Antibody-mediated suppression of V5.25.3+ T cells in multiple sclerosis Results from an MRI-monitored phase II clinical trialкод для вставкиСкачать
Antibody-Mediated Suppression of V␤5.2/ 5.3⫹ T Cells in Multiple Sclerosis: Results from an MRI-Monitored Phase II Clinical Trial Joep Killestein, MD,1 Tomas Olsson, MD, PhD,4 Erik Wallström, MD, PhD,4 Anders Svenningsson, MD, PhD,4 Mohsen Khademi, MD, PhD,4 Lance D. Blumhardt MD, FRCP,9 Jan Fagius, MD, PhD,5 Jan Hillert, MD, PhD,6 Anne-Marie Landtblom, MD, PhD,7 Charlotte Edenius, MD, PhD,8 Leopold Årfors, MD, PhD,8 Frederik Barkhof, MD, PhD,2,3 and Chris H. Polman, MD, PhD1 The objective of this study was to evaluate the safety and efficacy of the humanized antibody ATM-027 in a baseline versus treatment magnetic resonance imaging-monitored study. Expansion of V␤5.2/5.3ⴙ T cells has been demonstrated in the peripheral blood, cerebrospinal fluid, and brain lesions of MS patients. In a phase I study, ATM-027 depleted these cells in peripheral blood and, in parallel, T-cell MBP reactivity and IFN-␥ expression were reduced. We studied 59 patients with relapsing-remitting MS (47 on ATM-027 and 12 on placebo) stratified for HLA-DR2 status. Monthly intravenous injections were given for 6 months. Individual dose titration was employed to obtain depletion of the target T-cell level and downregulation of antigen receptor density as monitored by flow cytometry. Five monthly magnetic resonance imaging scans were performed before treatment to establish baseline activity, six during treatment, and three during follow-up. Additional immunological assessments were performed to elucidate the mechanism of action of ATM027. The treatment was safe and well tolerated, inducing consistent suppression of the target cell population. During run-in, active lesions were found in 78.7% (37/47) of patients treated with ATM-027. During treatment, the median number of lesions was reduced by 33% (p ⴝ 0.13) independent of DR2 status. The corresponding volume of enhancement was 221 mm3 at baseline, with a reduction of 10% during treatment. Decreased numbers of cells expressing interferon-␥ messenger RNA, and decreased T-cell reactivity to several myelin antigens were found in ATM-027 treated patients. In conclusion, consistent suppression of V␤ 5.2/5.3ⴙ T cells was achieved. However, the effect size on magnetic resonance imaging was considerably less than the targeted 60%. Ann Neurol 2002;51:467– 474 Multiple sclerosis (MS) is widely considered as an autoimmune disease characterized by an inflammatory attack on the myelin sheath, leading to demyelination and axonal damage. Although its cause remains unknown, it is currently believed that T cells reactive to myelin components provide the organ specificity of the pathogenic process.1 In particular, T cells recognizing the immunodominant peptides of myelin basic protein (MBP) are frequently detected in MS patients. Although MBP-reactive T cells are also found in healthy individuals, there is evidence that these T cells undergo in vivo activation and clonal expansion in peripheral blood, as well as in cerebrospinal fluid (CSF) in patients with MS, as opposed to healthy individuals.2–5 Expansion of V␤5.2/5.3⫹ T cells has been demonstrated in peripheral blood,6,7 cerebrospinal fluid (CSF),8,9 and brain lesions10 of MS patients. Although controversial, these findings have been most consistently found in patients with the human leukocyte antigen (HLA) haplotype DR2.7 Furthermore, it has been shown that these T cells can be activated by MBP, whereas depletion of V␤5.2/5.3⫹ T cells in vitro has been accompanied by a decrease in MBP reactivity.8 From the 1Department of Neurology, 2MS-MRI Center, and 3Image Analysis Center (IAC), VU Medical Center, Amsterdam, The Netherlands; 4Karolinska Hospital, Stockholm; 5Akademiska Hospital, Uppsala; 6Huddinge University Hospital, Huddinge; 7University Hospital, Linköping; 8AstraZeneca AB, AstraZeneca R & D Södertälje; and 9Queens Medical Center, Nottingham, UK. Published online Feb 19, 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.10146 Address correspondence to Dr Polman, VU Medical Center, Department of Neurology, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands. E-mail: firstname.lastname@example.org