ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 × Ob1)F1 Mice by Induction of IL-10-Secreting iTregs
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Antigen-specific immunotherapy could provide a targeted approach for the treatment of multiple sclerosis that removes the need for broad-acting immunomodulatory drugs. ATX-MS-1467 is a mixture of four peptides identified as the main immune-dominant disease-associated T-cell epitopes in myelin basic protein (MBP), an autoimmune target for activated autoreactive T cells in multiple sclerosis. Previous animal studies have shown that ATX-MS-1467 treatment prevented the worsening of signs of disease in experimental autoimmune encephalitis (EAE) in the humanized (DR2 × Ob1)F1 mouse in a dose-dependent fashion.
Methods and Results
Our study extends these observations to show that subcutaneous treatment with 100 µg of ATX-MS-1467 after induction of EAE in the same mouse model reversed established clinical disability (p < 0.0001) and histological markers of inflammation and demyelination (p < 0.001) compared with vehicle-treated animals; furthermore, in longitudinal magnetic resonance imaging analyses, disruption of blood–brain barrier integrity was reversed, compared with vehicle-treated animals (p < 0.05). Chronic treatment with ATX-MS-1467 was associated with an enduring shift from a pro-inflammatory to a tolerogenic state in the periphery, as shown by an increase in interleukin 10 secretion, relative to interleukin 2, interleukin 17 and interferon γ, a decrease in splenocyte proliferation and an increase in interleukin 10+ Foxp3− T cells in the spleen.
Our results suggest that ATX-MS-1467 can induce splenic iTregs and long-term tolerance to MBP with the potential to partially reverse the pathology of multiple sclerosis, particularly during the early stages of the disease.
EMD Serono, Inc., a business of Merck KGaA.
KeywordsATX-MS-1467 (DR2 × Ob1)F1 mouse Experimental autoimmune encephalitis Interleukin 10 iTregs Multiple sclerosis
Most currently prescribed treatments for multiple sclerosis (MS) have a broad mechanism of action, often leading to nonspecific immunomodulation and associated complications, such as infections, malignancy and disruption of natural regulatory mechanisms . Therefore, there is an unmet need for antigen-specific treatments targeting key regulators in the breakdown of tolerance to self-antigens, such as antigen-presenting cells. The aim of such treatments is to silence or reprogram autoreactive T cells in the periphery to a regulatory phenotype and thus create a tolerogenic state to the targeted protein. By directly targeting the pathogenic mechanisms of MS, immunological tolerance to environmental and self-antigens, such as myelin basic protein (MBP), can be reinstated without disrupting normal immune function, and the hypersensitivity associated with repeated antigen-specific immunotherapy can also be avoided [1, 2].
Several studies have shown antigen-specific peptide immunotherapy suppresses central nervous system (CNS) inflammation in rodent models of MS [3, 4, 5]. Specifically, peptide-specific down-regulation of T-cell responses with a soluble acetylated form of the N-terminal peptide of MBP (Ac1-9) was protective against experimental autoimmune encephalitis (EAE), a T cell-mediated inflammatory disease that has similarities to MS and is the most commonly used experimental model for this disease [6, 7].
Nearly two decades of research on tolerance to T cell-restricted epitopes, more specifically in the context of the study of EAE in a murine model of MS, has led to the generation of the concept of antigen-processing-independent T-cell epitopes (apitopes), which have the potential to be used in the treatment of several autoimmune diseases [7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18]. ATX-MS-1467 is a mixture of four peptides of human MBP (MBP epitope 30–44: PRHRDTGILDSIGRF; MBP epitope 83–99: ENPVVHFFKNIVTPRTP; MBP epitope 130–144: RASDYKSAHKGFKGV; MBP epitope 140–154: GFKGVDAQGTLSKIF). These have been identified as disease-associated epitopes in patients with MS who have the human leukocyte antigen (HLA) DR2 haplotypes DRB1*1501 and DQB1*0602 , of which the DRB1*1501 haplotype may explain around 50% of cases of MS . It is assumed that ATX-MS-1467 binds to naked MHC-II of immature dendritic cells  and engages the T-cell receptor of MBP-specific primed memory T cells, leading to the development of a state of peripheral tolerance to MBP .
Treatment with ATX-MS-1467 has previously been shown to prevent worsening of EAE scores in a dose-dependent manner in the humanized transgenic (DR2 × Ob1)F1 mouse model , despite the fact that the study did not investigate the effect of ATX-MS-1467 on established EAE. In addition, a phase 1 study in six adults with secondary progressive MS showed that treatment with ATX-MS-1467 was well tolerated. The immune response to MBP was neither dramatically enhanced nor inhibited, due to study group sizes being too small to allow conclusions to be drawn concerning the efficacy of this treatment in humans. IL-10 mRNA transcripts were, nevertheless, transiently induced by the treatment in one patient phenotyped as expressing DRB1*15, suggesting that the drug might be capable of inducing desirable cytokine changes .
