ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 × Ob1)F1 Mice by Induction of IL-10-Secreting iTregs

ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 × Ob1)F1 Mice by... Neurol Ther (2018) 7:103–128 https://doi.org/10.1007/s40120-018-0094-z ORIGINAL RESEARCH ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 3 Ob1)F1 Mice by Induction of IL-10-Secreting iTregs . . . Adriano Luı´s Soares De Souza Stefan Rudin Rui Chang . . . . Keith Mitchell Timothy Crandall Shuning Huang Ji-Kyung Choi . . . Shinji L. Okitsu Danielle L. Graham Blake Tomkinson Tammy Dellovade Received: January 8, 2018 / Published online: March 14, 2018 The Author(s) 2018. This article is an open access publication humanized (DR2 9 Ob1)F1 mouse in a dose- ABSTRACT dependent fashion. Methods and Results: Our study extends these Introduction: Antigen-specific immunotherapy observations to show that subcutaneous treat- could provide a targeted approach for the ment with 100 lg of ATX-MS-1467 after induc- treatment of multiple sclerosis that removes the tion of EAE in the same mouse model reversed need for broad-acting immunomodulatory established clinical disability (p \ 0.0001) and drugs. ATX-MS-1467 is a mixture of four pep- histological markers of inflammation and tides identified as the main immune-dominant demyelination (p \ 0.001) compared with disease-associated T-cell epitopes in myelin vehicle-treated animals; furthermore, in longi- basic protein (MBP), an autoimmune target for tudinal magnetic resonance imaging analyses, activated autoreactive T cells in multiple scle- disruption of blood–brain barrier integrity was rosis. Previous animal studies have shown that reversed, compared with vehicle-treated ani- ATX-MS-1467 treatment prevented the wors- mals (p \ 0.05). Chronic treatment with ATX- ening of signs of disease in experimental MS-1467 was associated with an enduring shift autoimmune encephalitis (EAE) in the from a pro-inflammatory to a tolerogenic state in the periphery, as shown by an increase in Enhanced content To view enhanced content for this interleukin 10 secretion, relative to interleukin article, go to https://doi.org/10.6084/m9.figshare. 2, interleukin 17 and interferon c, a decrease in splenocyte proliferation and an increase in ? - interleukin 10 Foxp3 T cells in the spleen. A. L. S. De Souza (&)  S. Rudin  R. Chang Conclusion: Our results suggest that ATX-MS- K. Mitchell  T. Crandall  S. Huang 1467 can induce splenic iTregs and long-term D. L. Graham  B. Tomkinson  T. Dellovade Neurology eTIP, Translational and Biomarker tolerance to MBP with the potential to partially Research Group, EMD Serono Research and reverse the pathology of multiple sclerosis, Development Institute, Inc., Billerica, MA, USA particularly during the early stages of the e-mail: adriano.souza@ufmg.br disease. J.-K. Choi Funding: EMD Serono, Inc., a business of Merck Athinoula A. Martinos Center for Biomedical KGaA. Imaging, Massachusetts General Hospital, Charlestown, MA, USA Keywords: ATX-MS-1467; (DR2 9 Ob1)F1 S. L. Okitsu mouse; Experimental autoimmune TIP Immunology, EMD Serono Research and Development Institute, Inc., Billerica, MA, USA 104 Neurol Ther (2018) 7:103–128 encephalitis; Interleukin 10; iTregs; Multiple epitope 140–154: GFKGVDAQGTLSKIF). These sclerosis have been identified as disease-associated epi- topes in patients with MS who have the human leukocyte antigen (HLA) DR2 haplotypes INTRODUCTION DRB1*1501 and DQB1*0602 [14], of which the DRB1*1501 haplotype may explain around 50% Most currently prescribed treatments for multi- of cases of MS [15]. It is assumed that ATX-MS- ple sclerosis (MS) have a broad mechanism of 1467 binds to naked MHC-II of immature den- action, often leading to nonspecific dritic cells [19] and engages the T-cell receptor immunomodulation and associated complica- of MBP-specific primed memory T cells, leading tions, such as infections, malignancy and dis- to the development of a state of peripheral tol- ruption of natural regulatory mechanisms [1]. erance to MBP [14]. Therefore, there is an unmet need for antigen- Treatment with ATX-MS-1467 has previously specific treatments targeting key regulators in been shown to prevent worsening of EAE scores the breakdown of tolerance to self-antigens, in a dose-dependent manner in the humanized such as antigen-presenting cells. The aim of transgenic (DR2 9 Ob1)F1 mouse model [14], such treatments is to silence or reprogram despite the fact that the study did not investi- autoreactive T cells in the periphery to a regu- gate the effect of ATX-MS-1467 on established latory phenotype and thus create a tolerogenic EAE. In addition, a phase 1 study in six adults state to the targeted protein. By directly target- with secondary progressive MS showed that ing the pathogenic mechanisms of MS, treatment with ATX-MS-1467 was well toler- immunological tolerance to environmental and ated. The immune response to MBP was neither self-antigens, such as myelin basic protein dramatically enhanced nor inhibited, due to (MBP), can be reinstated without disrupting study group sizes being too small to allow con- normal immune function, and the hypersensi- clusions to be drawn concerning the efficacy of tivity associated with repeated antigen-specific this treatment in humans. IL-10 mRNA tran- immunotherapy can also be avoided [1, 2]. scripts were, nevertheless, transiently induced Several studies have shown antigen-specific by the treatment in one patient phenotyped as peptide immunotherapy suppresses central expressing DRB1*15, suggesting that the drug nervous system (CNS) inflammation in rodent might be capable of inducing desirable cytokine models of MS [3–5]. Specifically, peptide-specific changes [14]. down-regulation of T-cell responses with a sol- The aim of this study is to extend the results uble acetylated form of the N-terminal peptide from the EAE by assessing the efficacy of late (at of MBP (Ac1-9) was protective against experi- the observed peak of disease) as well as early (in mental autoimmune encephalitis (EAE), a T the prodromal phase) treatment with ATX-MS- cell-mediated inflammatory disease that has 1467 for reversing the neurological damage and similarities to MS and is the most commonly deficits accompanying EAE in the (DR2 9 used experimental model for this disease [6, 7]. Ob1)F1 mouse. These findings will be corrobo- Nearly two decades of research on tolerance rated with an examination of treatment effect to T cell-restricted epitopes, more specifically in on blood–brain barrier (BBB) permeability, on the context of the study of EAE in a murine known markers of the shift from a pro-inflam- model of MS, has led to the generation of the matory to a tolerogenic state following acute concept of antigen-processing-independent and chronic treatment with ATX-MS-1467, T-cell epitopes (apitopes), which have the more specifically by looking at the effect of potential to be used in the treatment of several in vivo pharmacological treatment on: the autoimmune diseases [7–18]. ATX-MS-1467 is a ex vivo antigen-specific cytokine release from mixture of four peptides of human MBP (MBP splenocytes of EAE mice; the in vivo cytokine epitope 30–44: PRHRDTGILDSIGRF; MBP epi- production following antigenic stimulation; tope 83–99: ENPVVHFFKNIVTPRTP; MBP epi- and the accompanying changes on IL-10- tope 130–144: RASDYKSAHKGFKGV; MBP Neurol Ther (2018) 7:103–128 105 secreting FoxP3?/FoxP3- regulatory cell Table 1 Clinical score scale to assess extent of EAE subpopulations. Clinical score Outcome 0 Normal METHODS 0.5 Partially limp tail Animals 1.0 Completely limp tail 1.5 Slightly impaired righting reflex (DR2 9 Ob1)F1 transgenic mice homozygous 2.0 Delayed righting reflex for DRA*0101/DRB1*1501 (HLA-DR2) and MBP -specific human T-cell receptor (TCR) 84–102 2.5 Virtually absent righting reflex clone (Ob1) were generated in C57Bl/6 mice 3.0 Partial hind-limb paralysis [20] and maintained and bred at Charles River Laboratories (Wilmington, MA, USA); the F1 3.5 Complete unilateral hind-limb paralysis progeny of these animals were used in all 4.0* Complete bilateral hind-limb paralysis experiments. Before each experiment, expres- sion of human MHC and TCR were confirmed 4.5 Moribund in peripheral blood by phenotyping with fluo- 5.0 Death due to EAE rescein isothiocyanate (FITC)-labelled mouse anti-human HLA-DR, HLA-DP, HLA-DQ (BD EAE experimental autoimmune encephalitis *Humane endpoints and special care for mice with EAE Pharmingen, San Jose, CA, USA) and IOTest were also observed. Once animals reached a score of 4.0 anti-TCR VB2-PE clone MPB2D5 (Beckman they were observed at least twice a day to ensure they did Coulter, Indianapolis, IN, USA). Only double- not progress further and did not get worse: animals that positive mice with a frequency of human MHC improved to a score of 3.5 or lower were allowed to con- and TCR expression [ 15% in peripheral tinue in the study; if animals did not improve below a leukocytes were used in the studies. score of 4.0 within 72 h, they were euthanized. In addition, On arrival, mice were group-housed in an mice were also euthanized if they experienced a body Association for Assessment and Accreditation of weight loss of 20%. From the onset of paralysis, mice also Laboratory Animal Care-accredited vivarium received wet dietary supplement (Dietgel 76A, ClearH2O) with a 12:12 light–dark cycle and food and on the cage bottom and subcutaneous sterile Ringer’s water provided ad libitum. The mice were glucose injections, as needed, for fluid replacement allowed to acclimatize for at least 48 h before any experiments were started. All procedures were approved by the EMD Serono Institutional Induction of EAE Animal Care and Use Committees (IACUC) or by the Massachusetts General Hospital IACUC Ten to 14-week-old male or female (DR2 9 in the case of magnetic resonance imaging Ob1)F1 mice under isoflurane anesthesia were (MRI) studies. shaved and received a subcutaneous injection at Mice reaching a predefined clinical endpoint the tail base of 100 lL of an emulsion contain- for humane withdrawal from the study (Table 1) ing 200 lg (wet weight) of mouse spinal cord and mice that were not required for tissue homogenate (SCH, harvested in-house from sampling were euthanized by slow replacement syngeneic spinal cords) and heat-inactivated of the air in their cage by an air mixture con- Mycobacterium tuberculosis (400 lg/mouse; taining CO . Death by CO asphyxiation was 2 2 Difco, Oxford, Oxon, UK) prepared using equal confirmed by cervical displacement. All animal volumes of phosphate-buffered saline (PBS) and experiments complied with the National Insti- incomplete Freund’s adjuvant (Difco, Oxford, tutes of Health guide for the care and use of Oxon, UK). On the day of immunization [0 days laboratory animals. All efforts were made to post-induction (dpi)], and also 2 days later minimize the number of animals used and to (2 dpi), mice received an intraperitoneal optimize their well-being. 106 Neurol Ther (2018) 7:103–128 injection with 300 ng of pertussis toxin (ENZO, investigator scored the animals in each study, Farmingdale, NY, USA). and in any single day the same investigator scored all groups of animals. However, because clinical scoring sometimes progressed for sev- ATX-MS-1467 Treatment eral weeks, including weekends, up to four dif- ferent investigators potentially provided input All treatments with ATX-MS-1467 were per- into a single study. Each investigator’s scoring formed from a reconstituted sterile, freeze-dried was routinely calibrated, to ensure the results formulation of a mixture (1:1:1:1) of four syn- were comparable across different investigators. thetic peptides (MBP epitope 30–44: PRHRDTGILDSIGRF; MBP epitope 83–99: Magnetic Resonance Imaging ENPVVHFFKNIVTPRTP; MBP epitope 130–144: RASDYKSAHKGFKGV; MBP epitope 140–154: GFKGVDAQGTLSKIF). After reconstitution of EAE was induced on 0 dpi (n = 35). On 9 or the lyophilized formulation to the original 10 dpi, MRI was obtained, and those animals volume with sterile water, the solution was with radiological evidence of leakage in the further diluted with PBS to the required doses. cerebellum/brainstem seen on T1-weighted The concentration of the ATX-MS-1467 mixture gadolinium-enhanced (Gd ) MRI with a 9.4- was adjusted for a 100-lL injection volume, Tesla magnet scanner (Bruker Biospin, Billerica, administered subcutaneously in all cases. The MA, USA) were randomized into two groups for doses corresponded to the sum of each peptide treatment with either ATX-MS-1467 in the mixture; therefore, a dose of 100 lg (100 lg/mouse, n = 7) or vehicle (n = 7). Treat- contained 25 lg of each peptide. The treatment ment was given every other day, starting at regimen with ATX-MS-1467 varied; please refer 10 dpi (mice scanned on 9 dpi) or 11 dpi (mice to specific sections for the dose regimen used in scanned at 10 dpi). T1-weighted Gd MRI was each experiment. 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 HLA-Binding Peptide Treatment time (TR)/TE/flip angle = 100 ms/2.7 ms/50 degrees] was used to assess Gd leakage. In some experiments, HLA-binding peptide (HLAbp) was used as a control for ATX-MS- Histology 1467. Although HLAbp can bind directly to MHC on the surface of antigen-presenting cells without requiring antigen processing, the pep- Pathological analysis was done on samples of tide sequence of HLAbp has no similarity to the spinal cords from animals treated at 7 or 14 dpi epitopes involved in the adaptive immune with either ATX-MS-1467 or vehicle. Samples responses to MS or EAE. The peptide sequence were obtained at the termination date of these used (NPILLWQPIPVHTVPLSEDQ) was provided experiments (30 post-immunization). A 1.6 by Professor David Wraith (University of Bristol, g/mL 2,2,2-tribromoethanol (T48402, Sigma, St. UK). 