Received Aug 1, 2001, and in revised form Dec 7. Accepted for publication Dec 7, 2001. © 2002 Wiley-Liss, Inc. 467 On the basis of these observations, a humanized antibody (mAb), ATM-027, specifically targeting the V␤5.2/5.3⫹ T cells was developed. In the first study with ATM-027 in humans, on average 70% of the target cell population was depleted, whereas T-cell receptor (TCR) density in the remaining cells was downregulated.11 ATM-027 treatment decreased the number of interferon-␥ (IFN-␥), but not interleukin-10 (IL-10) mRNA-expressing peripheral blood mononuclear cells.12 Furthermore, target cell depletion was accompanied by a decrease in reactivity to several MBP peptides.12 In the present study, we present the results of a subsequent phase II clinical trial in which the safety, tolerability, and efficacy of multiple dose administration of ATM-027 for 6 months were evaluated in relapsing-remitting (RR) MS patients. Efficacy was measured by the number and volume of active brain lesions on magnetic resonance imaging (MRI) in a baseline versus treatment design. An important study objective was to evaluate whether depletion and TCR downregulation of the target T cells would result in a decrease of MRI lesion activity of at least 60%. Immunological assessments were performed to elucidate further the mechanism of action of ATM-027 in RRMS. Patients and Methods Study Design and Patient Characteristics This multi-center study involved six centers, in the Netherlands, United Kingdom, and Sweden. The study started with a 4-month run-in period, followed by a 6-month treatment period with monthly clinical and MRI examinations and immunological assessments. A 7-month follow-up period ended the study and included three additional MRI scans. Fiftynine RRMS patients were enrolled and stratified according to DR2 status. The sample size of at least 20 patients in each stratum was based on the assumption that a treatment effect of 60% should be detectable at a 5% significance level with a power of approximately 80%. Efficacy was measured as the percentage of change in number and volume of active MRI lesions from baseline. To allow for unbiased assessment of safety and tolerability, 6 additional patients in each stratum were randomly selected to receive placebo. According to the inclusion and exclusion criteria, patients had clinically definite MS;13 had at least two clinical relapses or one clinical relapse and one separate subclinical relapse as documented with at least one enhancing lesion on MRI during the 36 months before entry in the study and an Expanded Disability Status Scale (EDSS) score of ⱕ5.14 MRI changes highly suggestive of MS were required (either nine or more brain lesions on T2-weighted images or four or more T2 brain lesions, of which at least one showed gadolinium enhancement before inclusion). A level of at least 1 % V␤5.2/5.3⫹ T cells in peripheral blood (percentage of total CD3⫹ T cells) was also required. None of the patients received treatment with immunomodulatory agents, including interferon-␤, within 6 months before inclusion. 468 Annals of Neurology Vol 51 No 4 April 2002 Study Drug ATM-027 is a recombinant humanized IgG1 developed under license from Avant Immunotherapeutics. Purified bulk material was produced by Lonza Biologics plc (UK). The study drug was provided as a solution for intravenous injection (6ml) at three different concentrations: 1.0mg/ml, 2.5mg/ml, and 5.0mg/ml in isotonic 0.01mol/L citrate buffer. The placebo was a solution for injection of identical appearance, consisting of isotonic 0.01mol/L citrate buffer. ATM-027 and placebo were administered as a short (5minute) injection. Treatment Regimen The aim of the present study was to keep the total number of V␤5.2/5.3⫹ T cells at ⬍1.2 % of the total CD3⫹ T-cell population and TCR density on the remaining target cells of ⬍20% of normal (TCR-low). Target cells with a TCR density of 20 to 100% of normal were denoted TCR-high. No target cells with normal TCR expression were to be present in the circulation. All patients received 6 monthly injections. All patients randomized to ATM-027 received a starting dose of 6mg. Subsequent dosing was based on both the levels of target cells and their TCR expression, as measured