The aim of this study is to extend the results from the EAE by assessing the efficacy of late (at the observed peak of disease) as well as early (in the prodromal phase) treatment with ATX-MS-1467 for reversing the neurological damage and deficits accompanying EAE in the (DR2 × Ob1)F1 mouse. These findings will be corroborated with an examination of treatment effect on blood–brain barrier (BBB) permeability, on known markers of the shift from a pro-inflammatory to a tolerogenic state following acute and chronic treatment with ATX-MS-1467, more specifically by looking at the effect of in vivo pharmacological treatment on: the ex vivo antigen-specific cytokine release from splenocytes of EAE mice; the in vivo cytokine production following antigenic stimulation; and the accompanying changes on IL-10-secreting FoxP3+/FoxP3− regulatory cell subpopulations.
(DR2 × Ob1)F1 transgenic mice homozygous for DRA*0101/DRB1*1501 (HLA-DR2) and MBP84–102-specific human T-cell receptor (TCR) clone (Ob1) were generated in C57Bl/6 mice  and maintained and bred at Charles River Laboratories (Wilmington, MA, USA); the F1 progeny of these animals were used in all experiments. Before each experiment, expression of human MHC and TCR were confirmed in peripheral blood by phenotyping with fluorescein isothiocyanate (FITC)-labelled mouse anti-human HLA-DR, HLA-DP, HLA-DQ (BD Pharmingen, San Jose, CA, USA) and IOTest anti-TCR VB2-PE clone MPB2D5 (Beckman Coulter, Indianapolis, IN, USA). Only double-positive mice with a frequency of human MHC and TCR expression > 15% in peripheral leukocytes were used in the studies.
On arrival, mice were group-housed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited vivarium with a 12:12 light–dark cycle and food and water provided ad libitum. The mice were allowed to acclimatize for at least 48 h before any experiments were started. All procedures were approved by the EMD Serono Institutional Animal Care and Use Committees (IACUC) or by the Massachusetts General Hospital IACUC in the case of magnetic resonance imaging (MRI) studies.
Clinical score scale to assess extent of EAE
Partially limp tail
Completely limp tail
Slightly impaired righting reflex
Delayed righting reflex
Virtually absent righting reflex
Partial hind-limb paralysis
Complete unilateral hind-limb paralysis
Complete bilateral hind-limb paralysis
Death due to EAE
Induction of EAE
Ten to 14-week-old male or female (DR2 × Ob1)F1 mice under isoflurane anesthesia were shaved and received a subcutaneous injection at the tail base of 100 µL of an emulsion containing 200 µg (wet weight) of mouse spinal cord homogenate (SCH, harvested in-house from syngeneic spinal cords) and heat-inactivated Mycobacterium tuberculosis (400 µg/mouse; Difco, Oxford, Oxon, UK) prepared using equal volumes of phosphate-buffered saline (PBS) and incomplete Freund’s adjuvant (Difco, Oxford, Oxon, UK). On the day of immunization [0 days post-induction (dpi)], and also 2 days later (2 dpi), mice received an intraperitoneal injection with 300 ng of pertussis toxin (ENZO, Farmingdale, NY, USA).
All treatments with ATX-MS-1467 were performed from a reconstituted sterile, freeze-dried formulation of a mixture (1:1:1:1) of four synthetic peptides (MBP epitope 30–44: PRHRDTGILDSIGRF; MBP epitope 83–99: ENPVVHFFKNIVTPRTP; MBP epitope 130–144: RASDYKSAHKGFKGV; MBP epitope 140–154: GFKGVDAQGTLSKIF). After reconstitution of the lyophilized formulation to the original volume with sterile water, the solution was further diluted with PBS to the required doses. The concentration of the ATX-MS-1467 mixture was adjusted for a 100-µL injection volume, administered subcutaneously in all cases. The doses corresponded to the sum of each peptide in the mixture; therefore, a dose of 100 µg contained 25 µg of each peptide. The treatment regimen with ATX-MS-1467 varied; please refer to specific sections for the dose regimen used in each experiment.
HLA-Binding Peptide Treatment
In some experiments, HLA-binding peptide (HLAbp) was used as a control for ATX-MS-1467. Although HLAbp can bind directly to MHC on the surface of antigen-presenting cells without requiring antigen processing, the peptide sequence of HLAbp has no similarity to the epitopes involved in the adaptive immune responses to MS or EAE. The peptide sequence used (NPILLWQPIPVHTVPLSEDQ) was provided by Professor David Wraith (University of Bristol, UK).