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 Clinical Scoring, Animal Care obtained by subsequent dilution with PBS. At and Humane Endpoints predefined time points, mice were overdosed with anesthetic (400 mg/kg intraperitoneal Clinical score and body weight were monitored 2,2,2-tribromoethanol) and perfused through and recorded daily from 7 dpi by observers blind the left cardiac ventricle with saline followed by to treatment. Neurological disability and 10% neutral buffered formalin. The vertebrae humane endpoints were monitored daily using were partially removed to obtain the spinal a subjective 0–5-point clinical score scale cord, and the tissues were post-fixed with 10% (Table 1)[21]. Whenever possible, a single Neurol Ther (2018) 7:103–128 107 neutral buffered formalin for 48 h and then The H&E scoring was performed as set out in stored in PBS before dissecting out the spinal Table 2. CD3 and CD45R scoring were similar to cords. Spinal cords were cut into five segments, the H&E scoring, except that only immunos- spanning the cervical (one piece), thoracic (two tained cells were scored. For the LFB scoring, pieces) and lumbar (two pieces) regions, which each cross section of the spinal cord was ana- were processed for paraffin embedding. Histo- lyzed as four independent regions (ventral, logical analyses were done on 10 cross-sections dorsal, right lateral and left lateral). In all cases, of the spinal cord for each animal. These cross- the scores for each of the ten sections was sections were 7 lm thick and at least 280 lm averaged and entered as a single data point for apart. each mouse in the experiment. Hematoxylin and eosin (H&E) and luxol fast blue (LFB) staining were performed, as well as Ex-Vivo Antigen-Recall Assay immunohistochemistry staining against CD3 (Abcam, Cambridge, MA, USA) and CD45R (BD Mice were treated with either ATX-MS-1467 Pharmingen, San Jose, CA, USA) surface anti- (100 lg/mouse, three times per week) or vehicle gens using Leica BOND-III autostainer (Leica, for 7 days. At 0 dpi, mice were also immunized Wetzlar, Germany). High-quality images as if for induction of EAE, but these mice were obtained with a Nanozoomer 2.0 HT digital euthanized before they displayed signs of dis- slide scanner (Hamamatsu, Hamamatsu City, ease. Mice received the last dose of ATX-MS- Shizuoka, Japan) were semi-quantitatively 1467 or vehicle at 7 dpi, 1 h before sacrifice for scored for each stain by an experimenter who spleen removal and cell culture. On the day of was blinded to the treatment groups. sacrifice, mice were anaesthetized with 400 mg/ kg intraperitoneal 2,2,2-tribromoethanol, and Table 2 Histology scoring of spinal cord sections Score H&E/CD3 and CD45R LFB 0 No visible signs of inflammation No region presenting with demyelination 0.5 Immune cells found only in close association with meninges Only one region exhibiting demyelination spanning \ 50% of its area 1.0 Plus at least one clearly defined example of a perivascular cuff [at this Two regions exhibiting demyelination stage, perivascular cuffs have limited cellularity (\ 10 cells) and spanning \ 50% usually up to maximum of 3–4 perivascular cuffs can be found] 2.0 Plus discrete numbers of immune cells beyond the perivascular cuffs Three regions exhibiting demyelination (i.e., infiltrating into the parenchyma of the white matter) spanning \ 50% 3.0 Plus extensive cell clusters (C 20 cells each) throughout the parenchyma Four regions exhibiting demyelination of the white matter (i.e., not restricted to perivascular cuffs) spanning \ 50% 4.0 Plus major infiltrations (typically clusters of C 50 cells) all over the One region exhibiting demyelination white matter spanning [ 50% 5.0 Significant numbers of immune cells ([ 30) dispersed throughout the Two regions exhibiting demyelination grey matter, usually accompanied by massive hypercellular events in spanning [ 50% the white matter H&E hematoxylin and eosin, LFB luxol fast blue 108 Neurol Ther (2018) 7:103–128 their spleens were harvested and processed in a immunized with adjuvant in order to avoid gentle MACS Octo dissociator (Myltenyi Biotec, background inflammation caused by this agent. Birgisch Gladback, Germany). The splenocytes Serum samples were collected from untreated underwent several cycles of washing and cen- mice or mice that had received repeated treat- trifugation after lysis of red blood cells with red ment with ATX-MS-1467 (100 lg/mouse, three blood cell lysis buffer (Roche, Basel, Switzer- times per week) or HLAbp (100 lg/mouse three land) and were counted manually in a Neubauer times per week) receiving a total of 10 injec- chamber using trypan blue solution to exclude tions, followed or not by a wash-out period dead cells. The concentration of the cell sus- lasting from 2–42 days for assessment of the pension was adjusted to 3 9 10 cells/mL in concentrations of IL-2, IL-10, IL-17 and IFNc. RPMI medium (Gibco, Waltham, MA, USA) Samples were collected 2 h after the last injec- supplemented with fetal calf serum (10%), tion, which was experimentally predetermined penicillin–streptomycin (1:100; Gibco, Wal- as the time point at which the concentrations of tham, MA, USA), HEPES (final concentration most cytokines had reached a maximum. To 10 mM; Gibco, Waltham, MA, USA), MEM non- ascertain the in vivo cytokine secretion of essential amino acid solution (1:100; Sigma, St. (DR2 9 Ob1)F1 mice to an acute challenge with Louis, MO, USA), L-glutamine (final concentra- MBP, serum was collected 2 h after acute treat- tion 2 mM; Gibco, Waltham, MA, USA), sodium ment with MBP at doses ranging 30– pyruvate (final concentration 1 mM; A&E Sci- 1000 lg/mouse. To ascertain the effect of entific PAA, San Jose, CA, USA) and b-mercap- chronic dosing with ATX-MS-1467 on in vivo toethanol (final concentration 2.5 lM; Sigma, pro-inflammatory cytokine secretion, mice St, Louis, MO, USA). For cell culture, 100 lLof received repetitive treatment (from one up to the cell suspension was prepared as described, ten doses) with ATX-MS-1467 (100 lg/mouse, together with 100 lL of each appropriate stim- three times per week) with serum collected 2 h ulant (ATX-MS-1467 0.03–30 lg/mL final con- after the last dose. To ascertain the effect of centrations) diluted in medium and incubated chronic treatment with ATX-MS-1467 on the at 37 C for 48 or 72 h, corresponding to the ratio of IL-10-to-pro-inflammatory cytokines optimum time points for evaluation of cytokine after an acute challenge with MBP, mice or proliferative responses, respectively. Cytoki- received ten doses of ATX-MS-1467 nes were quantified in supernatants from 48-h (100 lg/mouse, three times per week) or ten splenocyte cultures with commercial enzyme- doses of HLAbp (100 lg/mouse, three times per linked immunosorbent assay (ELISA) kits week). At 2, 7, 21 or 42 days after the last (mouse IL-2 Duoset ELISA, mouse IL-10 Quan- injection with ATX-MS-1467 or HLAbp, mice tikine ELISA, mouse IL-17 Duoset ELISA and received a single injection of MBP mouse IFNc Duoset ELISA, all purchased from (300 lg/mouse) and serum was collected 2 h R&D Systems, Minneapolis, MN, USA). Spleno- later. Acute cytokine responses in serum were cyte proliferation was assessed by replacing quantified using a customized MCYTOMAG- 100 lL of the medium from wells that had been 70 K | MILLIPLEX MAP Mouse Cytokine/Che- stimulated for 72 h with 100 lLof H thymidine mokine Magnetic Bead Panel—Immunology solution and further incubating for 12 h. Multiplex Assay (Millipore, Billerica, MA, USA). Incorporation of H thymidine was quantified in a scintillator. The results were expressed as Effect of ATX-MS-1467 Treatment counts per minute (cpm). on Leukocyte Populations Antigen-Specific Cytokine Release In Vivo Flow cytometric analysis was used to assess the effect of treatment with ATX-MS-1467 The experiments that were conducted to assess (100 lg/mouse, three times per week) or vehicle antigen-specific cytokine release in vivo were for 2 weeks on cell surface and intracellular performed in mice that had not been markers associated with regulatory cell Neurol Ther (2018) 7:103–128 109 function. Two different staining panels were FlowJo version 10 (FlowJo, LLC, Ashland, OR, used (Table 3). USA). The first panel focused on cell surface mark- ers and was applied in freshly isolated spleno- Statistical Analyses cytes. The second panel included intracellular markers, IL-10 and the transcription factor In experiments with multiple groups, clinical forkhead box P3 (Foxp3). To enable better score data were analyzed via daily comparison identification of the IL-10-secreting cells, the of group averages using the Kruskal–Wallis test second panel was applied in splenocytes incu- followed by Dunn’s multiple comparison test. bated for 3 h with phorbol-myristate acetate Clinical score data were also integrated as area (PMA, 5 ng/mL; Sigma, St. Louis, MO, USA), under the curve, and the results analyzed with ionomycin (500 ng/mL; Sigma, St. Louis, MO, the Kruskal–Wallis test followed by Dunn’s USA) and the protein transport-inhibitor mon- multiple comparison test. MRI, EAE imaging ensin (4 lL/6 mL of cell culture; GolgiStop, BD and clinical score data were analyzed daily by Biosciences, San Jose, CA, USA) for 3 h. The Mann–Whitney test. The mean semi-quantita- splenocytes were isolated as previously descri- tive histological scores of spinal cord sections bed. B220 and CD8 staining were used as a were input as a single data point that was used negative control for the T lymphocyte subpop- to calculate group averages, which were com- ulations of interest. Samples were measured on pared by one-way analysis of variance (ANOVA) a LSRFortessa X20 flow cytometer (BD Bio- followed by the Bonferroni test. Data on con- science, San Jose, CA, USA) and analyzed in centration-dependent cytokines and Table 3 Antibody panels used for splenocyte staining Panel 1 (cell surface markers on freshly isolated Panel 2 (cell surface and intracellular markers) splenocytes) CD49b (BV421, 1:100, cat. # 63063, BD Pharmingen, San CD49b (BV421, 1:100, cat. # 63063] BD Pharmingen, San Jose, CA, USA) Jose, CA, USA) CD3 (V500, 1:100, cat. # 560771, BD Pharmingen, San CD4 (V500, 1:100, cat. # 560782] BD Pharmingen San Jose, CA, USA) Jose, CA, USA) CD4 (FITC, 1:100, cat. # 553729, BD Pharmingen, San LAG3 (PE, 1:100, cat. # 125207, Biolegend, San Diego, CA, Jose, CA, USA) USA) LAG3 (PE, 1:100, cat. # 125207, Biolegend, San Diego, CA, IL-10 (APC, 1:100, cat. # 554468, BD Pharmingen, San USA) Jose, CA, USA) B220 (PE-Cy7, 1:200, cat. # 552772, BD Pharmingen, San CD8 (APC-Cy-7, 1:100, cat. # NC0079947, Biolegend, San Jose, CA, USA) Diego, CA, USA) CD8 (BV605, 1:100, 563152, cat. # 563152, BD CD45R/B220 (BV605 1:100, cat. # 563708, BD Pharmingen, San Jose, CA, USA) Pharmingen, San Jose, CA, USA) FC block (CD16/CD32, unstained, 1:100, cat. # 553141, FC block (CD16/CD32, unstained, 1:100, cat. # 553141, BD Pharmingen, San Jose, CA, USA) BD Pharmingen, San Jose, CA, USA) FoxP3 (PerCP-Cy5.5 1:100, cat. # 35-5773-82, eBioscience, San Diego, CA, USA) CD45R/B220 and CD8 staining were used as a negative selection for the T lymphocyte subpopulations of interest. Listed are the specificities, fluorochromes, catalogue numbers and manufacturers, and dilutions for each of the antibodies 110 Neurol Ther (2018) 7:103–128 proliferation response ex vivo were analyzed observed efficacy. The average score for the with two-way ANOVA followed by the Bonfer- vehicle-treated groups, which varied from 2 to roni test. In vivo cytokine-release data were 3, depending on the marker being analyzed, analyzed with one-way ANOVA followed by could wrongly suggest that the level of tissue Dunnett’s multiple comparison test. damage is not compatible with overt disease pathology. However, due to the unpre- dictable location where the lesions occur in this RESULTS model, the average score is the result of both highly affected spinal cord sections and other Effect of ATX-MS-1467 on EAE Clinical sections that might have remained intact. Disability Indeed, it was a common feature of all vehicle- treated mice that one or multiple areas with We treated (DR2 9 Ob1)F1 mice with different intense cellular infiltration and demyelination doses of ATX-MS-1467, starting either 7 or were observed at various, albeit not all, sections 12 dpi and observed the clinical signs of disease. examined. In agreement with the attenuation Compared with vehicle, ATX-MS-1467 dose- in clinical score, treatment with ATX-MS-1467 dependently reduced clinical disability from the from the empirically determined disease onset empirically determined disease onset (7 dpi, (7 dpi) or peak of disease (14 dpi) time points Fig. 1a, c) and peak disease (12 dpi, Fig. 1b, d) reduced spinal cord inflammation, demyelina- time points, as assessed by the mean daily score tion and cellular infiltration, unsurprisingly and cumulative scores on the subjective 0–5- with the greatest effect for all outcomes repor- point clinical score scale during the treatment ted when treatment was initiated at disease period. As can be observed in Fig. 1a, d, if onset (p \ 0.001 for all measures; Fig. 2). The treatment starts early in the course of disease, it reduction in inflammation and demyelination is possible to completely prevent the appear- seen in the CNS was also accompanied by an ance of clinical signs. Even if the treatment is improvement in the clinical score, for which initiated when mice exhibit full-blown disease the greatest improvement was observed follow- (tail–hand–hind limb paralysis, matching an ing early (7 dpi) treatment (Fig. 2a). Treatment average score of 3), partial, albeit significant, with ATX-MS-1467 from the peak of disease recovery from the natural course of EAE was (14 dpi) partially reduced the histological seen in this mouse strain (Fig. 1b, d). The opti- markers indicative of inflammation (H&E score; mum dose (100 lg) significantly improved the Fig. 2b), demyelination (LFB score; Fig. 2c) and clinical score in mice showing advanced signs of T-cell infiltration (CD3 score; Fig. 2d; p \ 0.05) neurological disability, and this dose was selec- and histology scores indicative of B-cell infil- ted for subsequent experiments. tration (p \ 0.01; Fig. 2e). MRI was applied as a translational approach to investigate the efficacy of ATX-MS-1467 in Effect of ATX-MS-1467 on CNS Infiltration mice. Gd administered intravenously during the and Inflammation acute phase of disease allows the detection of leakage, which can be seen as bright areas of the We performed two independent experiments, spinal cord in T1-weighted scans, indicating with treatment started at either 7 or 14 dpi. This disruption of the BBB. Because BBB disruption allowed us to generate a large tissue bank to tends to be more active during the beginning of assess the effects of treatment with ATX-MS- the acute phase of disease, and tends to natu- 1467: spinal cord samples were obtained from rally subside at later phases, we opted for a 20–28 mice from each treatment group, with shorter window of treatment, in order to better each spinal cord being stained and analyzed at appreciate the drug effect. In a longitudinal MRI ten different levels for each staining. In