody conjugated with horseradish peroxidase at a dilution of 1:1500 (Sigma). A solution of 0.0125% tetramethylbenzidine/0.008% H2O2 in citrate buffer (pH 5.0) was used as a substrate, and colour development was stopped using 2N H2SO4. Optical densities (ODs) were measured using an ELISA reader (Bio-Rad, Hercules, CA, USA). Antibody titres were expressed in the form of an antibody binding index (ABI) as follows: mean OD of GA-bound wells/mean OD of control wells coated with HEL. In all experiments, the mean OD obtained from HEL-coated control wells was equivalent to the background OD in GA-coated wells (control sera obtained from healthy controls that had no antibody reactivity to GA). A specific response is defined as an ABI of 
4.
Inhibition assay
Peripheral blood mononuclear cells (PBMCs) derived from healthy individuals were cultured at 100 000 cells/well with GA at a predetermined concentration of 100 µg/ml or phytohemagglutinin (1 µg/ml) as a control. Cultures were set up in duplicate in the presence and absence of sera (1:100 dilution) pre-dialyzed against culture media. Cultures were retained for 3 days and were pulsed with [3H]-thymidine (Nycomed Amersham, Arlington Heights, IL, USA) at 1 µCi per well during the final 16 h of culture. Cells were then harvested using an automated cell harvester and [3H]-thymidine incorporation was measured in a ß-plate counter. Percent inhibition is calculated as: {1 – [mean counts per minute (CPM) of PBMC culture in the presence of GA serum/mean CPM of PBMC culture in the absence of GA serum] x 100%}.
Immunoblot analysis
GA was electrophoresed at the indicated amount using 10% SDS–PAGE. After blotting, nitrocellulose membranes were cut into strips and then blocked with 5% low-fat milk powder in Tris-buffered saline containing 0.1% Tween 20 (milk-TBST). The strips were then incubated with pre-treatment and post-treatment serum preparations at a dilution of 1:100 in mini-incubation trays for 1 h at room temperature. A goat anti-human IgG and IgM (H+L chains) coupled to horseradish peroxidase was used as a secondary antibody (100 ng/ml in 2% milk-TBST) and incubated with pre-washed strips for 45 min, followed by enhanced chemiluminescent visualization using a reagent kit purchased from Amersham (Piscataway, NJ, USA).
Ammonium sulphate precipitation of serum immunoglobulin
Sera were brought to 100% saturation with ammonia sulphate and were stirred continuously overnight at 4°C. The resulting solution was centrifuged at 120 000 g for 30 min, and the precipitates were dissolved in 1 ml of 100 mM sodium phosphate buffer (pH 7.3) and dialyzed subsequently against RPMI 1640 medium.
Generation of GA-specific T-cell lines
PBMCs were plated at 200 000 cells/well (for a total of 96 wells) in the presence of GA (100 µg/ml). Seven days later, all cultures were restimulated with GA in the presence of irradiated autologous PBMCs. After another week, each well was split into four aliquots (
104 cells per aliquot) and cultured in duplicate with 105 irradiated autologous PBMCs in the presence and absence of GA. Cultures were retained for 3 days and were pulsed with [3H]-thymidine at 1 µCi per well during the final 16 h of culture. Cells were then harvested using an automated cell harvester, and [3H]-thymidine incorporation was measured. A well/culture was defined as specific for GA when the CPM was >1000 and exceeded the reference CPM (in the absence of GA) by at least three times. Specific T-cell lines were restimulated and expanded by culturing 20 000 cells/well with 100 000 cells/well of irradiated autologous PBMCs in the presence of GA (100 µg/ml).
T-cell proliferation assay
GA-specific T-cell lines were derived from healthy individuals who were not exposed to GA and were cultured at 20 000 cells/well in the presence of irradiated autologous PBMC at 100 000 cells/well and GA at a predetermined concentration of 100 µg/ml. Cultures were set up in triplicate in the presence and absence of different dilutions of purified GA antibody. Cultures were retained for 3 days and [3H]-thymidine incorporation was measured as described above. The results were expressed in CPM.