Clinical Scoring, Animal Care and Humane Endpoints
Clinical score and body weight were monitored and recorded daily from 7 dpi by observers blind to treatment. Neurological disability and humane endpoints were monitored daily using a subjective 0–5-point clinical score scale (Table 1) . Whenever possible, a single investigator scored the animals in each study, and in any single day the same investigator scored all groups of animals. However, because clinical scoring sometimes progressed for several weeks, including weekends, up to four different investigators potentially provided input into a single study. Each investigator’s scoring was routinely calibrated, to ensure the results were comparable across different investigators.
Magnetic Resonance Imaging
EAE was induced on 0 dpi (n = 35). On 9 or 10 dpi, MRI was obtained, and those animals with radiological evidence of leakage in the cerebellum/brainstem seen on T1-weighted gadolinium-enhanced (Gd+) MRI with a 9.4-Tesla magnet scanner (Bruker Biospin, Billerica, MA, USA) were randomized into two groups for treatment with either ATX-MS-1467 (100 µg/mouse, n = 7) or vehicle (n = 7). Treatment was given every other day, starting at 10 dpi (mice scanned on 9 dpi) or 11 dpi (mice scanned at 10 dpi). T1-weighted Gd+ MRI was repeated after three doses (15 or 16 dpi). The T1-weighted gradient echo sequence with a short echo time (TE) and a large flip angle [repetition time (TR)/TE/flip angle = 100 ms/2.7 ms/50 degrees] was used to assess Gd leakage.
Pathological analysis was done on samples of spinal cords from animals treated at 7 or 14 dpi with either ATX-MS-1467 or vehicle. Samples were obtained at the termination date of these experiments (30 post-immunization). A 1.6 g/mL 2,2,2-tribromoethanol (T48402, Sigma, St. Louis, MO, USA) stock solution was prepared in 2-methyl-2-butanol (A1685, Sigma, St. Louis, MO, USA). A working solution at 20 mg/mL was obtained by subsequent dilution with PBS. At predefined time points, mice were overdosed with anesthetic (400 mg/kg intraperitoneal 2,2,2-tribromoethanol) and perfused through the left cardiac ventricle with saline followed by 10% neutral buffered formalin. The vertebrae were partially removed to obtain the spinal cord, and the tissues were post-fixed with 10% neutral buffered formalin for 48 h and then stored in PBS before dissecting out the spinal cords. Spinal cords were cut into five segments, spanning the cervical (one piece), thoracic (two pieces) and lumbar (two pieces) regions, which were processed for paraffin embedding. Histological analyses were done on 10 cross-sections of the spinal cord for each animal. These cross-sections were 7 µm thick and at least 280 µm apart.
Hematoxylin and eosin (H&E) and luxol fast blue (LFB) staining were performed, as well as immunohistochemistry staining against CD3 (Abcam, Cambridge, MA, USA) and CD45R (BD Pharmingen, San Jose, CA, USA) surface antigens using Leica BOND-III autostainer (Leica, Wetzlar, Germany). High-quality images obtained with a Nanozoomer 2.0 HT digital slide scanner (Hamamatsu, Hamamatsu City, Shizuoka, Japan) were semi-quantitatively scored for each stain by an experimenter who was blinded to the treatment groups.