this experiment (Fig. 3a), therapeutic dosing with experiment, we used histological evaluation ATX-MS-1467 (100 lg subcutaneous) every with subjective scoring (Table 2) to confirm the other day (three doses) from 10 dpi significantly Neurol Ther (2018) 7:103–128 111 Vehicle ATX-MS-1467 (3 μg) a ATX-MS-1467 (10 μg) b Vehicle ATX-MS-1467 (30 μg) ATX-MS-1467 (30 μg) ATX-MS-1467 (100 μg) ATX-MS-1467 (100 μg) 4 4 3 3 2 2 ** ** *** ** *** 1 1 ** ** ** *** ** 0 0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 22 Days post-immunization Days post-immunization c d 30 30 20 20 ** 10 10 **** 0 0 Vehicle 3 10 30 100 Vehicle 30 100 ATX-MS-1467 dose (μg) ATX-MS-1467 dose (μg) Fig. 1 Effect of dose response of ATX-MS-1467 from for the experiment shown B and D, the data represent a disease onset or peak of clinical disability in a mouse model pool from two independent experiments with a total of of EAE. EAE was induced at 0 dpi with spinal cord n = 21–22 mice. *, **, *** and **** indicate p \ 0.05, homogenate in (DR2 9 Ob1)F1 mice, and treatment was \ 0.01, \ 0.001 and \ 0.0001, respectively, by initiated with ATX-MS-1467 at 7 dpi (a, c) or at 12 dpi Kruskal–Wallis test followed by Dunn’s test versus vehicle (b, d) at doses ranging 3–100 lg, as indicated, subcuta- (PBS) group. dpi days post-injection, EAE experimental neously three times per week. The grey area in graphs a, autoimmune encephalomyelitis, PBS phosphate-buffered b indicates the treatment period. For the experiment saline, SEM standard error of the mean shown in a, c, the group sizes were n = 10–14 mice, and (p \ 0.05 on 13 and 15 dpi and p \ 0.01 on 14 (Fig. 3e) compared with baseline but not in and 16 dpi) reversed disruption to the BBB, as vehicle-treated mice [Fig. 3e; ATX-MS-1467- measured by Gd leakage, compared with vehi- treated mice: 77.4% reduction (Gd-leakage vol- cle-treated animals (Fig. 3c). In group analyses ume 1.1 ± 0.3 mm ) from baseline, p \ 0.01; at follow-up, Gd-leakage volume was signifi- vehicle-treated mice: 39.7% reduction (Gd- cantly reduced in ATX-MS-1467-treated mice leakage volume 4.45 ± 0.3 mm ) from baseline, Cumulative score Clinical score (mean ± SEM) (mean ± SEM) Cumulative score Clinical score (mean ± SEM) (mean ± SEM) 112 Neurol Ther (2018) 7:103–128 a Vehicle b Vehicle ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 14 ATX-MS-1467 from dpi 14 2.5 2.0 1.5 1.0 ### 0.5 ** ** *** * * Naїve Vehicle *** 0 2 4 6 8 1012141618 20 22 24 26 28 3032 ATX from dpi7 ATX from dpi14 Days post-immunization c d Vehicle Vehicle ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 14 ATX-MS-1467 from dpi 14 4 3 ### ### 0 0 Naїve Vehicle Naїve Vehicle ATX from dpi7 ATX from dpi14 ATX from dpi7 ATX from dpi14 Vehicle ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 14 2.5 2.0 ## 1.5 1.0 ### 0.5 0.0 Naїve Vehicle ATX from dpi7 ATX from dpi14 Cd45R score LFB score Clinical score (mean ± SEM) (mean ± SEM) (mean ± SEM) CD3 score H&E score (mean ± SEM) (mean ± SEM) Neurol Ther (2018) 7:103–128 113 bFig. 2 Effect of therapeutic treatment with ATX-MS- release of IL-10, which is indicative of a tolero- 1467 on spinal cord tissue damage in spinal cord genic state, would be expected. In vivo and homogenate-induced EAE in (DR2 9 Ob1)F1 mice. in vitro strategies were employed to demon- a The effect of 100-lg ATX-MS-1467 treatment starting strate whether these changes occurred in mice at 7 or 14 dpi on daily clinical scores and on terminal (day treated with ATX-MS-1467. For the in vitro 30) histological scores. The arrows indicate the beginning analysis, splenocytes were recovered from ani- of the treatment period in each group. *, ** and *** indicate mals treated four times between 0 to 7 dpi with p \ 0.05, \ 0.01 and \ 0.001, respectively, by ATX-MS-1467 (on days 0, 2, 4 and 7); the last Kruskal–Wallis test followed by Dunn’s test versus vehicle. administration took place 1 h prior to take H&E staining was applied to assess inflammation (b), luxol down. The spleens were processed, as described fast blue staining was applied to assess demyelination (c), in the methods section, and the cytokine con- and immunohistochemical staining for CD3 or CD45R centrations in the supernatant were assayed was applied to assess T-cell (d) and B-cell infiltration (e). (Fig. 4a–d). The cultures were stimulated in the The inserts show representative images for each group with presence of a range of concentrations of each the respective staining for which the semi-quantitative # ## ### antigen, to allow the assessment of the response evaluation is plotted. , and indicate p \ 0.05, over a broad range of concentrations. \ 0.01 and \ 0.001, respectively, by ANOVA followed It is important to differentiate the purpose of by Bonferroni versus mice treated with vehicle (PBS). The ATX-MS-1467 as a treatment (Fig. 4a–e filled data represent a pool from 2 independent experiments squares), when it was administered in vivo with with a total of 20–28 mice. Spinal cords from naı¨ve mice a vehicle-administered group as a control were also processed, stained and analyzed along with the (Fig. 4a–e filled circles), from its use here as an samples from the immunized/treated mice. The histolog- ical (as well as clinical) scores for naı¨ve mice was zero for antigen-specific stimulus for cellular prolifera- all cases; therefore, for the sake of simplicity, data from tion and cytokine release (x axis, various con- naı¨ve mice was not included in the graphs. dpi days post- centrations). The in vitro antigen recall assay induction, EAE experimental autoimmune encephalitis, showed that the in vivo treatment with ATX- H&E hematoxylin & eosin, LFB luxol fast blue, PBS MS-1467 caused a dose-dependent inhibition in phosphate-buffered saline, SEM standard error of the pro-inflammatory cytokine (IL-17 and IFNc) mean and IL-2 secretion and splenocyte proliferation. IL-10 secretion was significantly increased only after stimulation with concentrations of ATX- MS-1467 of 3 lg/mL (p \ 0.05) and above p = 0.23]. Figure 3b shows representative T1- (p \ 0.001) in the ex vivo antigen recall assay weighed MRI scans of the spinal cords of the (Fig. 4). mice in this study. Effect of ATX-MS-1467 Treatment Effect of ATX-MS-1467 Treatment on the In Vivo Cytokine Profile on the Anti-Inflammatory Milieu in Ex Vivo Splenocytes The effect of ATX-MS-1467 persists when the drug is cleared from the circulation. To extend ATX-MS-1467 binds to surface-expressed MHC- the results for ex vivo cytokine secretion, we II on antigen-presenting cells. Following bind- wanted to assess whether a favorable shift in ing, ATX-MS-1467 is hypothesized to drive cytokine balance also occurred in vivo. To either the differentiation of MBP-specific regu- achieve this, we performed three types of latory cells from naıve cells or to induce the experiments. conversion of effector into type 1 regulatory The first type (Fig. 5) aimed to define the cells (Tr1). In both cases, inhibition of the optimal dose of MBP needed to elicit robust release of the pro-inflammatory cytokines IL-17, serum cytokine changes. In the naı¨ve wild-type and IFNc from Th1 and Th17 T-cell subsets, mouse strain, MBP injection would not nor- which are known to promote the inflammatory mally induce cytokine release at levels that can processes in MS, along with an increase in the 114 Neurol Ther (2018) 7:103–128 PBS ATX-MS-1467 3.5 PBS ATX-MS-1467 3.0 2.5 Baseline Baseline 2.0 ** 1.5 ** 1.0 Follow-up Follow-up 0.5 Treatment 0 2 4 6 8 10 12 14 16 18 Days post-induction PBS cd 10 18 PBS ATX-MS-1467 0 0 Baseline MRI Follow-up MRI Baseline MRI Follow-up ATX-MS-1467 Baseline MRI Follow-up Fig. 3 Effect of ATX-MS-1467 on EAE-induced blood– leakage. Levels of Gd leakage volume at dosing onset and brain barrier leakage. The effect on clinical score of ATX- follow-up (c). Leakage volume for the individual mice with MS-1467 (n = 7) or vehicle (PBS; n = 7) given at 10 or vehicle (d) or ATX-MS-1467 treatment (e) were com- 11 dpi, depending on the timing of MRI; the grey area pared using a paired t test. *p \ 0.05, **p \ 0.01. dpi days shows the treatment period (a). Four serial images at post-induction, EAE experimental autoimmune baseline and follow-up for two animals (left: vehicle- encephalomyelitis, Gd gadolinium, MRI magnetic reso- treated mice; right: ATX-MS-1467-treated mice; b). The nance imaging, PBS phosphate-buffered saline, SEM yellow arrows highlight the areas with changes in Gd standard error of the mean Clinical score 2 2 Leakage volume (mm ) Leakage volume (mm ) (mean ± SEM) Leakage volume (mm ) Neurol Ther (2018) 7:103–128 115 be detected in serum. However (DR2 9 Ob1)F1 shows how a system is responding to the chal- mice were genetically engineered to constitu- lenge [22]. If treatment with ATX-MS-1467 does tionally express both MHC and TCR recogniz- indeed induce tolerance, the persistence of the ing one MBP epitope; therefore, circulating tolerogenic effect, as deduced from changes in effector and regulatory cells that respond to the ratios of IL-10-to-pro-inflammatory cytoki- MBP are already present in (DR2 9 Ob1)F1 nes, could be assessed by the changes in these mice, even when they have not been immu- ratios. From the second dose to the fifth dose, nized against this protein. Untreated (DR2 9 the concentration of IL-10 relative to IL-17 and Ob1)F1 mice responded to an acute challenge IFNc, respectively, was higher than in vehicle- with MBP with a dose-dependent increase in treated animals. However, the concentration of pro-inflammatory cytokines IL-17 (Fig. 5c) and IL-10 relative to IL-2 was lower for vehicle- IFNc (Fig. 5d) and also IL-2 (Fig. 5a) compared treated mice after one dose, and was not dif- with vehicle-treated mice. Although the con- ferent from vehicle-treated animals from the centration of IL-10 was also increased in second dose onwards (Fig. 6). response to low doses of MBP, this effect pla- Finally, a third type of experiment was done teaued at MBP concentrations of 100–300 lg to assess how mice reacted to the native antigen and then declined as the concentration of MBP (MBP) after they received a course of 10 injec- increased (Fig. 5b). tions with 300 lg of ATX-MS-1467, which was The second type of experiment on in vivo previously determined as the highest dose to cytokine release (Fig. 6) assessed how cytokine elicit the maximum response (Fig. 7). Because release shifted after repeat treatments. The all previous evidence indicates that ATX-MS- insert summarizes how the injections were 1467 treatment is associated with induction of performed in each group (Fig. 6a). A single dose Tregs, we hypothesized that the effect of the of ATX-MS-1467 elicited an increase in the treatment is long-lasting; therefore, the effects concentrations of all cytokines in serum should persist for at least as long as this pool of (Fig. 6b–e), and the concentrations returned to regulatory cells remained expanded, counter- baseline values within a few hours after reach- acting the natural emergence of Th1 or Th17 ing their peak at 2 h after the injection (results effector cells. To test this hypothesis, we chal- not shown). Similar to explanation given above lenged a group of mice with MBP following a for MBP-induced cytokine release, naı¨ve mice wash-out period of 2 days (equivalent to the 39/ with the transgenic mouse construct also weekly regimen treatment with ATX-MS-1467), respond to ATX-MS-1467 with quantifiable a group with wash-out periods of either 7, 21 or cytokine levels in serum without the need of 42 days from the last ATX-MS-1467 treatment, concomitant exposure with adjuvant. Further and also mice in which HLAbp was used as a doses of ATX-MS-1467 did change the serum IL- control for ATX-MS-1467. To help interpret this 2 (Fig. 6b), IL-17 (Fig. 6d) or IFNc (Fig. 6e) study (as depicted in Fig. 7), we compared the cytokine concentrations in comparison with cytokine levels (or ratios) between the third bar, vehicle for up to ten consecutive doses of ATX- i.e., mice treated with control peptide (HLA-bp) MS-1467. However, up to five consecutive doses then challenged with MBP, which could also be of ATX-MS-1467 were still capable of inducing regarded as ‘‘untolerized’’, against those levels in statistically significant drug-induced IL-10 the four last columns representing mice treated release in serum (Fig. 6d). The use of ratios of chronically with ATX-MS-1467 prior to MBP signature cytokines for the predicted response is challenge (performed from 2, 7, 21 or 43 days an accurate index of how an organism responds after the last treatment). When naıve mice were to antigens or disease: how the ratios change challenged with MBP (Fig. 7a–d; second column over time or how they compare between dif- in each plot) there was always a large induction ferent groups under controlled conditions of serum cytokines. The same was true for the 116 Neurol Ther (2018) 7:103–128 IL-2 IL-10 a b 300 140 Vehicle Vehicle *** ATX-MS-1467 ATX-MS-1467 *** *** 50 *** *** ** 0 0 0.03 0.1 0.3 1 3 10 30 0.03 0.1 0.3 1 3 10 30 ATX-MS-1467 in vitro (μg/mL) ATX-MS-1467 in vitro (μg/mL) IL-17 IFN-γ c d 600 1500 Vehicle Vehicle ATX-MS-1467 ATX-MS-1467 *** 100 *** *** 0 0 0.03 0.1 0.3 1 3 10 30 0.03 0.1 0.3 1 3 10 30 ATX-MS-1467 in vitro (μg/mL) ATX-MS-1467 in vitro (μg/mL) e Thymidine incorporation Vehicle ATX-MS-1467 30000 * 0.03 0.1 0.3 1 3 10 30 ATX-MS-1467 in vitro (μg/mL) Radioactivity (cpm) Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Neurol Ther (2018) 7:103–128 117 bFig. 4 Effect of preventive treatment with ATX-MS-1467 out of 7 days. However, the effects on the IL-10- on ex vivo splenocyte proinflammatory cytokine release to-IFNc ratio are more persistent; this ratio did and proliferation. (DR2 9 Ob1)F1 mice were immunized not return to baseline values until a wash-out with spinal cord homogenate and treated with 100 lg period of 42 days. This result indicates that, at ATX-MS-1467 or vehicle (PBS) on days 0, 2, 5 and 7. least with regard to this cytokine pair ratio, the Splenocytes were harvested at 7 dpi and stimulated in vitro effect of ATX-MS-1467 is long-lasting. On the with ATX-MS-1467 at 0.03, 0.1, 0.3, 1, 3, 10 and 30 lg/ other hand, the effect on the IL-10-to-IL-17 mL; cytokine concentrations were measured in the ratio was less persistent. supernatants (a–d). Splenocyte proliferation was also measured by H thymidine uptake (cpm) following Effect of ATX-MS-1467 Treatment in vitro stimulation with ATX-MS-1467 (e). *, ** and on Regulatory T Cell Populations *** indicate p \ 0.05,\ 0.01 and\ 