Cytokine measurements by ELISA
Culture supernatants were collected from the GA T-cell line culture 48 h after initiation of the culture and were measured for concentrations of cytokines using ELISA kits according to the manufacturer’s instructions (PharMingen, San Diego, CA, USA). Microtitre plates were coated overnight at 4°C with mouse monoclonal antibodies (capturing antibody). Wells were then blocked at 37°C for 2 h with PBS containing 2% BSA (Sigma) and were subsequently washed four times with 0.02% Tween 20 in a 0.9% NaCl solution. Samples were added and incubated for 2 h with a biotinylated detecting antibody (0.25 µg/ml of each monoclonal antibody, respectively) in 2% BSA/PBS/Tween 20. The remainder of the procedure for colour development is as described previously. The detection limits for all four cytokines were <15 pg/ml in all assays.
Statistical analysis
The percentage of patients who developed antibodies during treatment was calculated to determine the incidence of antibody-positive patients. Using the ABI, low titres of GA antibodies were defined as 4, whereas high titres were defined as 16–64. Student’s t-tests were used to analyse normally distributed variables. All reported P values are based on two-tailed statistical tests, with a significance level of 0.05.
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Incidence and binding properties of GA antibodies
All pre-treatment serum specimens derived from 42 patients included in this study and an additional panel of 12 serum specimens obtained from untreated patients with multiple sclerosis and healthy individuals tested negative for GA-specific antibodies (ABI = 1). Post-treatment, 48% of patients tested positive for GA-specific antibodies post-treatment, with 33% developing high antibody titres (ABI = 16–64) and 14% developing low titres (ABI = 4); 52% of patients tested negative for antibodies post-treatment (ABI = 1) (Table 2). A trend was noted that patients with higher ABIs (16–64) showed more deterioration in EDSS scores and more relapses from pre-treatment to post-treatment compared with patients with an ABI
4 and the same length of treatment (Table 2). However, in this retrospective study, the number of patients was not sufficient to detect any statistically significant effects.
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Fourteen serum specimens of high GA antibody titres were pre-screened for an inhibitory effect on the proliferation of PBMCs in response to GA. Six specimens exhibited a significant blocking effect (>60%), at a serum dilution of 1:100, on the ability of GA to induce the T-cell proliferation in PBMCs, while others had either no effect or an effect that was not specific for GA.
Six serum specimens that had an inhibitory effect on GA-induced T-cell proliferation were selected for detailed characterization. These paired specimens consisted of pre-treatment sera without detectable GA antibodies and post-treatment sera with high titres of GA antibodies (ABI = 64) from the same patients. They are designated as MS-1 through MS-6. As shown in Fig. 1, the selected post-treatment serum antibodies reacted specifically with GA but not with the control antigens, interferon ß or HEL. In contrast, paired serum specimens derived from the same patients before treatment had no detectable reactivity to GA. The specific reactivity of serum antibodies to GA was confirmed by immunoblot analysis (Fig. 2).
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Effects of GA antibodies on GA-specific T-cell lines
We then addressed whether serum GA antibodies would interfere with the effect of GA on GA-specific T-cell lines and on their cytokine production. We therefore generated a small panel of well-characterized T-cell lines that reacted specifically to GA and analysed the effect of GA antibodies on the selected T-cell lines. These T-cell lines were generated from a healthy individual and had the following characteristics: (i) specifically recognized GA but not HEL (used as a control antigen); (ii) exhibited high stimulation indices (i.e. specific proliferation to GA/proliferation to a control antigen) of >20; and (iii) had a cytokine profile consistent with the Th0 phenotype. In addition, to avoid potential interference caused by unknown serum factors in the cell functional assays, immunoglobulin fractions were purified by ammonia sulphate precipitation and dialyzed thoroughly against culture media to remove other serum factors. The precipitated antibody fractions were tested to confirm the specific reactivity to GA by ELISA prior to use in the functional assays. As shown in Fig. 3, antibodies purified from post-treatment sera significantly inhibited the proliferation of three representative T-cell lines specific for GA at antibody dilutions of 1:5 to 1:10 (roughly equivalent to 1:50 to 1:100 serum dilutions). In contrast, paired antibodies purified from pre-treatment sera of the same patients did not demonstrate a similar effect.
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Furthermore, GA antibodies also were found to revert the in vitro effect of GA-induced cytokine production of GA-specific T-cell lines. As shown in Fig. 4, GA stimulation of the T-cell lines resulted in up-regulation of interleukin-10 (IL-10) and IL-4, and down-regulation of tumour necrosis factor-
(TNF-) and IL-12, which are the two prominent pro-inflammatory cytokines associated with multiple sclerosis. These regulatory effects of GA were substantially reverted by the addition of GA antibodies.