by flow cytometry 3 days before the next dose was scheduled. If the total level of V␤5.2/5.3⫹ T cells was ⬎1.2% or the level of V␤5.2/5.3⫹ T cells with TCR-high density was ⬎0.3%, the patient was to receive another dose of ATM-027. If not, patients received placebo. A secondary aim of the dose regimen was to find a dose that depleted target cells for more than 4 weeks. Thus, if the starting dose of 6mg did not result in target cell depletion for 4 weeks, the dose was increased to 15mg at the next injection. A final dose adjustment up to 30mg was permitted (Fig 1). Safety Assessments All adverse events were recorded continuously throughout the study. Safety laboratory measurements were performed monthly throughout the run-in and treatment period. Physical examinations were performed both before and after each injection, as well as during follow-up. A standard electrocardiogram was performed at enrollment and at the end of the treatment period. MRI Acquisition and Analysis Protocol MRI evaluation of MS lesions was in accordance with published guidelines for exploratory trials in established MS.15 Starting at enrollment and ending 4 months after the end of the treatment period, lesion activity was assessed by 14 monthly gadolinium-enhanced brain MRI scans for the number and volume of enhanced lesions. Five scans were obtained before treatment (⫺4, ⫺3, ⫺2, ⫺1, and baseline) and six scans during treatment (1, 2, 3, 4, 5, and 6), with an additional three scans during extended follow-up (7, 8, and 9). MR imaging included contiguous 3mm slices with a 1mm in-plane resolution using T1-weighted spin-echo images and dual-echo T2-weighted turbo spin-echo images obtained after injection of 0.2mmol/kg gadolinium DTPA. Evaluation of all scans performed was done at the Image Fig 1. Possible treatment regimen scenarios for the 47 ATM-027 treated multiple sclerosis patients. The maximum doses administered to achieve the aims of the individual dose titration were 6mg in 21 patients, 15mg in 19 patients, and 30mg in 7 patients. Analysis Center, VU Medical Center, Amsterdam, and was blinded according to patient identification, HLA-DR2 status, and treatment. The order of the scans was also blinded to the evaluators by assigning a random code to each scan; this allowed all scans of a given patient to be analyzed without knowledge of the order of scanning. Enhancing lesions were marked and counted according to established guidelines16 by two raters in consensus; their lesion volume was measured using local thresholding software (Show Images). Blood and CSF Sampling Blood samples were collected throughout the study for (1) flow cytometry assessment of T-lymphocyte subpopulations expressing V␤5.2/5.3 T-cell receptors; (2) analysis of ATM027 and treatment-induced antibodies; and (3) exploratory immunological assessments. CSF sampling was optional. CSF samples for V␤5.2/5.3 phenotyping were collected 1 month before the start of treatment and 6 months after the first injection. Target Cell Analysis in Peripheral Blood and CSF The target V␤5.2/5.3⫹ T-cell subset was analyzed by twocolor flow cytometry in blood and CSF. Analyses were performed at the Department of Hematology, VU Medical Center, Amsterdam, or at the Flow Cytometry Unit, Nova Medical AB, Clinical Trials Center, St Görans Hospital, Stockholm. Dose dependent presence of ATM-027 on the target cell surface following in vivo treatment precluded the use of a fluorochrome-conjugated primary mAb for proper target cell analysis. Thus, the V␤5.2/5.3⫹ T cells were analyzed by indirect staining, using ATM-027 as the primary antibody to saturate all target TCR molecules on the cell surface, followed by a FITC-conjugated F(ab)2 fragment of goat anti-human IgG, Fc specific (Immunotech, France). This reagent does not cross-react with mouse mAb and thus will not bind to the CD3 mAb (PerCP-conjugated, Becton Dickinson Immunocytometry Systems, CA) used concomitantly in the same tube. By this procedure, the staining al- ways revealed the total cell surface expression of the TCR V␤5.2/5.3, even after in vivo exposure to ATM-027. The results are presented as the proportion of target cells within the CD3⫹ T-cell population. Analysis of ATM-027 and Anti-ATM-027 Concentrations of ATM-027 