0.001, respectively, by two-way ANOVA followed by Bonferroni’s post-tests The mechanism of tolerance induction can be (n = 5–6). ANOVA analysis of variance, cpm counts per minute, dpi days post-induction, IFN interferon, IL investigated by quantification of the intracel- interleukin, PBS phosphate-buffered saline lular and/or extracellular markers unique to each of these Treg subsets (i.e., state-specific markers and subsets). We relied on two different approaches to define the induced regulatory T mice that received ten doses of HLA-bp cells (iTreg) subset: one was the co-expression of (Fig. 7a–d; third column in each plot). However, LAG3 and CD49 in CD4 lymphocytes; the for all cytokines tested, the serum cytokine other was the presence or absence of FOXP3 in concentrations were always lower for the groups IL-10 secreting CD4 lymphocytes. In the second of mice that received a course of ATX-MS-1467 ? ? approach, we assumed that the IL-10 FOXP3 than those mice that received HLA-bp treat- CD4 lymphocytes are natural T regulatory cells ment prior to the challenge, regardless of the ? - (nTregs), and the remaining IL-10 FOXP3 are, duration of the wash-out period. Furthermore, by exclusion, iTregs. Staining was performed in the effector cytokines tended towards a slight splenocytes of mice that had been treated increase after longer wash-out periods, whereas in vivo with ATX-MS-1467 or HLA-bp for the opposite occurred for IL-10. This suggests a 2 weeks. Figure 8a, b shows the representative down-regulation of the pool of IL-10-secreting gating leading to assessment of the frequency of cells and a re-emergence of the pool of effector ? ? ? LAG3 CD49b CD4 lymphocytes from an cytokine-secreting cells with time. From this HLA-bp-treated mouse and an ATX-MS-1467- observation, one question arises: at which point treated mouse, respectively. Figure 8c shows does the balance of regulatory/effector cytoki- mean data for both groups. The results indicate nes return to that seen in the absence of toler- that chronic treatment with ATX-MS-1467 ization (equivalent to the HLB-bp group, third expanded the iTreg compartment in the spleen column in each plot)? This can be assessed by compared with mice treated with HLAbp. The replotting the data as cytokine ratios (Fig. 7f–h). spleens of mice treated with ATX-MS-1467 had The results vary depending on the analyzed ? ? a higher proportion of LAG3 C49b cells com- cytokine ratio. For the IL-10-to-IL-2 ratio, pared with HLAbp-treated mice (p \ 0.05). Fig- tolerization with ATX-MS-1467 can still induce ure 8d–g shows the gating and the means for higher ratios at wash-out periods of up to the upper-left and upper-right quadrants, 7 days, but the ratios return to untolerized ? - ? ? denoting IL-10 FOXP3 and IL-10 FOXP3 levels (HLA-bp treated) after a wash-out of lymphocytes, respectively, for each group. In 21 days (Fig. 7f). For the IL-10-to-IL-17 ratio, the conclusion, in vivo ATX-MS-1467 treatment return to baseline values occurs very quickly ? - increased the pool of IL-10 Foxp3 (iTregs), but (Fig. 7g); the ratio has normalized after a wash- ? ? it had no effect on IL-10 Foxp3 (nTregs). 118 Neurol Ther (2018) 7:103–128 IL-2 IL-10 a b 2500 100 *** *** *** 2000 80 ** ** 1500 60 1000 40 ** 500 20 0 0 Vehicle 30 100 300 1000 Vehicle 30 100 300 1000 MBP dose (μg) MBP dose (μg) IL-17 IFN-γ c d 400 150 0 0 Vehicle 30 100 300 1000 Vehicle 30 100 300 1000 MBP dose (μg) MBP dose (μg) Fig. 5 Response of (DR2 9 Ob1)F1 mice to an acute *, ** and *** indicate p \ 0.05, \ 0.01 and \ 0.001, challenge with MBP. Dose-dependent secretion of cytoki- respectively, versus the vehicle (PBS)-treated group nes in the serum of (DR2 9 Ob1)F1 mice 2 h after (n = 4–5). ANOVA analysis of variance, IFN interferon, subcutaneous injection of MBP (a–d). Data were analyzed IL interleukin, MBP myelin basic protein, PBS phosphate- by ANOVA followed by Dunnett’s multi-comparison test. buffered saline or immediately after (dpi 8) the appearance of DISCUSSION the first signs of disease [14]. To study this effect in more detail, mice were treated with four Progressive axonal loss secondary to immune doses of ATX-MS-1467 at the onset and the peak cell infiltration into the CNS, inflammation- of disease. As shown here, therapeutic treat- mediated damage and demyelination, and ment with ATX-MS-1467, both early and late diminishing repair are widely considered to be after EAE induction in (DR2 9 Ob1)F1 mice, the pathological correlates of clinical disability reduced inflammation and T-cell and B-cell in MS [23]. In the preclinical study published by infiltration in the spinal cord, reduced Streeter et al., treatment with ATX-MS-1467 demyelination and reduced BBB disruption. prevented the increase of neurological disability These effects were more profound in the ani- when the drug was administered prior to (dpi 4) mals treated early in the course of EAE than in Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Neurol Ther (2018) 7:103–128 119 those treated later, thus demonstrating the an anti-inflammatory cytokine profile was seen neuroprotective benefit of early intervention in treated mice in response to repeated doses of with ATX-MS-1467 in this animal model of MS. ATX-MS-1467 but not in mice treated with Although the exact association between the vehicle or in response to HLAbp, which binds to severity of disability and the extent of damage the MHC but contains none of the epitopes in the CNS is not fully understood [24], these involved in the autoimmune response to MBP. results show good agreement between the Consideration of ATX-MS-1467 as a tolero- abatement of inflammatory markers and genic approach presupposes that a rebalance of demyelination and improvement in clinical antigen-specific immunity occurs sometime score, particularly when treatment is started after the initial treatment. It also presupposes soon after the onset of EAE. that because the immune system is, at least Autoantigen T-cell responses to MBP are temporally, reprogramed against a particular involved in the pathogenesis of MS [25–28]. antigen, the effect of ATX-MS-1467 persists T-cell responses to MBP were initially identified when the drug is cleared from the circulation. in eight regions of MBP, of which five (MBP The induction of tolerance in type-1 helper 30–44, MBP 83–99, MBP 130–144, MBP 140–154 (Th1) cells after repeated peptide immunization and MBP 156–170) are the regions most fre- of MBP-specific-TCR transgenic mice led to a quently recognized by T cells. ATX-MS-1467 is a reduction in the capacity of these cells to pro- mixture of four of these peptides (MBP 30–44 liferate and a shift to an IL-10-secreting phe- [ATX-MS1]; MBP 131–145 [ATX-MS4]; 140–154 notype [7]. IL-10 suppresses dendritic cell [ATX-MS6]; MBP 83–99 [ATX-MS7]), which act maturation and prevents Th1-cell differentia- as apitopes and suppress EAE in a humanized tion, which is proposed as the negative arm of a mouse model [14]. feedback loop to limit Th1-driven Direct targeting of key regulators in the immunopathology [11]. As shown here, with development and maintenance of autoreactiv- repeated ATX-MS-1467 treatment, the concen- ity aims to silence or reprogram autoreactive T trations of the pro-inflammatory cytokines IL- cells in the periphery back to a regulatory phe- 17 and IFNc diminished relative to the con- notype, and thus promote a tolerogenic state to centration of IL-10, but the relation between IL- the targeted protein. Such targeted treatment 2 and IL-10 was less clear. IL-2 is secreted by may help to avoid many of the bystander effects effector cells but not by regulatory cells, seen with broad suppression of the immune whereas it is removed, via receptor-mediated system. An ex vivo antigen recall assay and internalization, by both subtypes [29]. IL-2 is a in vivo quantification of cytokine concentra- broad regulator of Th-cell differentiation, nota- tions in response to an acute challenge with bly in the differentiation of Th cells into both MBP and also in response to chronic dosing effector and cytolytic T cells, and is vital for the with ATX-MS-1467 were used to investigate the normal development of iTregs [29]. Therefore, effect of ATX-MS-1467 on the immune envi- IL-2 may have a gateway role in the early ronment. ATX-MS-1467 elicited a dose-depen- development of tolerance that diminishes with dent switch to an IL-10-dominant environment the development of a tolerogenic state follow- after chronic treatment. Following repeated ing repeated exposure. Such a role would be exposure to ATX-MS-1467, there was a positive consistent with the results reported here, as well and robust shift in the ratio of anti-inflamma- as with other reports in which single peptide tory to pro-inflammatory cytokines, which exposure led to an initial variable peripheral persisted for as long as the mice were exposed to T-cell death that was transient and incomplete. ATX-MS-1467. Some aspects of the effect Taken in isolation, the reduction in ex vivo induced by ATX-MS-1467 remained, at least secretion of IL-2 from splenocytes of mice trea- partially, beyond 21 days, such as the shift in IL- ted in vivo with ATX-MS-1467 does not allow us 10/IFNc ratio; however, this effect was not as to conclude whether the treatment led to an persistent as the effect on the ratio of other increase in MBP-specific Tregs, a reduction in cytokine pairs (e.g., IL-10-to-IL-17). The shift to MBP-specific effector Tregs or both, because, in 120 Neurol Ther (2018) 7:103–128 Week -3 Week -2 Week -1 Week 0 Legend: Vehicle vehicle ATX-MS-1467 (100μg three times/week) 1x 2x 2h 3x Cytokine 4x quantification 5x 7x 10x b c IL-2 IL-10 600 120 0 0 Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Number of treatments with ATX-MS-1467 Number of treatments with ATX-MS-1467 d e IL-17 IFN-γ 300 100 0 0 Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Number of treatments with ATX-MS-1467 Number of treatments with ATX-MS-1467 fg IL-10/IL-2 ratio IL-10/IL-17 ratioh IL-10/IFN-γ ratio 2.5 2.5 5 2.0 2.0 4 1.5 1.5 3 1.0 1.0 2 0.5 0.5 1 0.0 0.0 0 Number of treatments with Number of treatments with Number of treatments with ATX-MS-1467 ATX-MS-1467 ATX-MS-1467 **** **** *** ** **** **** *** ** ** *** ** *** Concentration (pg/mL) Concentration (pg/mL) Ratio Ratio Concentration (pg/mL) Concentration (pg/mL) Ratio Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Neurol Ther (2018) 7:103–128 121 bFig. 6 Effect of chronic dosing with ATX-MS-1467 on tolerogenic state and the recruitment of resting in vivo proinflammatory cytokine secretion. (DR2 9 Th cells from more than one compartment. This Ob1)F1 mice received from 1 to 10 treatments with is also in keeping with the more consistent 100 lg of ATX-MS-1467 or vehicle (PBS) on days 0, 2, 5, response seen following repeated exposure to 7, 9, 12, 14, 16, 18 and 20. The top panel shows the overall peptides, as compared with the response to dosing schedule (a). Serum cytokine concentrations were single exposure, which was transient, incom- assayed 2 h after the last injection. The bottom panel plete and resulted in only variable peripheral shows the absolute concentrations of each cytokine in T-cell apoptosis [11]. serum (b–e) used to generate ratios of interleukin 10 Analysis of the phenotype of the IL-10-se- versus the other cytokines, shown in the insert (f–h). Data creting cells showed that the splenic cell com- were analyzed by ANOVA followed by Dunnett’s multi- partment contained iTregs that were derived comparison test. *, **, *** and **** indicate p \ 0.05, from peripheral compartments. iTregs that co- \ 0.01, \ 0.001 and \ 0.0001, respectively, versus the ? ? express the LAG3 CD49 phenotype represent vehicle (PBS)-treated group. ANOVA analysis of variance, a population of cells that migrate to the bone IFN interferon, IL interleukin, PBS phosphate-buffered marrow following the effector stage of an saline 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 each case, the observed result (i.e., reduced IL-2 treatment also led to greater secretion of IL-10 concentration in culture supernatant) was to be from iTregs that lacked the transcription factor ? – ? expected. Nevertheless, any of these treatment Foxp3 (IL-10 Foxp3 ) but not from IL-10 Fox- effects would have a beneficial therapeutic p3 nTregs. This indicated that the main source impact. By comparison, multiple doses of Ac of IL-10 is from iTregs that originated in the 1–9 of MBP completely protected TCR-trans- periphery during the effector phase of the initial genic mice inoculated with myelin, leading to immune response to ATX-MS-1467, as opposed down-regulation of the capacity of CD4 T cells to nTregs of thymic origin [30]. The presence of to proliferate or to produce IL-2, IFNc and IL-4, ? ? ? – iTregs with LAG3 CD49b and IL-10 Foxp3 but increased the production of IL-10. This phenotypes in the spleens of ATX-MS-1467- effect was completely reversed by neutralization treated mice might indicate an overlapping cell of IL-10 [7]. population that express IL-10 transiently and at Treg cells are a crucial subpopulation of T low concentrations during differentiation [31]. cells that suppress the activity of several The results presented here raise some inter- immune-cell lineages and are thought to have esting questions for further investigation in diverse and overlapping roles in the suppression preclinical studies and clinical trials in humans of foreign-stimulated and autoantigen-stimu- that were outside the scope of this manuscript. lated immune reactions. Pharmacological Future investigators should look at performing induction of tolerance may, theoretically, occur the ex vivo assay in co-cultures of purified by expansion of the nTreg compartment, by dendritic cells and T cells, instead of whole- way of differentiation, expansion of induced spleen cultures. Furthermore, ex vivo data could Tregs (iTregs, also called type 1 regulatory T also be obtained only from leukocytes that cells), or by both mechanisms. Chronic treat- infiltrated the CNS, which is more relevant for ment with ATX-MS-1467 preferentially induced diseases such as EAE, and a wider complement differentiation of naıve T cells into iTregs and of cytokines (such as IL-6 and transforming there is also evidence of re-education of the growth factor beta) could be measured. The immune system, with sparing of normal cellular regulatory effect of IL-10 could be further con- immune surveillance. These findings are con- firmed in the EAE mouse model by the use of IL- sistent with the