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Correlation of high titres of GA antibody with serum cytokine profile
To further delineate the in vivo effect of GA antibodies on serum cytokine production, we examined the change in serum cytokine profile before and after GA treatment in patients with high GA antibody titres as compared with that in patients with low titres. To this end, serum cytokine profiles of two groups were compared: 12 multiple sclerosis patients who were on GA treatment for 3–5 years and exhibited low titres of GA antibodies (ABI = 4) and 13 multiple sclerosis patients who were treated for the same length of time and who had high GA antibody titres (ABI = 16–64). As shown in Fig. 5, a significant increase in the serum concentrations of IL-10 and a reduction in TNF-
and IL-12 were observed in post-treatment specimens in the low GA antibody titre group. This effect also has been reported by other investigators (Li et al., 1998; Hussien et al., 2001). In contrast, a reversed cytokine profile was seen in the high antibody titre group as evident by high concentrations of TNF- and IL-12 and a low concentration of IL-10 in post-treatment specimens versus pre-treatment specimens (Fig. 5). These results suggest that the in vivo effect of GA is reverted by GA antibodies in patients who developed high titres of GA antibodies.
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Discussion |
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GA has been available in the USA as a therapeutic option for the treatment of relapsing-remitting multiple sclerosis since 1995. Administration of GA may induce the development of substantial ‘reactive antibody’ responses in multiple sclerosis patients within 1 year of treatment initiation (Brenner et al., 2001
; Farina et al., 2002). However, there is no clear evidence as to whether the development of GA-reactive antibodies during prolonged treatment is associated with reduced therapeutic efficacy. The findings described in the present study provide, for the first time, important experimental evidence for considering the potential blocking effect of serum antibodies induced by treatment with GA. GA-specific antibodies were shown to reverse GA-stimulated proliferation of GA-specific T-cells, up-regulation of the anti-inflammatory cytokines IL-10 and IL-4, and down-regulation of the pro-inflammatory cytokines TNF- and IL-12 in T-cells.
The results of this study raise a number of important issues. First, what is the potential mechanism of action that underlies the blocking/neutralizing effect of GA antibodies on the regulatory properties of the drug? Although the present study involved only a T-cell functional analysis and was not designed to investigate molecular interactions or to address mechanistic issues, several possibilities exist that may explain the findings. For example, GA antibodies may bind to and block certain amino acid groups on the polymer, which act as functional epitopes capable of activating GA-specific T-cells (e.g. Th2 or Th0 subsets). This possible action may explain the loss of the stimulatory effect of GA on T-cells and the diminution of its intrinsic regulatory properties on T-cell-mediated cytokine production when GA antibodies were added into the system. Alternatively, antibody reactivity to GA may interfere with the binding of GA to the molecules of the major histocompatibility complex, a proposed mechanism by which GA may competitively impair the presentation of self-myelin antigens/peptides to T-cells (Racke et al., 1992; Teitelbaum et al., 1992; Fridkis-Hareli et al., 1994; Fridkis-Hareli and Strominger, 1998). It is also possible that GA antibodies may facilitate rapid internalization and intracellular degradation of GA through Fc and Fc receptors commonly expressed on B-cells, macrophages and other blood cells, resulting in substantially reduced bioavailability of GA in the system. Further investigations are needed to delineate the mechanism whereby GA antibodies block the regulatory effects of GA on T-cells.
The findings also raise an important clinical issue as to whether the occurrence and high titres of GA antibodies may impair the treatment effect of GA in multiple sclerosis patients. A trend demonstrating a potential correlation between high GA antibody titres and clinical deterioration in EDSS scores and relapse rate was noted in the present study. However, this retrospective study was not originally designed to address the clinical relevance of elevated titres of GA antibodies and, hence, the number of patients examined was not sufficient to detect a statistically significant effect. It should be cautioned that development of antibody responses to GA is dynamic in multiple sclerosis patients during treatment, and that GA antibodies have been reported to have intrinsic properties that potentially promote myelin repair in a murine model of demyelinating disease (Ure and Rodriguez, 2002). Therefore, the issue regarding clinical consequences of GA antibodies induced by GA treatment is complex, and the outcome may depend mainly on interplay between the blocking effects on immune regulation induced by GA and the direct beneficial effects of the antibodies. In conclusion, the clinical relevance of GA antibodies must be evaluated in clinical trials.
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Acknowledgements |
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We wish to thank Mrs De La Rosa for patient and sample coordination. This work was supported by a grant from the Richardson Foundation and a fellowship grant from the Egyptian government to H.H.S. The study was not related to any pharmaceutical companies.
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References |
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