Histology scoring of spinal cord sections
H&E/CD3 and CD45R
No visible signs of inflammation
No region presenting with demyelination
Immune cells found only in close association with meninges
Only one region exhibiting demyelination spanning < 50% of its area
Plus at least one clearly defined example of a perivascular cuff [at this stage, perivascular cuffs have limited cellularity (< 10 cells) and usually up to maximum of 3–4 perivascular cuffs can be found]
Two regions exhibiting demyelination spanning < 50%
Plus discrete numbers of immune cells beyond the perivascular cuffs (i.e., infiltrating into the parenchyma of the white matter)
Three regions exhibiting demyelination spanning < 50%
Plus extensive cell clusters (≥ 20 cells each) throughout the parenchyma of the white matter (i.e., not restricted to perivascular cuffs)
Four regions exhibiting demyelination spanning < 50%
Plus major infiltrations (typically clusters of ≥ 50 cells) all over the white matter
One region exhibiting demyelination spanning > 50%
Significant numbers of immune cells (> 30) dispersed throughout the grey matter, usually accompanied by massive hypercellular events in the white matter
Two regions exhibiting demyelination spanning > 50%
Ex-Vivo Antigen-Recall Assay
Mice were treated with either ATX-MS-1467 (100 µg/mouse, three times per week) or vehicle for 7 days. At 0 dpi, mice were also immunized as if for induction of EAE, but these mice were euthanized before they displayed signs of disease. Mice received the last dose of ATX-MS-1467 or vehicle at 7 dpi, 1 h before sacrifice for spleen removal and cell culture. On the day of sacrifice, mice were anaesthetized with 400 mg/kg intraperitoneal 2,2,2-tribromoethanol, and their spleens were harvested and processed in a gentle MACS Octo dissociator (Myltenyi Biotec, Birgisch Gladback, Germany). The splenocytes underwent several cycles of washing and centrifugation after lysis of red blood cells with red blood cell lysis buffer (Roche, Basel, Switzerland) and were counted manually in a Neubauer chamber using trypan blue solution to exclude dead cells. The concentration of the cell suspension was adjusted to 3 × 106 cells/mL in RPMI medium (Gibco, Waltham, MA, USA) supplemented with fetal calf serum (10%), penicillin–streptomycin (1:100; Gibco, Waltham, MA, USA), HEPES (final concentration 10 mM; Gibco, Waltham, MA, USA), MEM non-essential amino acid solution (1:100; Sigma, St. Louis, MO, USA), l-glutamine (final concentration 2 mM; Gibco, Waltham, MA, USA), sodium pyruvate (final concentration 1 mM; A&E Scientific PAA, San Jose, CA, USA) and β-mercaptoethanol (final concentration 2.5 µM; Sigma, St, Louis, MO, USA). For cell culture, 100 µL of the cell suspension was prepared as described, together with 100 µL of each appropriate stimulant (ATX-MS-1467 0.03–30 µg/mL final concentrations) diluted in medium and incubated at 37 °C for 48 or 72 h, corresponding to the optimum time points for evaluation of cytokine or proliferative responses, respectively. Cytokines were quantified in supernatants from 48-h splenocyte cultures with commercial enzyme-linked immunosorbent assay (ELISA) kits (mouse IL-2 Duoset ELISA, mouse IL-10 Quantikine ELISA, mouse IL-17 Duoset ELISA and mouse IFNγ Duoset ELISA, all purchased from R&D Systems, Minneapolis, MN, USA). Splenocyte proliferation was assessed by replacing 100 μL of the medium from wells that had been stimulated for 72 h with 100 μL of 3H thymidine solution and further incubating for 12 h. Incorporation of 3H thymidine was quantified in a scintillator. The results were expressed as counts per minute (cpm).
Antigen-Specific Cytokine Release In Vivo
The experiments that were conducted to assess antigen-specific cytokine release in vivo were performed in mice that had not been immunized with adjuvant in order to avoid background inflammation caused by this agent. Serum samples were collected from untreated mice or mice that had received repeated treatment with ATX-MS-1467 (100 µg/mouse, three times per week) or HLAbp (100 µg/mouse three times per week) receiving a total of 10 injections, followed or not by a wash-out period lasting from 2–42 days for assessment of the concentrations of IL-2, IL-10, IL-17 and IFNγ. Samples were collected 2 h after the last injection, which was experimentally predetermined as the time point at which the concentrations of most cytokines had reached a maximum. To ascertain the in vivo cytokine secretion of (DR2 × Ob1)F1 mice to an acute challenge with MBP, serum was collected 2 h after acute treatment with MBP at doses ranging 30–1000 µg/mouse. To ascertain the effect of chronic dosing with ATX-MS-1467 on in vivo pro-inflammatory cytokine secretion, mice received repetitive treatment (from one up to ten doses) with ATX-MS-1467 (100 µg/mouse, three times per week) with serum collected 2 h after the last dose. To ascertain the effect of chronic treatment with ATX-MS-1467 on the ratio of IL-10-to-pro-inflammatory cytokines after an acute challenge with MBP, mice received ten doses of ATX-MS-1467 (100 µg/mouse, three times per week) or ten doses of HLAbp (100 µg/mouse, three times per week). At 2, 7, 21 or 42 days after the last injection with ATX-MS-1467 or HLAbp, mice received a single injection of MBP (300 µg/mouse) and serum was collected 2 h later. Acute cytokine responses in serum were quantified using a customized MCYTOMAG-70 K | MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel—Immunology Multiplex Assay (Millipore, Billerica, MA, USA).