development of a devolved 10-neutralizing antibodies or by adoptive- 122 Neurol Ther (2018) 7:103–128 Legend: Vehicle HLAbp (100 μg, 3x/weekly) ATX-MS-1467 (100μg, 3x/weekly) MBP (300μg) Chronic Wash out Challenge Week -9 Week -8 Week -7 Week -6 Week -5 Week -4 Week -3 Week -2 Week -1 Week 0 treatment period (days) None n.a. PBS None n.a. MBP Variable wash-out between 2-42 days HLAbp Variable MBP 2h Cytokine ATX-MS-1467 2 MBP quantification 7 days ATX-MS-1467 7 MBP 21 days ATX-MS-1467 21 MBP 42 days ATX-MS-1467 42 MBP b IL-2 c IL-10 4000 160 **** 3000 **** ** *** * ## ## ## ## ## ## ### 0 0 Chronic treatment none none H LAbp ATX-MS-1467 Chronic treatment none none H LAbp ATX-MS-1467 † † Wash-out (days) none none 2-42 2 7 21 42 Wash-out (days) none none 2-42 2 7 21 42 Challenge PBS MBP Challenge PBS MBP d IL-17 e IFN-γ 500 500 ** ** 400 400 300 300 200 200 100 100 # # # # 0 0 Chronic treatment none none H LAbp ATX-MS-1467 Chronic treatment none none H LAbp ATX-MS-1467 † † Wash-out (days) none none 2-42 2 7 21 42 Wash-out (days) none none 2-42 2 7 21 42 Challenge PBS MBP Challenge PBS MBP f IL-10/IL-2 ratio g IL-10/IL-17 ratio h IL-10/IFN-γ ratio 1.4 8 8 #### #### **** 1.2 #### 6 6 **** 1.0 0.8 #### 4 4 ## 0.6 *** 0.4 2 2 *** *** 0.2 *** *** *** 0.0 0 0 Chronic none none H LAbp ATX-MS-1467 Chronic none none H LAbp ATX-MS-1467 Chronic none none H LAbp ATX-MS-1467 treatment treatment treatment Wash-out none none 2-42 2 7 21 42 Wash-out none none 2-42 2 7 21 42 Wash-out none none 2-42 2 7 21 42 † † † (days) (days) (days) Challenge PBS MBP Challenge PBS MBP Challenge PBS MBP Ratio Concentration (pg/mL) Concentration (pg/mL) Ratio Concentration (pg/mL) Concentration (pg/mL) Ratio Neurol Ther (2018) 7:103–128 123 bFig. 7 Effect of chronic treatment with ATX-MS-1467 ATX-MS-1467 has so far been restricted to ani- on the ratio of interleukin 10-to-proinflammatory cytoki- mals designed to model the MS-associated HLA- nes after an acute challenge with myelin basic protein. The DR2b allele; therefore, the generalizability of length of the wash-out period is indicated by the arrows in the treatment for patients who do not have the schematic representation of the study design (a). The genetic susceptibility and the efficacy in absolute serum cytokine concentrations (b–e) were used to patients with different forms of MS will need to generate ratios of interleukin 10 versus the other cytokines be established. shown in the insert (f–h). Data were analyzed by ANOVA Furthermore, in the absence of a model that followed by Dunnett’s multicomparison test. *, **, *** and responds to all the peptides in the ATX-MS- **** indicates p \ 0.05, \ 0.01, \ 0.001 and \ 0.0001, 1467 sequence, the model used in this study # ## ### respectively, versus naı¨ve mice (first column). , , and was constructed around only a small region of #### indicate p \ 0.05, \ 0.01, \ 0.001 and \ 0.0001, MBP: MS7 overlapping the MBP amino acids respectively, versus mice that were challenged with MBP 81–102 [14]. This means that the efficacy of the without any prior tolerization (HLAbp column only), drug in this model is restricted to this region, n = 6–8. After 10 doses of ATX-MS-1467. ANOVA rather than the whole mix of peptides in ATX- analysis of variance, HLAbp human leukocyte antigen- MS-1467. It would be interesting to fully assess binding peptide, IFN interferon, IL interleukin, MBP the efficacy of ATX-MS-1467 in a non-trans- myelin basic protein, na not applicable, PBS phosphate- genic model, in which the MBP sequences in buffered saline the drug are part of the encephalitic T-cell repertoire of the animal. We have already reported preliminary findings that ATX-MS- transfer experiments using purified iTregs, to 1467, given before the onset of EAE, delayed gain further validation of the regulatory the onset (p \ 0.04) and severity (p \ 0.002) in potential of the iTregs. non-transgenic Lewis rats [32]. A phase 1 first- Although these current results show that in-human study in six patients with secondary ATX-MS-1467 can induce tolerance in (DR2 9 progressive MS showed that ATX-MS-1467 was Ob1)F1 mice in which EAE has been induced, safe and well tolerated [14]. This study also this effect diminished with a 7-day wash-out showed a trend towards a transient induction period in the case of IL-10/IL-17. However, the of immunotolerance 1 month after chronic effect on the IL-10-to-IFNc ratio was more per- dosing with ATX-MS-1467. A phase 2 trial of sistent. This observation is consistent with the ATX-MS-1467 in patients with relapsing/ inhibition of antigen-specific IFNc release, but remitting MS to evaluate the clinical, biologi- not of IL-17 release, in vitro from the spleno- cal and radiological effects and the safety pro- cytes of mice treated with ATX-MS-1467. These file of ATX-MS-1467 for up to 36 weeks has also data suggest that, in this model, ATX-MS-1467 been completed [33]. is a more robust treatment to curb Th1-type responses than Th17, although this falling off may also be indicative of improved efficacy and CONCLUSIONS a reduction in the need for high levels of cytokines over time. This finding could have a ATX-MS-1467 improved clinical disability and substantial effect on the regularity of dosing histological markers of inflammation and required in patients to achieve and maintain a demyelination. It also restored the integrity of state of tolerance to MBP. Studies in patients the BBB following induction of EAE in the with MS will also determine whether the humanized (DR2 9 Ob1)F1 mouse. The greatest improvements in disability can be reproduced effectiveness was seen when treatment was and whether the magnitude of the treatment given at the onset of EAE, but the effect is still effect will be sufficient to make this viable for significant and reproducible with dosing at the the treatment of patients beyond the early peak of disease. Chronic treatment with ATX- stages of the disease. Experimental testing of MS-1467 led to the development of tolerance to 124 Neurol Ther (2018) 7:103–128 a c HLAbp ATX-MS-1467 1.2 1.0 0.8 0.6 0.4 0.2 0.0 de f g 5 3 HLAbp HLAbp ** ATX-MS-1467 ATX-MS-1467 0 0 + - % IL-10 from Foxp3 CD4 lymphocytes + + % IL-10 from Foxp3 CD4 lymphocytes + + + % LAG3 CD49b from CD4 lymphocytes Neurol Ther (2018) 7:103–128 125 bFig. 8 Effect of chronic treatment with ATX-MS-1467 involved in previous project management activ- on the splenic iTreg population of (DR2 9 Ob1)F1 mice. ities and model validation relevant to this work. For a–c, the spleens were harvested and fresh cells stained as described in the methods section. a, b are representative Funding. This study was funded by EMD ? ? plots showing the populations of LAG3 CD49b cells Serono, Inc., a business of Merck KGaA, Darm- ? - - ? (CD3 CD8 B220 ) from total CD4 lymphocytes, for stadt, Germany. At the time of the study, most which the averages are shown in c. For d–g, the harvested of the authors were employees of EMD Serono, splenocytes were stimulated in vitro for 3 h in the presence Inc.; therefore, the sponsor was involved in the of PMA, ionomycin and monensin, to allow intracellular study design, the collection, analysis and accumulation and subsequent staining of IL-10. The - ? interpretation of the data and the decision to contributions of Foxp3 and Foxp3 cells to the total IL ? submit the study for publication. All authors 10-producing CD4 lymphocytes is shown in d and e, had full access to all of the data in this study which are representative dot plots of the groups of mice and take complete responsibility for the integ- treated with HLAbp (n = 8) or ATX-MS-1467 (n = 8), rity of the data and accuracy of the data respectively. The averaged data are shown in f and g. * and analysis. ** indicate p \ 0.05 and\ 0.01, respectively, by unpaired t test with Welch’s correction versus the HLAbp group Medical Writing and/or Editorial Assis- (n = 8). Foxp3 forkhead box p3, HLAbp human leukocyte tance. Medical writing support was provided by antigen-binding peptide, IL interleukin, iTreg induced Steven Goodrick, inScience Communications, regulatory T cells, PMA phorbol myristate acetate UK, and funded by Merck KGaA, Darmstadt, Germany. MBP in this model, accompanied by a shift to Authorship. All named authors meet the an immunotolerant state. The results of this International Committee of Medical Journal study indicate that ATX-MS-1467 is a potential Editors (ICMJE) criteria for authorship for this candidate in the management of MS. article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. ACKNOWLEDGEMENTS Author Contributions. Adriano Luı´s Soares de Souza wrote the manuscript, designed and ATX-MS-1467 is owned and being developed by performed the research and analyzed the data. Apitope Technology (Bristol) Limited. All of the Shuning Huang performed the research, MRI data presented here was generated under a scans and analysis. Ji-Kyung Choi performed license from Apitope Technology (Bristol) Lim- the MRI scans and analysis. Timothy Crandall ited. The authors are grateful to David Wraith performed the experiments. Stefan Rudin (University of Bristol) and Keith F. Martin (Api- designed and performed the research and ana- tope, Chepstow, Monmouthshire, UK) for sci- lyzed the data. Blake Tomkinson devised the entific exchange. The authors would like to strategy for the experiments and contributed to thank Professor Joseph Mandeville (Athinoula A. the research design. Rui Chang contributed to Martinos Center for Biomedical Imaging, Mas- the histological analysis and analyzed the data. sachusetts General Hospital, Charlestown, MA, Keith Mitchell contributed to antigen-specific USA) who helped to design the study and pro- proliferation assays and analyzed the data. vided guidance on the data analysis, and Fre´deric Shinji Okitsu contributed to the flow-cytometry Bernard (EMD Serono, Billerica, MA, USA), experiments and analyzed the data. Danielle Anneli Savinainen (EMD Serono, Billerica, MA, Graham designed the research and analyzed the USA) and Dongzi Yu (EMD Serono, Billerica, MA data. Tammy Dellovade wrote the manuscript, USA) for supporting the in vivo studies. The designed the research and analyzed the data. authors would also like to thank Paul Smith Images were acquired by Shuning Huang and Ji- (Merck, Geneva, Switzerland) and Alla Zozoulya Kyung Choi. Gadolinium leakage was evaluated (EMD Serono, Billerica, MA, USA) for being 126 Neurol Ther (2018) 7:103–128 independently by Shuning Huang, Ji-Kyung Attribution-NonCommercial 4.0 International Choi and Joe Mandeville. All authors critically License (http://creativecommons.org/licenses/ reviewed the content of the manuscript and by-nc/4.0/), which permits any noncommer- approved the final version for publication. cial use, distribution, and reproduction in any medium, provided you give appropriate credit Disclosures. Adriano Luı´s Soares de Souza is to the original author(s) and the source, provide a former employee of EMD Serono, Inc, a busi- a link to the Creative Commons license, and ness of Merck KGaA, Darmstadt, Germany. Ste- indicate if changes were made. fan Rudin is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Rui Chang is a former employee of REFERENCES EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. 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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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Abstract

Neurol Ther (2018) 7:103–128 https://doi.org/10.1007/s40120-018-0094-z ORIGINAL RESEARCH ATX-MS-1467 Induces Long-Term Tolerance to Myelin Basic Protein in (DR2 3 Ob1)F1 Mice by Induction of IL-10-Secreting iTregs . . . Adriano Luı´s Soares De Souza Stefan Rudin Rui Chang . . . . Keith Mitchell Timothy Crandall Shuning Huang Ji-Kyung Choi . . . Shinji L. Okitsu Danielle L. Graham Blake Tomkinson Tammy Dellovade Received: January 8, 2018 / Published online: March 14, 2018 The Author(s) 2018. This article is an open access publication humanized (DR2 9 Ob1)F1 mouse in a dose- ABSTRACT dependent fashion. Methods and Results: Our study extends these Introduction: Antigen-specific immunotherapy observations to show that subcutaneous treat- could provide a targeted approach for the ment with 100 lg of ATX-MS-1467 after induc- treatment of multiple sclerosis that removes the tion of EAE in the same mouse model reversed need for broad-acting immunomodulatory established clinical disability (p \ 0.0001) and drugs. ATX-MS-1467 is a mixture of four pep- histological markers of inflammation and tides identified as the main immune-dominant demyelination (p \ 0.001) compared with disease-associated T-cell epitopes in myelin vehicle-treated animals; furthermore, in longi- basic protein (MBP), an autoimmune target for tudinal magnetic resonance imaging analyses, activated autoreactive T cells in multiple scle- disruption of blood–brain barrier integrity was rosis. Previous animal studies have shown that reversed, compared with vehicle-treated ani- ATX-MS-1467 treatment prevented the wors- mals (p \ 0.05). Chronic treatment with ATX- ening of signs of disease in experimental MS-1467 was associated with an enduring shift autoimmune encephalitis (EAE) in the from a pro-inflammatory to a tolerogenic state in the periphery, as shown by an increase in Enhanced content To view enhanced content for this interleukin 10 secretion, relative to interleukin article, go to https://doi.org/10.6084/m9.figshare. 2, interleukin 17 and interferon c, a decrease in splenocyte proliferation and an increase in ? - interleukin 10 Foxp3 T cells in the spleen. A. L. S. De Souza (&)  S. Rudin  R. Chang Conclusion: Our results suggest that ATX-MS- K. Mitchell  T. Crandall  S. Huang 1467 can induce splenic iTregs and long-term D. L. Graham  B. Tomkinson  T. Dellovade Neurology eTIP, Translational and Biomarker tolerance to MBP with the potential to partially Research Group, EMD Serono Research and reverse the pathology of multiple sclerosis, Development Institute, Inc., Billerica, MA, USA particularly during the early stages of the e-mail: adriano.souza@ufmg.br disease. J.