were determined in plasma and CSF, using a competitive immunoassay with Europiumlabeled ATM-027 as a tracer. Analysis of treatment-induced anti-ATM-027 antibodies in plasma was performed using a double antigen sandwich immunoassay. Autoantigen Reactivity Measured with ELISPOT for Detection of Single Cells Secreting IFN-␥ Because of the necessity of using fresh cells for both this assay and the assay described in the next section, only patients studied in Sweden were included. The ELISPOT assay was performed essentially as described earlier.17 The antigens used were peptides of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), acetylcholine receptor (AChR-␣, used as the negative control peptide), and purified protein derivative (used as the positive control antigen). Background control cultures received no antigen. The following peptides were selected based on their previous documented role as T-cell epitopes either in human MS or in experimental autoimmune encephalomyelitis: MBP 1–20 (ASQKRPSQRHGSKYLATAST) MBP 80 –102 (TQDENPVVHFFKNIVTPRTPPPS) MBP 131–153 (ASDYKSAHKGFKGVDAQGTLSKI) MBP 142–167 (KGVDAQGTLSKIFKLGGRDSRSGSPM) MOG 14 –39 (ALVGDEVELPCRISPGKNATGMEVGW) MOG 63– 87 (PEYRGRTELLKDAIGEGKVTLRIRN) AChR-␣ 60 – 80 (WVDYNLKWNPDDYGGVKKIHI) The mean number of spots, corresponding to single cells that had secreted IFN-␥, was expressed as the number of IFN-␥-secreting cells per 105 peripheral blood lymphocytes Killestein et al: Suppression of V␤5.2/5.3⫹ T Cells in Multiple Sclerosis 469 (PBL) initially added to each well. The number of specific IFN-␥-secreting cells was calculated by subtracting the number of IFN-␥-secreting cells obtained in control wells containing media alone from the total number of IFN-␥secreting cells obtained for each antigen stimulation. Cellular Cytokine mRNA Expression A mixture of four different synthetic oligonucleotide probes complementary to mRNA for tumor necrosis factor-␣ (TNF␣), IFN-␥, IL-10, and TGF-␤ was used with sequences and methodology as described previously.18 A probe complementary to an IFN-␥ antisense probe was used as negative control. Data were expressed as numbers of cytokine mRNApositive cells per 105 plated PBL. Statistics The main efficacy variables were the within-patient changes in the mean number and mean volume of enhanced brain lesions during the run-in period compared with the treatment period. For the proof of concept (PoC) analysis only those patients who fulfilled the following two criteria: (1) target cell depletion successful, and (2) at least one active lesion detected during run-in. The Wilcoxon signed-rank test was used to test the hypothesis of no treatment effect based on the median of the within-patient changes. The effect size expressed as a percentage of the run-in level was calculated as the median of the distribution of individual percentage changes. Corresponding 95% confidence levels were derived from the 2.5 and 97.5 percentiles in the bootstrap distribution for the median of the distribution of individual percentage changes using 10,000 bootstrap samples. This method was chosen because the symmetry assumption for a HodgesLehmann procedure for estimation associated with the Wilcoxon signed-rank test was clearly not fulfilled for the MRI variables. For comparison of MRI variables between HLADR2⫹ and DR2⫺ subgroups, the two-sample Wilcoxon rank-sum test was used. Exploratory variables were analyzed by use of the Wilcoxon signed rank test with the associated Hodges-Lehmann estimates and 95% confidence intervals. Results Patient characteristics of all patients included are given in Table 1. Forty-seven patients were treated with ATM-027, 27 of them being HLA-DR2⫹ and 20 HLA- DR2⫺. Twelve patients were treated with placebo, six of each stratum. Thirteen patients were ex- cluded from the PoC analysis, two because target cell depletion was incomplete and 11 due to lack of enhanced lesions during run-in. Safety and Tolerability of ATM-027 The study drug was well tolerated. Two patients discontinued treatment because of adverse events and another two did not take part in the follow-up. A total number of 13 serious adverse events (SAEs) were reported during the study, three of which were judged by the investigator to have a possible causal