Effect of ATX-MS-1467 Treatment on Leukocyte Populations
Antibody panels used for splenocyte staining
Panel 1 (cell surface markers on freshly isolated splenocytes)
Panel 2 (cell surface and intracellular markers)
CD49b (BV421, 1:100, cat. # 63063, BD Pharmingen, San Jose, CA, USA)
CD49b (BV421, 1:100, cat. # 63063] BD Pharmingen, San Jose, CA, USA)
CD3 (V500, 1:100, cat. # 560771, BD Pharmingen, San Jose, CA, USA)
CD4 (V500, 1:100, cat. # 560782] BD Pharmingen San Jose, CA, USA)
CD4 (FITC, 1:100, cat. # 553729, BD Pharmingen, San Jose, CA, USA)
LAG3 (PE, 1:100, cat. # 125207, Biolegend, San Diego, CA, USA)
LAG3 (PE, 1:100, cat. # 125207, Biolegend, San Diego, CA, USA)
IL-10 (APC, 1:100, cat. # 554468, BD Pharmingen, San Jose, CA, USA)
B220 (PE-Cy7, 1:200, cat. # 552772, BD Pharmingen, San Jose, CA, USA)
CD8 (APC-Cy-7, 1:100, cat. # NC0079947, Biolegend, San Diego, CA, USA)
CD8 (BV605, 1:100, 563152, cat. # 563152, BD Pharmingen, San Jose, CA, USA)
CD45R/B220 (BV605 1:100, cat. # 563708, BD Pharmingen, San Jose, CA, USA)
FC block (CD16/CD32, unstained, 1:100, cat. # 553141, BD Pharmingen, San Jose, CA, USA)
FC block (CD16/CD32, unstained, 1:100, cat. # 553141, BD Pharmingen, San Jose, CA, USA)
FoxP3 (PerCP-Cy5.5 1:100, cat. # 35-5773-82, eBioscience, San Diego, CA, USA)
The first panel focused on cell surface markers and was applied in freshly isolated splenocytes. The second panel included intracellular markers, IL-10 and the transcription factor forkhead box P3 (Foxp3). To enable better identification of the IL-10-secreting cells, the second panel was applied in splenocytes incubated for 3 h with phorbol-myristate acetate (PMA, 5 ng/mL; Sigma, St. Louis, MO, USA), ionomycin (500 ng/mL; Sigma, St. Louis, MO, USA) and the protein transport-inhibitor monensin (4 µL/6 mL of cell culture; GolgiStop, BD Biosciences, San Jose, CA, USA) for 3 h. The splenocytes were isolated as previously described. B220 and CD8 staining were used as a negative control for the T lymphocyte subpopulations of interest. Samples were measured on a LSRFortessa X20 flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed in FlowJo version 10 (FlowJo, LLC, Ashland, OR, USA).
In experiments with multiple groups, clinical score data were analyzed via daily comparison of group averages using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. Clinical score data were also integrated as area under the curve, and the results analyzed with the Kruskal–Wallis test followed by Dunn’s multiple comparison test. MRI, EAE imaging and clinical score data were analyzed daily by Mann–Whitney test. The mean semi-quantitative histological scores of spinal cord sections were input as a single data point that was used to calculate group averages, which were compared by one-way analysis of variance (ANOVA) followed by the Bonferroni test. Data on concentration-dependent cytokines and proliferation response ex vivo were analyzed with two-way ANOVA followed by the Bonferroni test. In vivo cytokine-release data were analyzed with one-way ANOVA followed by Dunnett’s multiple comparison test.
Effect of ATX-MS-1467 on EAE Clinical Disability
Effect of ATX-MS-1467 on CNS Infiltration and Inflammation
Effect of ATX-MS-1467 Treatment on the Anti-Inflammatory Milieu in Ex Vivo Splenocytes
It is important to differentiate the purpose of ATX-MS-1467 as a treatment (Fig. 4a–e filled squares), when it was administered in vivo with a vehicle-administered group as a control (Fig. 4a–e filled circles), from its use here as an antigen-specific stimulus for cellular proliferation and cytokine release (x axis, various concentrations). The in vitro antigen recall assay showed that the in vivo treatment with ATX-MS-1467 caused a dose-dependent inhibition in pro-inflammatory cytokine (IL-17 and IFNγ) and IL-2 secretion and splenocyte proliferation. IL-10 secretion was significantly increased only after stimulation with concentrations of ATX-MS-1467 of 3 µg/mL (p < 0.05) and above (p < 0.001) in the ex vivo antigen recall assay (Fig. 4).
Effect of ATX-MS-1467 Treatment on the In Vivo Cytokine Profile
The effect of ATX-MS-1467 persists when the drug is cleared from the circulation. To extend the results for ex vivo cytokine secretion, we wanted to assess whether a favorable shift in cytokine balance also occurred in vivo. To achieve this, we performed three types of experiments.