-K. Choi Funding: EMD Serono, Inc., a business of Merck Athinoula A. Martinos Center for Biomedical KGaA. Imaging, Massachusetts General Hospital, Charlestown, MA, USA Keywords: ATX-MS-1467; (DR2 9 Ob1)F1 S. L. Okitsu mouse; Experimental autoimmune TIP Immunology, EMD Serono Research and Development Institute, Inc., Billerica, MA, USA 104 Neurol Ther (2018) 7:103–128 encephalitis; Interleukin 10; iTregs; Multiple epitope 140–154: GFKGVDAQGTLSKIF). These sclerosis have been identified as disease-associated epi- topes in patients with MS who have the human leukocyte antigen (HLA) DR2 haplotypes INTRODUCTION DRB1*1501 and DQB1*0602 [14], of which the DRB1*1501 haplotype may explain around 50% Most currently prescribed treatments for multi- of cases of MS [15]. It is assumed that ATX-MS- ple sclerosis (MS) have a broad mechanism of 1467 binds to naked MHC-II of immature den- action, often leading to nonspecific dritic cells [19] and engages the T-cell receptor immunomodulation and associated complica- of MBP-specific primed memory T cells, leading tions, such as infections, malignancy and dis- to the development of a state of peripheral tol- ruption of natural regulatory mechanisms [1]. erance to MBP [14]. Therefore, there is an unmet need for antigen- Treatment with ATX-MS-1467 has previously specific treatments targeting key regulators in been shown to prevent worsening of EAE scores the breakdown of tolerance to self-antigens, in a dose-dependent manner in the humanized such as antigen-presenting cells. The aim of transgenic (DR2 9 Ob1)F1 mouse model [14], such treatments is to silence or reprogram despite the fact that the study did not investi- autoreactive T cells in the periphery to a regu- gate the effect of ATX-MS-1467 on established latory phenotype and thus create a tolerogenic EAE. In addition, a phase 1 study in six adults state to the targeted protein. By directly target- with secondary progressive MS showed that ing the pathogenic mechanisms of MS, treatment with ATX-MS-1467 was well toler- immunological tolerance to environmental and ated. The immune response to MBP was neither self-antigens, such as myelin basic protein dramatically enhanced nor inhibited, due to (MBP), can be reinstated without disrupting study group sizes being too small to allow con- normal immune function, and the hypersensi- clusions to be drawn concerning the efficacy of tivity associated with repeated antigen-specific this treatment in humans. IL-10 mRNA tran- immunotherapy can also be avoided [1, 2]. scripts were, nevertheless, transiently induced Several studies have shown antigen-specific by the treatment in one patient phenotyped as peptide immunotherapy suppresses central expressing DRB1*15, suggesting that the drug nervous system (CNS) inflammation in rodent might be capable of inducing desirable cytokine models of MS [3–5]. Specifically, peptide-specific changes [14]. down-regulation of T-cell responses with a sol- The aim of this study is to extend the results uble acetylated form of the N-terminal peptide from the EAE by assessing the efficacy of late (at of MBP (Ac1-9) was protective against experi- the observed peak of disease) as well as early (in mental autoimmune encephalitis (EAE), a T the prodromal phase) treatment with ATX-MS- cell-mediated inflammatory disease that has 1467 for reversing the neurological damage and similarities to MS and is the most commonly deficits accompanying EAE in the (DR2 9 used experimental model for this disease [6, 7]. Ob1)F1 mouse. These findings will be corrobo- Nearly two decades of research on tolerance rated with an examination of treatment effect to T cell-restricted epitopes, more specifically in on blood–brain barrier (BBB) permeability, on the context of the study of EAE in a murine known markers of the shift from a pro-inflam- model of MS, has led to the generation of the matory to a tolerogenic state following acute concept of antigen-processing-independent and chronic treatment with ATX-MS-1467, T-cell epitopes (apitopes), which have the more specifically by looking at the effect of potential to be used in the treatment of several in vivo pharmacological treatment on: the autoimmune diseases [7–18]. ATX-MS-1467 is a ex vivo antigen-specific cytokine release from mixture of four peptides of human MBP (MBP splenocytes of EAE mice; the in vivo cytokine epitope 30–44: PRHRDTGILDSIGRF; MBP epi- production following antigenic stimulation; tope 83–99: ENPVVHFFKNIVTPRTP; MBP epi- and the accompanying changes on IL-10- tope 130–144: RASDYKSAHKGFKGV; MBP Neurol Ther (2018) 7:103–128 105 secreting FoxP3?/FoxP3- regulatory cell Table 1 Clinical score scale to assess extent of EAE subpopulations. Clinical score Outcome 0 Normal METHODS 0.5 Partially limp tail Animals 1.0 Completely limp tail 1.5 Slightly impaired righting reflex (DR2 9 Ob1)F1 transgenic mice homozygous 2.0 Delayed righting reflex for DRA*0101/DRB1*1501 (HLA-DR2) and MBP -specific human T-cell receptor (TCR) 84–102 2.5 Virtually absent righting reflex clone (Ob1) were generated in C57Bl/6 mice 3.0 Partial hind-limb paralysis [20] and maintained and bred at Charles River Laboratories (Wilmington, MA, USA); the F1 3.5 Complete unilateral hind-limb paralysis progeny of these animals were used in all 4.0* Complete bilateral hind-limb paralysis experiments. Before each experiment, expres- sion of human MHC and TCR were confirmed 4.5 Moribund in peripheral blood by phenotyping with fluo- 5.0 Death due to EAE rescein isothiocyanate (FITC)-labelled mouse anti-human HLA-DR, HLA-DP, HLA-DQ (BD EAE experimental autoimmune encephalitis *Humane endpoints and special care for mice with EAE Pharmingen, San Jose, CA, USA) and IOTest were also observed. Once animals reached a score of 4.0 anti-TCR VB2-PE clone MPB2D5 (Beckman they were observed at least twice a day to ensure they did Coulter, Indianapolis, IN, USA). Only double- not progress further and did not get worse: animals that positive mice with a frequency of human MHC improved to a score of 3.5 or lower were allowed to con- and TCR expression [ 15% in peripheral tinue in the study; if animals did not improve below a leukocytes were used in the studies. score of 4.0 within 72 h, they were euthanized. In addition, On arrival, mice were group-housed in an mice were also euthanized if they experienced a body Association for Assessment and Accreditation of weight loss of 20%. From the onset of paralysis, mice also Laboratory Animal Care-accredited vivarium received wet dietary supplement (Dietgel 76A, ClearH2O) with a 12:12 light–dark cycle and food and on the cage bottom and subcutaneous sterile Ringer’s water provided ad libitum. The mice were glucose injections, as needed, for fluid replacement allowed to acclimatize for at least 48 h before any experiments were started. All procedures were approved by the EMD Serono Institutional Induction of EAE Animal Care and Use Committees (IACUC) or by the Massachusetts General Hospital IACUC Ten to 14-week-old male or female (DR2 9 in the case of magnetic resonance imaging Ob1)F1 mice under isoflurane anesthesia were (MRI) studies. shaved and received a subcutaneous injection at Mice reaching a predefined clinical endpoint the tail base of 100 lL of an emulsion contain- for humane withdrawal from the study (Table 1) ing 200 lg (wet weight) of mouse spinal cord and mice that were not required for tissue homogenate (SCH, harvested in-house from sampling were euthanized by slow replacement syngeneic spinal cords) and heat-inactivated of the air in their cage by an air mixture con- Mycobacterium tuberculosis (400 lg/mouse; taining CO . Death by CO asphyxiation was 2 2 Difco, Oxford, Oxon, UK) prepared using equal confirmed by cervical displacement. All animal volumes of phosphate-buffered saline (PBS) and experiments complied with the National Insti- incomplete Freund’s adjuvant (Difco, Oxford, tutes of Health guide for the care and use of Oxon, UK). On the day of immunization [0 days laboratory animals. All efforts were made to post-induction (dpi)], and also 2 days later minimize the number of animals used and to (2 dpi), mice received an intraperitoneal optimize their well-being. 106 Neurol Ther (2018) 7:103–128 injection with 300 ng of pertussis toxin (ENZO, investigator scored the animals in each study, Farmingdale, NY, USA). and in any single day the same investigator scored all groups of animals. However, because clinical scoring sometimes progressed for sev- ATX-MS-1467 Treatment eral weeks, including weekends, up to four dif- ferent investigators potentially provided input All treatments with ATX-MS-1467 were per- into a single study. Each investigator’s scoring formed from a reconstituted sterile, freeze-dried was routinely calibrated, to ensure the results formulation of a mixture (1:1:1:1) of four syn- were comparable across different investigators. thetic peptides (MBP epitope 30–44: PRHRDTGILDSIGRF; MBP epitope 83–99: Magnetic Resonance Imaging ENPVVHFFKNIVTPRTP; MBP epitope 130–144: RASDYKSAHKGFKGV; MBP epitope 140–154: GFKGVDAQGTLSKIF). After reconstitution of EAE was induced on 0 dpi (n = 35). On 9 or the lyophilized formulation to the original 10 dpi, MRI was obtained, and those animals volume with sterile water, the solution was with radiological evidence of leakage in the further diluted with PBS to the required doses. cerebellum/brainstem seen on T1-weighted The concentration of the ATX-MS-1467 mixture gadolinium-enhanced (Gd ) MRI with a 9.4- was adjusted for a 100-lL injection volume, Tesla magnet scanner (Bruker Biospin, Billerica, administered subcutaneously in all cases. The MA, USA) were randomized into two groups for doses corresponded to the sum of each peptide treatment with either ATX-MS-1467 in the mixture; therefore, a dose of 100 lg (100 lg/mouse, n = 7) or vehicle (n = 7). Treat- contained 25 lg of each peptide. The treatment ment was given every other day, starting at regimen with ATX-MS-1467 varied; please refer 10 dpi (mice scanned on 9 dpi) or 11 dpi (mice to specific sections for the dose regimen used in scanned at 10 dpi). T1-weighted Gd MRI was each experiment. 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 HLA-Binding Peptide Treatment time (TR)/TE/flip angle = 100 ms/2.7 ms/50 degrees] was used to assess Gd leakage. In some experiments, HLA-binding peptide (HLAbp) was used as a control for ATX-MS- Histology 1467. Although HLAbp can bind directly to MHC on the surface of antigen-presenting cells without requiring antigen processing, the pep- Pathological analysis was done on samples of tide sequence of HLAbp has no similarity to the spinal cords from animals treated at 7 or 14 dpi epitopes involved in the adaptive immune with either ATX-MS-1467 or vehicle. Samples responses to MS or EAE. The peptide sequence were obtained at the termination date of these used (NPILLWQPIPVHTVPLSEDQ) was provided experiments (30 post-immunization). A 1.6 by Professor David Wraith (University of Bristol, g/mL 2,2,2-tribromoethanol (T48402, Sigma, St. UK). 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 Clinical Scoring, Animal Care obtained by subsequent dilution with PBS. At and Humane Endpoints predefined time points, mice were overdosed with anesthetic (400 mg/kg intraperitoneal Clinical score and body weight were monitored 2,2,2-tribromoethanol) and perfused through and recorded daily from 7 dpi by observers blind the left cardiac ventricle with saline followed by to treatment. Neurological disability and 10% neutral buffered formalin. The vertebrae humane endpoints were monitored daily using were partially removed to obtain the spinal a subjective 0–5-point clinical score scale cord, and the tissues were post-fixed with 10% (Table 1)[21]. Whenever possible, a single Neurol Ther (2018) 7:103–128 107 neutral buffered formalin for 48 h and then The H&E scoring was performed as set out in stored in PBS before dissecting out the spinal Table 2. CD3 and CD45R scoring were similar to cords. Spinal cords were cut into five segments, the H&E scoring, except that only immunos- spanning the cervical (one piece), thoracic (two tained cells were scored. For the LFB scoring, pieces) and lumbar (two pieces) regions, which each cross section of the spinal cord was ana- were processed for paraffin embedding. Histo- lyzed as four independent regions (ventral, logical analyses were done on 10 cross-sections dorsal, right lateral and left lateral). In all cases, of the spinal cord for each animal. These cross- the scores for each of the ten sections was sections were 7 lm thick and at least 280 lm averaged and entered as a single data point for apart. each mouse in the experiment. Hematoxylin and eosin (H&E) and luxol fast blue (LFB) staining were performed, as well as Ex-Vivo Antigen-Recall Assay immunohistochemistry staining against CD3 (Abcam, Cambridge, MA, USA) and CD45R (BD Mice were treated with either ATX-MS-1467 Pharmingen, San Jose, CA, USA) surface anti- (100 lg/mouse, three times per week) or vehicle gens using Leica BOND-III autostainer (Leica, for 7 days. At 0 dpi, mice were also immunized Wetzlar, Germany). High-quality images as if for induction of EAE, but these mice were obtained with a Nanozoomer 2.0 HT digital euthanized before they displayed signs of dis- slide scanner (Hamamatsu, Hamamatsu City, ease. Mice received the last dose of ATX-MS- Shizuoka, Japan) were semi-quantitatively 1467 or vehicle at 7 dpi, 1 h before sacrifice for scored for each stain by an experimenter who spleen removal and cell culture. On the day of was blinded to the treatment groups. sacrifice, mice were anaesthetized with 400 mg/ kg intraperitoneal 2,2,2-tribromoethanol, and Table 2 Histology scoring of spinal cord sections Score H&E/CD3 and CD45R LFB 0 No visible signs of inflammation No region presenting with demyelination 0.5 Immune cells found only in close association with meninges Only one region exhibiting demyelination spanning \ 50% of its area 1.0 Plus at least one clearly defined example of a perivascular cuff [at this Two regions exhibiting demyelination stage, perivascular cuffs have limited cellularity (\ 10 cells) and spanning \ 50% usually up to maximum of 3–4 perivascular cuffs can be found] 2.0 Plus discrete numbers of immune cells beyond the perivascular cuffs Three regions exhibiting demyelination (i.e., infiltrating into the parenchyma of the white matter) spanning \ 50% 3.0 Plus extensive cell clusters (C 20 cells each) throughout the parenchyma Four regions exhibiting demyelination of the white matter (i.e., not restricted to perivascular cuffs) spanning \ 50% 4.0 Plus major infiltrations (typically clusters of C 50 cells) all over the One region exhibiting demyelination white matter spanning [ 50% 5.0 Significant numbers of immune cells ([ 30) dispersed throughout the Two regions exhibiting demyelination grey matter, usually accompanied by massive hypercellular events in spanning [ 50% the white matter H&E hematoxylin and eosin, LFB luxol fast blue 108 Neurol Ther (2018) 7:103–128 their spleens were harvested and processed in a immunized with adjuvant in order to avoid gentle MACS Octo dissociator (Myltenyi Biotec, background inflammation caused by this agent. Birgisch Gladback, Germany). The splenocytes Serum samples were collected from untreated underwent several cycles of washing and cen- mice or mice that had received repeated treat- trifugation after lysis of red blood cells with red ment with ATX-MS-1467 (100 lg/mouse, three blood cell lysis buffer (Roche, Basel, Switzer- times per week) or HLAbp (100 lg/mouse three land) and were counted manually in a Neubauer times per week) receiving a total of 10 injec- chamber using trypan blue solution to exclude tions, followed or not by a wash-out period dead cells. The concentration of the cell sus- lasting from 2–42 days for assessment of the pension was adjusted to 3 9 10 cells/mL in concentrations of IL-2, IL-10, IL-17 and IFNc. RPMI medium (Gibco, Waltham, MA, USA) Samples were collected 2 h after the last injec- supplemented with fetal calf serum (10%), tion, which was experimentally predetermined penicillin–streptomycin (1:100; Gibco, Wal- as the time point at which the concentrations of tham, MA, USA), HEPES (final concentration most cytokines had reached a maximum. To 10 mM; Gibco, Waltham, MA, USA), MEM non- ascertain the in vivo cytokine secretion of essential amino acid solution (1:100; Sigma, St. (DR2 9 Ob1)F1 mice to an acute challenge with Louis, MO, USA), L-glutamine (final concentra- MBP, serum was collected 2 h after acute treat- tion 2 mM; Gibco, Waltham, MA, USA), sodium ment with MBP at doses ranging 30– pyruvate (final concentration 1 mM; A&E Sci- 1000 lg/mouse. To ascertain the effect of entific PAA, San Jose, CA, USA) and b-mercap- chronic dosing with ATX-MS-1467 on in vivo toethanol (final concentration 2.5 lM; Sigma, pro-inflammatory cytokine secretion, mice St, Louis, MO, USA). For cell culture, 100 lLof received repetitive treatment (from one up to the cell suspension was prepared as described, ten doses) with ATX-MS-1467 (100 lg/mouse, together with 100 lL of each appropriate stim- three times per week) with serum collected 2 h ulant (ATX-MS-1467 0.03–30 lg/mL final con- after the last dose. To ascertain the effect of centrations) diluted in medium and incubated chronic treatment with ATX-MS-1467 on the at 37 C for 48 or 72 h, corresponding to the ratio of IL-10-to-pro-inflammatory cytokines optimum time points for evaluation of cytokine after an acute challenge with MBP, mice or proliferative responses, respectively. Cytoki- received ten doses of ATX-MS-1467 nes were quantified in supernatants from 48-h (100 lg/mouse, three times per week) or ten splenocyte cultures with commercial enzyme- doses of HLAbp (100 lg/mouse, three times per linked immunosorbent assay (ELISA) kits week). At 2, 7, 21 or 42 days after the last (mouse IL-2 Duoset ELISA, mouse IL-10 Quan- injection with ATX-MS-1467 or HLAbp, mice tikine ELISA, mouse IL-17 Duoset ELISA and received a single injection of MBP mouse IFNc Duoset ELISA, all purchased from (300 lg/mouse) and serum was collected 2 h R&D Systems, Minneapolis, MN, USA). Spleno- later. Acute cytokine responses in serum were cyte proliferation was assessed by replacing quantified using a customized MCYTOMAG- 100 lL of the medium from wells that had been 70 K | MILLIPLEX MAP Mouse Cytokine/Che- stimulated for 72 h with 100 lLof H thymidine mokine Magnetic Bead Panel—Immunology solution and further incubating for 12 h. Multiplex Assay (Millipore, Billerica, MA, USA). Incorporation of H thymidine was quantified in a scintillator. The results were expressed as Effect of ATX-MS-1467 Treatment counts per minute (cpm). on Leukocyte Populations Antigen-Specific Cytokine Release In Vivo Flow cytometric analysis was used to assess the effect of treatment with ATX-MS-1467 The experiments that were conducted to assess (100 lg/mouse, three times per week) or vehicle antigen-specific cytokine release in vivo were for 2 weeks on cell surface and intracellular performed in mice that had not been markers associated with regulatory cell Neurol Ther (2018) 7:103–128 109 function. Two different staining panels were FlowJo version 10 (FlowJo, LLC, Ashland, OR, used (Table 3). USA). The first panel focused on cell surface mark- ers and was applied in freshly isolated spleno- Statistical Analyses cytes. The second panel included intracellular markers, IL-10 and the transcription factor In experiments with multiple groups, clinical forkhead box P3 (Foxp3). To enable better score data were analyzed via daily comparison identification of the IL-10-secreting cells, the of group averages using the Kruskal–Wallis test second panel was applied in splenocytes incu- followed by Dunn’s multiple comparison test. bated for 3 h with phorbol-myristate acetate Clinical score data were also integrated as area (PMA, 5 ng/mL; Sigma, St. Louis, MO, USA), under the curve, and the results analyzed with ionomycin (500 ng/mL; Sigma, St. Louis, MO, the Kruskal–Wallis test followed by Dunn’s USA) and the protein transport-inhibitor mon- multiple comparison test. MRI, EAE imaging ensin (4 lL/6 mL of cell culture; GolgiStop, BD and clinical score data were analyzed daily by Biosciences, San Jose, CA, USA) for 3 h. The Mann–Whitney test. The mean semi-quantita- splenocytes were isolated as previously descri- tive histological scores of spinal cord sections bed. B220 and CD8 staining were used as a were input as a single data point that was used negative control for the T lymphocyte subpop- to calculate group averages, which were com- ulations of interest. Samples were measured on pared by one-way analysis of variance (ANOVA) a LSRFortessa X20 flow cytometer (BD Bio- followed by the Bonferroni test. Data on con- science, San Jose, CA, USA) and analyzed in centration-dependent cytokines and Table 3 Antibody panels used for splenocyte staining Panel 1 (cell surface markers on freshly isolated Panel 2 (cell surface and intracellular markers) splenocytes) CD49b (BV421, 1:100, cat. # 63063, BD Pharmingen, San CD49b (BV421, 1:100, cat. # 63063] BD Pharmingen, San Jose, CA, USA) Jose, CA, USA) CD3 (V500, 1:100, cat. # 560771, BD Pharmingen, San CD4 (V500, 1:100, cat. # 560782] BD Pharmingen San Jose, CA, USA) Jose, CA, USA) CD4 (FITC, 1:100, cat. # 553729, BD Pharmingen, San LAG3 (PE, 1:100, cat. # 125207, Biolegend, San Diego, CA, Jose, CA, USA) USA) LAG3 (PE, 1:100, cat. # 125207, Biolegend, San Diego, CA, IL-10 (APC, 1:100, cat. # 554468, BD Pharmingen, San USA) Jose, CA, USA) B220 (PE-Cy7, 1:200, cat. # 552772, BD Pharmingen, San CD8 (APC-Cy-7, 1:100, cat. # NC0079947, Biolegend, San Jose, CA, USA) Diego, CA, USA) CD8 (BV605, 1:100, 563152, cat. # 563152, BD CD45R/B220 (BV605 1:100, cat. # 563708, BD Pharmingen, San Jose, CA, USA) Pharmingen, San Jose, CA, USA) FC block (CD16/CD32, unstained, 1:100, cat. # 553141, FC block (CD16/CD32, unstained, 1:100, cat. # 553141, BD Pharmingen, San Jose, CA, USA) BD Pharmingen, San Jose, CA, USA) FoxP3 (PerCP-Cy5.5 1:100, cat. # 35-5773-82, eBioscience, San Diego, CA, USA) CD45R/B220 and CD8 staining were used as a negative selection for the T lymphocyte subpopulations of interest. Listed are the specificities, fluorochromes, catalogue numbers and manufacturers, and dilutions for each of the antibodies 110 Neurol Ther (2018) 7:103–128 proliferation response ex vivo were analyzed observed efficacy. The average score for the with two-way ANOVA followed by the Bonfer- vehicle-treated groups, which varied from 2 to roni test. In vivo cytokine-release data were 3, depending on the marker being analyzed, analyzed with one-way ANOVA followed by could wrongly suggest that the level of tissue Dunnett’s multiple comparison test. damage is not compatible with overt disease pathology. However, due to the unpre- dictable location where the lesions occur in this RESULTS model, the average score is the result of both highly affected spinal cord sections and other Effect of ATX-MS-1467 on EAE Clinical sections that might have remained intact. Disability Indeed, it was a common feature of all vehicle- treated mice that one or multiple areas with We treated (DR2 9 Ob1)F1 mice with different intense cellular infiltration and demyelination doses of ATX-MS-1467, starting either 7 or were observed at various, albeit not all, sections 12 dpi and observed the clinical signs of disease. examined. In agreement with the attenuation Compared with vehicle, ATX-MS-1467 dose- in clinical score, treatment with ATX-MS-1467 dependently reduced clinical disability from the from the empirically determined disease onset empirically determined disease onset (7 dpi, (7 dpi) or peak of disease (14 dpi) time points Fig. 1a, c) and peak disease (12 dpi, Fig. 1b, d) reduced spinal cord inflammation, demyelina- time points, as assessed by the mean daily score tion and cellular infiltration, unsurprisingly and cumulative scores on the subjective 0–5- with the greatest effect for all outcomes repor- point clinical score scale during the treatment ted when treatment was initiated at disease period. As can be observed in Fig. 1a, d, if onset (p \ 0.001 for all measures; Fig. 2). The treatment starts early in the course of disease, it reduction in inflammation and demyelination is possible to completely prevent the appear- seen in the CNS was also accompanied by an ance of clinical signs. Even if the treatment is improvement in the clinical score, for which initiated when mice exhibit full-blown disease the greatest improvement was observed follow- (tail–hand–hind limb paralysis, matching an ing early (7 dpi) treatment (Fig. 2a). Treatment average score of 3), partial, albeit significant, with ATX-MS-1467 from the peak of disease recovery from the natural course of EAE was (14 dpi) partially reduced the histological seen in this mouse strain (Fig. 1b, d). The opti- markers indicative of inflammation (H&E score; mum dose (100 lg) significantly improved the Fig. 2b), demyelination (LFB score; Fig. 2c) and clinical score in mice showing advanced signs of T-cell infiltration (CD3 score; Fig. 2d; p \ 0.05) neurological disability, and this dose was selec- and histology scores indicative of B-cell infil- ted for subsequent experiments. tration (p \ 0.01; Fig. 2e). MRI was applied as a translational approach to investigate the efficacy of ATX-MS-1467 in Effect of ATX-MS-1467 on CNS Infiltration mice. Gd administered intravenously during the and Inflammation acute phase of disease allows the detection of leakage, which can be seen as bright areas of the We performed two independent experiments, spinal cord in T1-weighted scans, indicating with treatment started at either 7 or 14 dpi. This disruption of the BBB. Because BBB disruption allowed us to generate a large tissue bank to tends to be more active during the beginning of assess the effects of treatment with ATX-MS- the acute phase of disease, and tends to natu- 1467: spinal cord samples were obtained from rally subside at later phases, we opted for a 20–28 mice from each treatment group, with shorter window of treatment, in order to better each spinal cord being stained and analyzed at appreciate the drug effect. In a longitudinal MRI ten different levels for each staining. In this experiment (Fig. 3a), therapeutic dosing with experiment, we used histological evaluation ATX-MS-1467 (100 lg subcutaneous) every with subjective scoring (Table 2) to confirm the other day (three doses) from 10 dpi significantly Neurol Ther (2018) 7:103–128 111 Vehicle ATX-MS-1467 (3 μg) a ATX-MS-1467 (10 μg) b Vehicle ATX-MS-1467 (30 μg) ATX-MS-1467 (30 μg) ATX-MS-1467 (100 μg) ATX-MS-1467 (100 μg) 4 4 3 3 2 2 ** ** *** ** *** 1 1 ** ** ** *** ** 0 0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 22 Days post-immunization Days post-immunization c d 30 30 20 20 ** 10 10 **** 0 0 Vehicle 3 10 30 100 Vehicle 30 100 ATX-MS-1467 dose (μg) ATX-MS-1467 dose (μg) Fig. 1 Effect of dose response of ATX-MS-1467 from for the experiment shown B and D, the data represent a disease onset or peak of clinical disability in a mouse model pool from two independent experiments with a total of of EAE. EAE was induced at 0 dpi with spinal cord n = 21–22 mice. *, **, *** and **** indicate p \ 0.05, homogenate in (DR2 9 Ob1)F1 mice, and treatment was \ 0.01, \ 0.001 and \ 0.0001, respectively, by initiated with ATX-MS-1467 at 7 dpi (a, c) or at 12 dpi Kruskal–Wallis test followed by Dunn’s test versus vehicle (b, d) at doses ranging 3–100 lg, as indicated, subcuta- (PBS) group. dpi days post-injection, EAE experimental neously three times per week. The grey area in graphs a, autoimmune encephalomyelitis, PBS phosphate-buffered b indicates the treatment period. For the experiment saline, SEM standard error of the mean shown in a, c, the group sizes were n = 10–14 mice, and (p \ 0.05 on 13 and 15 dpi and p \ 0.01 on 14 (Fig. 3e) compared with baseline but not in and 16 dpi) reversed disruption to the BBB, as vehicle-treated mice [Fig. 3e; ATX-MS-1467- measured by Gd leakage, compared with vehi- treated mice: 77.4% reduction (Gd-leakage vol- cle-treated animals (Fig. 3c). In group analyses ume 1.1 ± 0.3 mm ) from baseline, p \ 0.01; at follow-up, Gd-leakage volume was signifi- vehicle-treated mice: 39.7% reduction (Gd- cantly reduced in ATX-MS-1467-treated mice leakage volume 4.45 ± 0.3 mm ) from baseline, Cumulative score Clinical score (mean ± SEM) (mean ± SEM) Cumulative score Clinical score (mean ± SEM) (mean ± SEM) 112 Neurol Ther (2018) 7:103–128 a Vehicle b Vehicle ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 14 ATX-MS-1467 from dpi 14 2.5 2.0 1.5 1.0 ### 0.5 ** ** *** * * Naїve Vehicle *** 0 2 4 6 8 1012141618 20 22 24 26 28 3032 ATX from dpi7 ATX from dpi14 Days post-immunization c d Vehicle Vehicle ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 14 ATX-MS-1467 from dpi 14 4 3 ### ### 0 0 Naїve Vehicle Naїve Vehicle ATX from dpi7 ATX from dpi14 ATX from dpi7 ATX from dpi14 Vehicle ATX-MS-1467 from dpi 7 ATX-MS-1467 from dpi 14 2.5 2.0 ## 1.5 1.0 ### 0.5 0.0 Naїve Vehicle ATX from dpi7 ATX from dpi14 Cd45R score LFB score Clinical score (mean ± SEM) (mean ± SEM) (mean ± SEM) CD3 score H&E score (mean ± SEM) (mean ± SEM) Neurol Ther (2018) 7:103–128 113 bFig. 2 Effect of therapeutic treatment with ATX-MS- release of IL-10, which is indicative of a tolero- 1467 on spinal cord tissue damage in spinal cord genic state, would be expected. In vivo and homogenate-induced EAE in (DR2 9 Ob1)F1 mice. in vitro strategies were employed