relationship with the study drug (Table 2). Most adverse events (AEs) were classified as being of mild or moderate intensity and could not be clearly related to study drug administration, neither to the time of dosing or to the dose given. Seven patients of a total of 47 receiving active treatment reported flu-like symptoms, occurring at variable intervals in relation to the injection of study drug. There were no treatment-related changes in physical examination, supine or standing heart rate, supine or standing blood pressure, body temperature, blood chemistry, or electrocardiograms. V␤5.2/5.3⫹ T-Cell Depletion The plasma concentrations of ATM-027 increased with increasing doses in a proportional fashion. Target T-cell depletion and TCR down regulation were achieved in all patients on treatment (Fig 2). In patients in the active treatment group, levels of V␤5.2/ 5.3⫹ T cells were reduced from approximately 2.4% before start of treatment, to slightly below 1% during the treatment (see Fig 2). This level remained low also during the follow-up phase, whereas the receptor density recovered within 5 months after the end of treatment (data not shown). The level of V␤5.2/5.3⫹ T cells in CSF was reduced in all treated patients for whom CSF was available (n ⫽ 6, data not shown). Only two patients developed anti-ATM-027 indicating a low immunogenicity of ATM-027. MRI and Clinical Parameters During run-in, active lesions were found in 78.7% (37/47) of patients treated with ATM-027. The mean Table 1. Patient Characteristics Treatment ATM-027 HLA DR2 type Mean age (SD) Sex M/F (%) Mean EDSS (baseline) (SD) Mean no. of relapses 36 months before entry in study (SD) ⫹ n ⫽ 27 38.4 (6.8) 30/70 2.3 (1.4) 3.6 (1.8) ⫺ n ⫽ 20 37.3 (7.0) 35/65 2.3 (1.2) 3.2 (1.0) Subtotal (n ⫽ 47) 37.9 (6.8) 32/68 2.3 (1.3) 3.4 (1.5) Placebo ⫹n⫽6 41.2 (9.9) 33/67 2.4 (1.0) 2.8 (1.3) ⫺n⫽6 40.1 (6.9) 17/83 2.5 (0.9) 2.7 (0.8) Subtotal (n ⫽ 12) Total (n ⫽ 59) 40.7 (8.2) 25/75 2.5 (0.9) 2.8 (1.1) 38.5 (7.1) 31/69 2.4 (1.2) 3.3 (1.4) EDSS ⫽ Expanded Disability Status Score; HLA ⫽ human leukocyte antigen; SD ⫽ standard deviation; M/F ⫽ male/female. 470 Annals of Neurology Vol 51 No 4 April 2002 number of enhanced lesions in the PoC population decreased from 5.06 (SD: 6.60) during run-in, to 3.89 (SD: 4.65) during treatment, whereas the mean volume of enhanced lesions decreased from 531 mm3 (SD: 753) to 466 mm3 (SD: 616). No statistically significant difference in any parameter was detected (Table 3). The observed effect size was in the range of 10 to 30% compared with the targeted 60% response (see Fig 2). The Wilcoxon signed-rank test for paired comparisons was used to investigate whether there were any changes in the efficacy variables from the run-in period to the different time points during treatment phase and the follow-up period. The effect was not statistically significant in any of the analyses performed. Nor was there any trend toward increasing effect at the later stage of the treatment period that could indicate a delayed response. Furthermore, individual data did not suggest any correlation between degree of target cell depletion and effect on MRI. No changes of MR parameters in placebo treated patients were found. The number of relapses occurring in the different periods of study was 21 during run-in (16 ATM-027, 5 placebo), 29 during treatment (24 ATM-027, 5 placebo) and 24 during follow-up (18 ATM-027, 6 placebo). The duration of this trial was too short for a clinically meaningful change in EDSS to occur. Differences between HLA-DR2 Haplotypes No statistical significance was detected in the change from baseline to treatment between the two HLA-DR2 types (Fig 3). Table 2. Serious Adverse Events in Patients Treated with ATM-027 and Placebo Serious Adverse Events Run-in period Positive cervical smear test Tension headache Treatment period (n ⫽ 4) Postoperative bleeding MS relapse (n ⫽ 3) Follow-up period (n ⫽ 7) Traumatic injury Deep-venous thrombosis Arm fracture MS relapse (n ⫽ 3)a Tonsillitis Treatment Group (n ⫽ 2) n.a. n.a. Autoantigen Reactivity Measured with ELISPOT Peptides from MOG and MBP induced increased numbers of single cells secreting IFN-␥ as compared with the control peptide ACHR, confirming an increased