Effect of ATX-MS-1467 Treatment on Regulatory T Cell Populations
Progressive axonal loss secondary to immune cell infiltration into the CNS, inflammation-mediated damage and demyelination, and diminishing repair are widely considered to be the pathological correlates of clinical disability in MS . In the preclinical study published by Streeter et al., treatment with ATX-MS-1467 prevented the increase of neurological disability when the drug was administered prior to (dpi 4) or immediately after (dpi 8) the appearance of the first signs of disease . To study this effect in more detail, mice were treated with four doses of ATX-MS-1467 at the onset and the peak of disease. As shown here, therapeutic treatment with ATX-MS-1467, both early and late after EAE induction in (DR2 × Ob1)F1 mice, reduced inflammation and T-cell and B-cell infiltration in the spinal cord, reduced demyelination and reduced BBB disruption. These effects were more profound in the animals treated early in the course of EAE than in those treated later, thus demonstrating the neuroprotective benefit of early intervention with ATX-MS-1467 in this animal model of MS. Although the exact association between the severity of disability and the extent of damage in the CNS is not fully understood , these results show good agreement between the abatement of inflammatory markers and demyelination and improvement in clinical score, particularly when treatment is started soon after the onset of EAE.
Autoantigen T-cell responses to MBP are involved in the pathogenesis of MS [25, 26, 27, 28]. T-cell responses to MBP were initially identified in eight regions of MBP, of which five (MBP 30–44, MBP 83–99, MBP 130–144, MBP 140–154 and MBP 156–170) are the regions most frequently recognized by T cells. ATX-MS-1467 is a mixture of four of these peptides (MBP 30–44 [ATX-MS1]; MBP 131–145 [ATX-MS4]; 140–154 [ATX-MS6]; MBP 83–99 [ATX-MS7]), which act as apitopes and suppress EAE in a humanized mouse model .
Direct targeting of key regulators in the development and maintenance of autoreactivity aims to silence or reprogram autoreactive T cells in the periphery back to a regulatory phenotype, and thus promote a tolerogenic state to the targeted protein. Such targeted treatment may help to avoid many of the bystander effects seen with broad suppression of the immune system. An ex vivo antigen recall assay and in vivo quantification of cytokine concentrations in response to an acute challenge with MBP and also in response to chronic dosing with ATX-MS-1467 were used to investigate the effect of ATX-MS-1467 on the immune environment. ATX-MS-1467 elicited a dose-dependent switch to an IL-10-dominant environment after chronic treatment. Following repeated exposure to ATX-MS-1467, there was a positive and robust shift in the ratio of anti-inflammatory to pro-inflammatory cytokines, which persisted for as long as the mice were exposed to ATX-MS-1467. Some aspects of the effect induced by ATX-MS-1467 remained, at least partially, beyond 21 days, such as the shift in IL-10/IFNγ ratio; however, this effect was not as persistent as the effect on the ratio of other cytokine pairs (e.g., IL-10-to-IL-17). The shift to an anti-inflammatory cytokine profile was seen in treated mice in response to repeated doses of ATX-MS-1467 but not in mice treated with vehicle or in response to HLAbp, which binds to the MHC but contains none of the epitopes involved in the autoimmune response to MBP.
Consideration of ATX-MS-1467 as a tolerogenic approach presupposes that a rebalance of antigen-specific immunity occurs sometime after the initial treatment. It also presupposes that because the immune system is, at least temporally, reprogramed against a particular antigen, the effect of ATX-MS-1467 persists when the drug is cleared from the circulation. The induction of tolerance in type-1 helper (Th1) cells after repeated peptide immunization of MBP-specific-TCR transgenic mice led to a reduction in the capacity of these cells to proliferate and a shift to an IL-10-secreting phenotype . IL-10 suppresses dendritic cell maturation and prevents Th1-cell differentiation, which is proposed as the negative arm of a feedback loop to limit Th1-driven immunopathology . As shown here, with repeated ATX-MS-1467 treatment, the concentrations of the pro-inflammatory cytokines IL-17 and IFNγ diminished relative to the concentration of IL-10, but the relation between IL-2 and IL-10 was less clear. IL-2 is secreted by effector cells but not by regulatory cells, whereas it is removed, via receptor-mediated internalization, by both subtypes . IL-2 is a broad regulator of Th-cell differentiation, notably in the differentiation of Th cells into both effector and cytolytic T cells, and is vital for the normal development of iTregs . Therefore, IL-2 may have a gateway role in the early development of tolerance that diminishes with the development of a tolerogenic state following repeated exposure. Such a role would be consistent with the results reported here, as well as with other reports in which single peptide exposure led to an initial variable peripheral T-cell death that was transient and incomplete. Taken in isolation, the reduction in ex vivo secretion of IL-2 from splenocytes of mice treated in vivo with ATX-MS-1467 does not allow us to conclude whether the treatment led to an increase in MBP-specific Tregs, a reduction in MBP-specific effector Tregs or both, because, in each case, the observed result (i.e., reduced IL-2 concentration in culture supernatant) was to be expected. Nevertheless, any of these treatment effects would have a beneficial therapeutic impact. By comparison, multiple doses of Ac 1–9 of MBP completely protected TCR-transgenic mice inoculated with myelin, leading to down-regulation of the capacity of CD4+ T cells to proliferate or to produce IL-2, IFNγ and IL-4, but increased the production of IL-10. This effect was completely reversed by neutralization of IL-10 .
Treg cells are a crucial subpopulation of T cells that suppress the activity of several immune-cell lineages and are thought to have diverse and overlapping roles in the suppression of foreign-stimulated and autoantigen-stimulated immune reactions. Pharmacological induction of tolerance may, theoretically, occur by expansion of the nTreg compartment, by way of differentiation, expansion of induced Tregs (iTregs, also called type 1 regulatory T cells), or by both mechanisms. Chronic treatment with ATX-MS-1467 preferentially induced differentiation of naïve T cells into iTregs and there is also evidence of re-education of the immune system, with sparing of normal cellular immune surveillance. These findings are consistent with the development of a devolved tolerogenic state and the recruitment of resting Th cells from more than one compartment. This is also in keeping with the more consistent response seen following repeated exposure to peptides, as compared with the response to single exposure, which was transient, incomplete and resulted in only variable peripheral T-cell apoptosis .
Analysis of the phenotype of the IL-10-secreting cells showed that the splenic cell compartment contained iTregs that were derived from peripheral compartments. iTregs that co-express the LAG3+CD49+ phenotype represent a population of cells that migrate to the bone marrow following the effector stage of an immune response and provide a pool of long-lived resting cells that form the basis of the memory response to an antigen. ATX-MS-1467 treatment also led to greater secretion of IL-10 from iTregs that lacked the transcription factor Foxp3 (IL-10+Foxp3–) but not from IL-10+Foxp3+ nTregs. This indicated that the main source of IL-10 is from iTregs that originated in the periphery during the effector phase of the initial immune response to ATX-MS-1467, as opposed to nTregs of thymic origin . The presence of iTregs with LAG3+CD49b+ and IL-10+Foxp3– phenotypes in the spleens of ATX-MS-1467-treated mice might indicate an overlapping cell population that express IL-10 transiently and at low concentrations during differentiation .
The results presented here raise some interesting questions for further investigation in preclinical studies and clinical trials in humans that were outside the scope of this manuscript. Future investigators should look at performing the ex vivo assay in co-cultures of purified dendritic cells and T cells, instead of whole-spleen cultures. Furthermore, ex vivo data could also be obtained only from leukocytes that infiltrated the CNS, which is more relevant for diseases such as EAE, and a wider complement of cytokines (such as IL-6 and transforming growth factor beta) could be measured. The regulatory effect of IL-10 could be further confirmed in the EAE mouse model by the use of IL-10-neutralizing antibodies or by adoptive-transfer experiments using purified iTregs, to gain further validation of the regulatory potential of the iTregs.
Although these current results show that ATX-MS-1467 can induce tolerance in (DR2 × Ob1)F1 mice in which EAE has been induced, this effect diminished with a 7-day wash-out period in the case of IL-10/IL-17. However, the effect on the IL-10-to-IFNγ ratio was more persistent. This observation is consistent with the inhibition of antigen-specific IFNγ release, but not of IL-17 release, in vitro from the splenocytes of mice treated with ATX-MS-1467. These data suggest that, in this model, ATX-MS-1467 is a more robust treatment to curb Th1-type responses than Th17, although this falling off may also be indicative of improved efficacy and a reduction in the need for high levels of cytokines over time. This finding could have a substantial effect on the regularity of dosing required in patients to achieve and maintain a state of tolerance to MBP. Studies in patients with MS will also determine whether the improvements in disability can be reproduced and whether the magnitude of the treatment effect will be sufficient to make this viable for the treatment of patients beyond the early stages of the disease. Experimental testing of ATX-MS-1467 has so far been restricted to animals designed to model the MS-associated HLA-DR2b allele; therefore, the generalizability of the treatment for patients who do not have genetic susceptibility and the efficacy in patients with different forms of MS will need to be established.