autoreactivity to myelin antigens in RRMS. Most V␤5.2/5.3⫹ depleted patients displayed lower reactivity to peptides from both MOG and MBP, as measured by the number of single cells secreting IFN-␥ during treatment compared with baseline. Changes from mean baseline values for the different autoantigens used are given in Table 4. Although no changes were observed in the placebo group, the numbers obtained were too small to allow meaningful interpretations. Cellular mRNA Cytokine Expression Changes in TNF-␣, IL-4, IL-10, and IFN-␥ mRNA expression are given in Table 5. A transient decrease was found in the number of IFN-␥ mRNA expressing cells during treatment. Similar patterns were found for TNF-␣. IL-4 remained constant throughout the (pre-) treatment and posttreatment phase, whereas most patients showed a slight transient increase in the number of IL-10 mRNA-expressing cells during treatment. Discussion In this phase II clinical trial of the humanized antibody ATM-027, suppression of target cells, ie, V␤5.2/5.3⫹ T cells in combination with a TCR downregulation of the remaining target cells was achieved. No safety concerns emerged during the trial and the study drug was generally well tolerated. However, the efficacy results of the study gave clear evidence that the target of 60% reduction in MRI activity could not be attained. An effect size on the MRI variables in the range of 10 – Fig 2. Mean number of enhancing lesions (⫾SEM) and mean percentage of V␤5.2/5.3⫹ T cells (⫾SEM) during run-in and treatment phase in ATM-027 proof of concept population (n ⫽ 37). ATM-027 Placebo (n ⫽ 2); ATM-027 (n ⫽ 1) Placebo ATM-027 ATM-027 ATM-027 (n ⫽ 2); placebo (n ⫽ 1) ATM-027 a A total number of 13 serious adverse events were reported, three of which were judged as having a possible causal relationship with the study drug (MS relapse, requiring hospital admittance) MS ⫽ multiple sclerosis. Killestein et al: Suppression of V␤5.2/5.3⫹ T Cells in Multiple Sclerosis 471 Table 3. Number of Enhanced Lesions, Proof of Concept Analysis Set, ATM-027-Treated Patients (n ⫽ 37) Variable Period Mean (SD) No. of lesions Volume (mm3) Run-in Treatment Run-in Treatment 5.06 (6.60) 3.89 (4.65) 531 (753) 466 (616) Mean Change (SD) Median (Range) Median Change (Range) pa Effect Size (95% CL)b 2.00 (0–25) ⫺1.18 (4.98) 2.00 (0–21) ⫺0.50 (⫺17–9) 0.127 ⫺33.3% (⫺45.1–33.3) 221 (12–2998) ⫺65 (697) 253 (0–2771) ⫺6 (⫺2365–1336) 0.558 ⫺10.3% (⫺52.1–67.0) Within-patient change of mean number and mean volume of brain lesions between the run-in period and the treatment period a Wilcoxon signed-rank test. Calculated from 10,000 bootstrap samples. b SD ⫽ standard deviation. 30% can not be definitely excluded. However, given the run-in versus treatment design of the study, such an effect might also reflect the natural course of the disease (regression to the mean). Given the sample size and the number of enhancing lesions observed during run-in, the current study has been sufficiently powered to conclude that the likelihood of a false-negative result is minimal. The exploratory immunological assays performed here include phenomena of possible importance in MS. The results suggest that IFN-␥ expression is connected to V␤5.2/5.3 usage. Whether a decreased in vivo production of IFN-␥ would be beneficial or detrimental to MS patients remains an open question. However, since there are several arguments suggesting that IFN-␥ may be harmful in MS,3 the observed decrease in number of cells expressing mRNA of this cytokine might have been beneficial. However, a decrease was not seen in all patients and was not related to a significant effect on MRI. Fig 3. Mean number of enhancing lesions (⫾SEM) in ATM027-treated multiple sclerosis patients (proof of concept population, n ⫽ 37, patients stratified according to human leukocyte antigen (HLA)-DR2 status). No statistical difference was found in the change from baseline to treatment between the 2 HLA-DR2 types. 