Furthermore, in the absence of a model that responds to all the peptides in the ATX-MS-1467 sequence, the model used in this study was constructed around only a small region of MBP: MS7 overlapping the MBP amino acids 81–102 . This means that the efficacy of the drug in this model is restricted to this region, rather than the whole mix of peptides in ATX-MS-1467. It would be interesting to fully assess the efficacy of ATX-MS-1467 in a non-transgenic model, in which the MBP sequences in the drug are part of the encephalitic T-cell repertoire of the animal. We have already reported preliminary findings that ATX-MS-1467, given before the onset of EAE, delayed the onset (p < 0.04) and severity (p < 0.002) in non-transgenic Lewis rats . A phase 1 first-in-human study in six patients with secondary progressive MS showed that ATX-MS-1467 was safe and well tolerated . This study also showed a trend towards a transient induction of immunotolerance 1 month after chronic dosing with ATX-MS-1467. A phase 2 trial of ATX-MS-1467 in patients with relapsing/remitting MS to evaluate the clinical, biological and radiological effects and the safety profile of ATX-MS-1467 for up to 36 weeks has also been completed .
ATX-MS-1467 improved clinical disability and histological markers of inflammation and demyelination. It also restored the integrity of the BBB following induction of EAE in the humanized (DR2 × Ob1)F1 mouse. The greatest effectiveness was seen when treatment was given at the onset of EAE, but the effect is still significant and reproducible with dosing at the peak of disease. Chronic treatment with ATX-MS-1467 led to the development of tolerance to MBP in this model, accompanied by a shift to an immunotolerant state. The results of this study indicate that ATX-MS-1467 is a potential candidate in the management of MS.
ATX-MS-1467 is owned and being developed by Apitope Technology (Bristol) Limited. All of the data presented here was generated under a license from Apitope Technology (Bristol) Limited. The authors are grateful to David Wraith (University of Bristol) and Keith F. Martin (Apitope, Chepstow, Monmouthshire, UK) for scientific exchange. The authors would like to thank Professor Joseph Mandeville (Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Charlestown, MA, USA) who helped to design the study and provided guidance on the data analysis, and Fréderic Bernard (EMD Serono, Billerica, MA, USA), Anneli Savinainen (EMD Serono, Billerica, MA, USA) and Dongzi Yu (EMD Serono, Billerica, MA USA) for supporting the in vivo studies. The authors would also like to thank Paul Smith (Merck, Geneva, Switzerland) and Alla Zozoulya (EMD Serono, Billerica, MA, USA) for being involved in previous project management activities and model validation relevant to this work.
This study was funded by EMD Serono, Inc., a business of Merck KGaA, Darmstadt, Germany. At the time of the study, most of the authors were employees of EMD Serono, Inc.; therefore, the sponsor was involved in the study design, the collection, analysis and interpretation of the data and the decision to submit the study for publication. All authors had full access to all of the data in this study and take complete responsibility for the integrity of the data and accuracy of the data analysis.
Medical Writing and/or Editorial Assistance
Medical writing support was provided by Steven Goodrick, inScience Communications, UK, and funded by Merck KGaA, Darmstadt, Germany.
All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.
Adriano Luís Soares de Souza wrote the manuscript, designed and performed the research and analyzed the data. Shuning Huang performed the research, MRI scans and analysis. Ji-Kyung Choi performed the MRI scans and analysis. Timothy Crandall performed the experiments. Stefan Rudin designed and performed the research and analyzed the data. Blake Tomkinson devised the strategy for the experiments and contributed to the research design. Rui Chang contributed to the histological analysis and analyzed the data. Keith Mitchell contributed to antigen-specific proliferation assays and analyzed the data. Shinji Okitsu contributed to the flow-cytometry experiments and analyzed the data. Danielle Graham designed the research and analyzed the data. Tammy Dellovade wrote the manuscript, designed the research and analyzed the data. Images were acquired by Shuning Huang and Ji-Kyung Choi. Gadolinium leakage was evaluated independently by Shuning Huang, Ji-Kyung Choi and Joe Mandeville. All authors critically reviewed the content of the manuscript and approved the final version for publication.
Adriano Luís Soares de Souza is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Stefan Rudin is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Rui Chang is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Keith Mitchell is an employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Timothy Crandall is an employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Shuning Huang is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Ji-Kyung Choi is an employee of Harvard Medical School at the Massachusetts General Hospital. The Massachusetts General Hospital received sponsorship from EMD Serono, Inc to allow access to magnetic resonance scanners and expertise in acquiring/analysing data, particularly for the MRI studies shown in this study. Shinji L. Okitsu is an employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Danielle L. Graham is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Blake Tomkinson is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Tammy Dellovade is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany.
Compliance with Ethics Guidelines
All procedures conducted were approved by the EMD Serono Institutional Animal Care and Use Committees (IACUC) or by the Massachusetts General Hospital IACUC in the case of MRI studies. All animal experiments complied with the National Institutes of Health guide for the care and use of laboratory animals. All efforts were made to minimize the number of animals used and to optimize their well-being.
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