472 Annals of Neurology Vol 51 No 4 April 2002 So far, different strategies have been applied to selectively suppress autoreactive T cells in MS. Recently, treatment of MS patients with a humanized antileukocyte mAb (CAMPATH-1H) suppressed MRIdocumented disease activity.19 The depleted lymphocyte pool was reconstituted with cells having decreased IFN-␥ secretion, in line with the findings of our study. However, one-third of the patients developed Campath-1H-mediated thyroid autoimmunity.20 Unlike Campath-1H, another trial using an anti-CD4⫹ T-cell-depleting mAb did not suppress MRIdocumented disease activity.21 It was suggested that treatment with CD4 mAb did not eliminate those cells most strongly involved in the disease process, ie, primed, IFN-␥ producing T-helper (TH)-1 cells.22 More recently, two clinical trials testing the ability of an altered peptide ligand (APL) derived from MBP to reduce disease activity in MS have been reported. Kappos and colleagues23 reported that enhancing lesions were reduced in MS patients receiving low doses of APL. However, this trial was terminated because of high incidence of immediate type hypersensitivity reactions. Bielekova and colleagues24 reported that APL treatment led to an increase in exacerbation rate in 3 of 8 patients studied. T-cell populations specific for MBP expanded in these 3 patients. Another approach to deplete circulating MBP-reactive T cells in MS has been vaccination with irradiated T cells reactive to MBP,25,26 or a TCR peptide vaccine from the V␤5.2 sequence.27 In our study, only a small subpopulation of T cells was depleted. It is very unlikely that this form of depletion would render the individual more susceptible to serious side effects, as was the case in some of the more general T-cell-directed trials described above.20,23,24 Although it has been reported that MBP responsive TCR V␤5⫹ genes are used more frequently in MS patients than in non-MS patients, other studies did not detect any expansion within the V␤5⫹ T cells in MS patients (for review see Offner and colleagues28). Enrichment of T cells expressing various other TCR V␤ Table 4. Means of Percentage Changes from Baseline in MBP and MOG Peptides, AChR-␣ and PPD Reactivity (ELISPOT, Number of Single Cells Secreting Interferon-␥/100,000 cells), Estimated with Hodges-Lehmann and Corresponding 95% Confidence Interval Difference n H-L Lower Limit Upper Limit 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 18 18 18 18 18 18 18 17 18 18 18 18 18 18 18 18 ⫺0.3 ⫺1.8 ⫺1.8 ⫺1.5 ⫺3.8 ⫺3.5 ⫺2.5 ⫺3.0 ⫺2.0 ⫺3.3 ⫺2.0 ⫺2.3 ⫺1.0 ⫺0.8 ⫺1.5 ⫺6.8 (⫺2.5 (⫺3.5 (⫺4.8 (⫺7.0 (⫺7.8 (⫺7.3 (⫺4.3 (⫺6.5 (⫺6.3 (⫺6.5 (⫺5.5 (⫺5.8 (⫺2.5 (⫺3.0 (⫺18.5 (⫺21.0 0.5) 0.0) ⫺0.5) 0.0) ⫺0.5) ⫺0.8) ⫺1.0) ⫺1.0) ⫺0.8) ⫺1.0) ⫺0.5) 0.0) ⫺0.3) 0.3) 2.5) 2.5) Variable MBP 1–20 MBP 80–102 MBP 131–153 MBP 142–167 MOG 14–39 MOG 63–87 AChR-␣ PPD MBP ⫽ myelin basic protein; MOG ⫽ myelin oligodendrocyte glycoprotein; AChR-␣ ⫽ acetylcholine receptor-␣; PPD ⫽ purified protein derivative; BL ⫽ baseline; H-L ⫽ Hodges-Lehman. chains has also been reported.9,28 –30 Moreover, different V␤ segments are to some extent functionally interchangeable, suggesting that the observed biases in TCR-V␤ expression in response to antigen reflect a preferred, rather than a required, combination of ␤ gene segments.28 Furthermore, lack of restriction of TCR-␤ variable gene usage has been reported in CSF lymphocytes in acute optic neuritis.31 This may imply either that MS is not a V␤ restricted disease event at onset or that the autoimmune response has widened before the disease becomes clinically manifest (epitope spreading). In conclusion, the results from this phase II study show that treatment with ATM-027, resulting in profound downregulation of V␤5.2/5.3⫹ T cells, is safe and well tolerated. Decreased numbers of cells expressTable 5. Means of the Percentage Changes from Baseline in TNF-␣, IL-4, IL-10, and IFN-␥ mRNA Expression, Estimated with Hodges-Lehmann and Corresponding 95% Confidence Interval Variable Difference n H-L Lower Limit Upper Limit TNF-␣ 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 54 days–BL 166 days–BL 23 22 20 22 23 22 23 22 ⫺1.0 ⫺3.0 0.0 0.0 1.0 2.0 ⫺5.0 ⫺7.0 (⫺3.0 (⫺6.0 (⫺1.0 (⫺1.0 (⫺1.0 (⫺1.0 (⫺12.0 (⫺12.0 1.0) 0.0) 2.0) 1.0) 4.0) 4.0) 0.0) ⫺2.0) IL-4 IL-10 IFN-␥ TNF-␣ ⫽ tumor necrosis factor; IL-4 ⫽ interleukin-4; IFN-␥ ⫽ interferon-␥; H-L ⫽ Hodges-Lehmann. ing IFN-␥ mRNA and decreased T-cell reactivity to several myelin antigens were found in ATM-027 treated patients. 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