to demon- a The effect of 100-lg ATX-MS-1467 treatment starting strate whether these changes occurred in mice at 7 or 14 dpi on daily clinical scores and on terminal (day treated with ATX-MS-1467. For the in vitro 30) histological scores. The arrows indicate the beginning analysis, splenocytes were recovered from ani- of the treatment period in each group. *, ** and *** indicate mals treated four times between 0 to 7 dpi with p \ 0.05, \ 0.01 and \ 0.001, respectively, by ATX-MS-1467 (on days 0, 2, 4 and 7); the last Kruskal–Wallis test followed by Dunn’s test versus vehicle. administration took place 1 h prior to take H&E staining was applied to assess inflammation (b), luxol down. The spleens were processed, as described fast blue staining was applied to assess demyelination (c), in the methods section, and the cytokine con- and immunohistochemical staining for CD3 or CD45R centrations in the supernatant were assayed was applied to assess T-cell (d) and B-cell infiltration (e). (Fig. 4a–d). The cultures were stimulated in the The inserts show representative images for each group with presence of a range of concentrations of each the respective staining for which the semi-quantitative # ## ### antigen, to allow the assessment of the response evaluation is plotted. , and indicate p \ 0.05, over a broad range of concentrations. \ 0.01 and \ 0.001, respectively, by ANOVA followed It is important to differentiate the purpose of by Bonferroni versus mice treated with vehicle (PBS). The ATX-MS-1467 as a treatment (Fig. 4a–e filled data represent a pool from 2 independent experiments squares), when it was administered in vivo with with a total of 20–28 mice. Spinal cords from naı¨ve mice a vehicle-administered group as a control were also processed, stained and analyzed along with the (Fig. 4a–e filled circles), from its use here as an samples from the immunized/treated mice. The histolog- ical (as well as clinical) scores for naı¨ve mice was zero for antigen-specific stimulus for cellular prolifera- all cases; therefore, for the sake of simplicity, data from tion and cytokine release (x axis, various con- naı¨ve mice was not included in the graphs. dpi days post- centrations). The in vitro antigen recall assay induction, EAE experimental autoimmune encephalitis, showed that the in vivo treatment with ATX- H&E hematoxylin & eosin, LFB luxol fast blue, PBS MS-1467 caused a dose-dependent inhibition in phosphate-buffered saline, SEM standard error of the pro-inflammatory cytokine (IL-17 and IFNc) mean and IL-2 secretion and splenocyte proliferation. IL-10 secretion was significantly increased only after stimulation with concentrations of ATX- MS-1467 of 3 lg/mL (p \ 0.05) and above p = 0.23]. Figure 3b shows representative T1- (p \ 0.001) in the ex vivo antigen recall assay weighed MRI scans of the spinal cords of the (Fig. 4). mice in this study. Effect of ATX-MS-1467 Treatment Effect of ATX-MS-1467 Treatment on the In Vivo Cytokine Profile on the Anti-Inflammatory Milieu in Ex Vivo Splenocytes The effect of ATX-MS-1467 persists when the drug is cleared from the circulation. To extend ATX-MS-1467 binds to surface-expressed MHC- the results for ex vivo cytokine secretion, we II on antigen-presenting cells. Following bind- wanted to assess whether a favorable shift in ing, ATX-MS-1467 is hypothesized to drive cytokine balance also occurred in vivo. To either the differentiation of MBP-specific regu- achieve this, we performed three types of latory cells from naıve cells or to induce the experiments. conversion of effector into type 1 regulatory The first type (Fig. 5) aimed to define the cells (Tr1). In both cases, inhibition of the optimal dose of MBP needed to elicit robust release of the pro-inflammatory cytokines IL-17, serum cytokine changes. In the naı¨ve wild-type and IFNc from Th1 and Th17 T-cell subsets, mouse strain, MBP injection would not nor- which are known to promote the inflammatory mally induce cytokine release at levels that can processes in MS, along with an increase in the 114 Neurol Ther (2018) 7:103–128 PBS ATX-MS-1467 3.5 PBS ATX-MS-1467 3.0 2.5 Baseline Baseline 2.0 ** 1.5 ** 1.0 Follow-up Follow-up 0.5 Treatment 0 2 4 6 8 10 12 14 16 18 Days post-induction PBS cd 10 18 PBS ATX-MS-1467 0 0 Baseline MRI Follow-up MRI Baseline MRI Follow-up ATX-MS-1467 Baseline MRI Follow-up Fig. 3 Effect of ATX-MS-1467 on EAE-induced blood– leakage. Levels of Gd leakage volume at dosing onset and brain barrier leakage. The effect on clinical score of ATX- follow-up (c). Leakage volume for the individual mice with MS-1467 (n = 7) or vehicle (PBS; n = 7) given at 10 or vehicle (d) or ATX-MS-1467 treatment (e) were com- 11 dpi, depending on the timing of MRI; the grey area pared using a paired t test. *p \ 0.05, **p \ 0.01. dpi days shows the treatment period (a). Four serial images at post-induction, EAE experimental autoimmune baseline and follow-up for two animals (left: vehicle- encephalomyelitis, Gd gadolinium, MRI magnetic reso- treated mice; right: ATX-MS-1467-treated mice; b). The nance imaging, PBS phosphate-buffered saline, SEM yellow arrows highlight the areas with changes in Gd standard error of the mean Clinical score 2 2 Leakage volume (mm ) Leakage volume (mm ) (mean ± SEM) Leakage volume (mm ) Neurol Ther (2018) 7:103–128 115 be detected in serum. However (DR2 9 Ob1)F1 shows how a system is responding to the chal- mice were genetically engineered to constitu- lenge [22]. If treatment with ATX-MS-1467 does tionally express both MHC and TCR recogniz- indeed induce tolerance, the persistence of the ing one MBP epitope; therefore, circulating tolerogenic effect, as deduced from changes in effector and regulatory cells that respond to the ratios of IL-10-to-pro-inflammatory cytoki- MBP are already present in (DR2 9 Ob1)F1 nes, could be assessed by the changes in these mice, even when they have not been immu- ratios. From the second dose to the fifth dose, nized against this protein. Untreated (DR2 9 the concentration of IL-10 relative to IL-17 and Ob1)F1 mice responded to an acute challenge IFNc, respectively, was higher than in vehicle- with MBP with a dose-dependent increase in treated animals. However, the concentration of pro-inflammatory cytokines IL-17 (Fig. 5c) and IL-10 relative to IL-2 was lower for vehicle- IFNc (Fig. 5d) and also IL-2 (Fig. 5a) compared treated mice after one dose, and was not dif- with vehicle-treated mice. Although the con- ferent from vehicle-treated animals from the centration of IL-10 was also increased in second dose onwards (Fig. 6). response to low doses of MBP, this effect pla- Finally, a third type of experiment was done teaued at MBP concentrations of 100–300 lg to assess how mice reacted to the native antigen and then declined as the concentration of MBP (MBP) after they received a course of 10 injec- increased (Fig. 5b). tions with 300 lg of ATX-MS-1467, which was The second type of experiment on in vivo previously determined as the highest dose to cytokine release (Fig. 6) assessed how cytokine elicit the maximum response (Fig. 7). Because release shifted after repeat treatments. The all previous evidence indicates that ATX-MS- insert summarizes how the injections were 1467 treatment is associated with induction of performed in each group (Fig. 6a). A single dose Tregs, we hypothesized that the effect of the of ATX-MS-1467 elicited an increase in the treatment is long-lasting; therefore, the effects concentrations of all cytokines in serum should persist for at least as long as this pool of (Fig. 6b–e), and the concentrations returned to regulatory cells remained expanded, counter- baseline values within a few hours after reach- acting the natural emergence of Th1 or Th17 ing their peak at 2 h after the injection (results effector cells. To test this hypothesis, we chal- not shown). Similar to explanation given above lenged a group of mice with MBP following a for MBP-induced cytokine release, naı¨ve mice wash-out period of 2 days (equivalent to the 39/ with the transgenic mouse construct also weekly regimen treatment with ATX-MS-1467), respond to ATX-MS-1467 with quantifiable a group with wash-out periods of either 7, 21 or cytokine levels in serum without the need of 42 days from the last ATX-MS-1467 treatment, concomitant exposure with adjuvant. Further and also mice in which HLAbp was used as a doses of ATX-MS-1467 did change the serum IL- control for ATX-MS-1467. To help interpret this 2 (Fig. 6b), IL-17 (Fig. 6d) or IFNc (Fig. 6e) study (as depicted in Fig. 7), we compared the cytokine concentrations in comparison with cytokine levels (or ratios) between the third bar, vehicle for up to ten consecutive doses of ATX- i.e., mice treated with control peptide (HLA-bp) MS-1467. However, up to five consecutive doses then challenged with MBP, which could also be of ATX-MS-1467 were still capable of inducing regarded as ‘‘untolerized’’, against those levels in statistically significant drug-induced IL-10 the four last columns representing mice treated release in serum (Fig. 6d). The use of ratios of chronically with ATX-MS-1467 prior to MBP signature cytokines for the predicted response is challenge (performed from 2, 7, 21 or 43 days an accurate index of how an organism responds after the last treatment). When naıve mice were to antigens or disease: how the ratios change challenged with MBP (Fig. 7a–d; second column over time or how they compare between dif- in each plot) there was always a large induction ferent groups under controlled conditions of serum cytokines. The same was true for the 116 Neurol Ther (2018) 7:103–128 IL-2 IL-10 a b 300 140 Vehicle Vehicle *** ATX-MS-1467 ATX-MS-1467 *** *** 50 *** *** ** 0 0 0.03 0.1 0.3 1 3 10 30 0.03 0.1 0.3 1 3 10 30 ATX-MS-1467 in vitro (μg/mL) ATX-MS-1467 in vitro (μg/mL) IL-17 IFN-γ c d 600 1500 Vehicle Vehicle ATX-MS-1467 ATX-MS-1467 *** 100 *** *** 0 0 0.03 0.1 0.3 1 3 10 30 0.03 0.1 0.3 1 3 10 30 ATX-MS-1467 in vitro (μg/mL) ATX-MS-1467 in vitro (μg/mL) e Thymidine incorporation Vehicle ATX-MS-1467 30000 * 0.03 0.1 0.3 1 3 10 30 ATX-MS-1467 in vitro (μg/mL) Radioactivity (cpm) Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Neurol Ther (2018) 7:103–128 117 bFig. 4 Effect of preventive treatment with ATX-MS-1467 out of 7 days. However, the effects on the IL-10- on ex vivo splenocyte proinflammatory cytokine release to-IFNc ratio are more persistent; this ratio did and proliferation. (DR2 9 Ob1)F1 mice were immunized not return to baseline values until a wash-out with spinal cord homogenate and treated with 100 lg period of 42 days. This result indicates that, at ATX-MS-1467 or vehicle (PBS) on days 0, 2, 5 and 7. least with regard to this cytokine pair ratio, the Splenocytes were harvested at 7 dpi and stimulated in vitro effect of ATX-MS-1467 is long-lasting. On the with ATX-MS-1467 at 0.03, 0.1, 0.3, 1, 3, 10 and 30 lg/ other hand, the effect on the IL-10-to-IL-17 mL; cytokine concentrations were measured in the ratio was less persistent. supernatants (a–d). Splenocyte proliferation was also measured by H thymidine uptake (cpm) following Effect of ATX-MS-1467 Treatment in vitro stimulation with ATX-MS-1467 (e). *, ** and on Regulatory T Cell Populations *** indicate p \ 0.05,\ 0.01 and\ 0.001, respectively, by two-way ANOVA followed by Bonferroni’s post-tests The mechanism of tolerance induction can be (n = 5–6). ANOVA analysis of variance, cpm counts per minute, dpi days post-induction, IFN interferon, IL investigated by quantification of the intracel- interleukin, PBS phosphate-buffered saline lular and/or extracellular markers unique to each of these Treg subsets (i.e., state-specific markers and subsets). We relied on two different approaches to define the induced regulatory T mice that received ten doses of HLA-bp cells (iTreg) subset: one was the co-expression of (Fig. 7a–d; third column in each plot). However, LAG3 and CD49 in CD4 lymphocytes; the for all cytokines tested, the serum cytokine other was the presence or absence of FOXP3 in concentrations were always lower for the groups IL-10 secreting CD4 lymphocytes. In the second of mice that received a course of ATX-MS-1467 ? ? approach, we assumed that the IL-10 FOXP3 than those mice that received HLA-bp treat- CD4 lymphocytes are natural T regulatory cells ment prior to the challenge, regardless of the ? - (nTregs), and the remaining IL-10 FOXP3 are, duration of the wash-out period. Furthermore, by exclusion, iTregs. Staining was performed in the effector cytokines tended towards a slight splenocytes of mice that had been treated increase after longer wash-out periods, whereas in vivo with ATX-MS-1467 or HLA-bp for the opposite occurred for IL-10. This suggests a 2 weeks. Figure 8a, b shows the representative down-regulation of the pool of IL-10-secreting gating leading to assessment of the frequency of cells and a re-emergence of the pool of effector ? ? ? LAG3 CD49b CD4 lymphocytes from an cytokine-secreting cells with time. From this HLA-bp-treated mouse and an ATX-MS-1467- observation, one question arises: at which point treated mouse, respectively. Figure 8c shows does the balance of regulatory/effector cytoki- mean data for both groups. The results indicate nes return to that seen in the absence of toler- that chronic treatment with ATX-MS-1467 ization (equivalent to the HLB-bp group, third expanded the iTreg compartment in the spleen column in each plot)? This can be assessed by compared with mice treated with HLAbp. The replotting the data as cytokine ratios (Fig. 7f–h). spleens of mice treated with ATX-MS-1467 had The results vary depending on the analyzed ? ? a higher proportion of LAG3 C49b cells com- cytokine ratio. For the IL-10-to-IL-2 ratio, pared with HLAbp-treated mice (p \ 0.05). Fig- tolerization with ATX-MS-1467 can still induce ure 8d–g shows the gating and the means for higher ratios at wash-out periods of up to the upper-left and upper-right quadrants, 7 days, but the ratios return to untolerized ? - ? ? denoting IL-10 FOXP3 and IL-10 FOXP3 levels (HLA-bp treated) after a wash-out of lymphocytes, respectively, for each group. In 21 days (Fig. 7f). For the IL-10-to-IL-17 ratio, the conclusion, in vivo ATX-MS-1467 treatment return to baseline values occurs very quickly ? - increased the pool of IL-10 Foxp3 (iTregs), but (Fig. 7g); the ratio has normalized after a wash- ? ? it had no effect on IL-10 Foxp3 (nTregs). 118 Neurol Ther (2018) 7:103–128 IL-2 IL-10 a b 2500 100 *** *** *** 2000 80 ** ** 1500 60 1000 40 ** 500 20 0 0 Vehicle 30 100 300 1000 Vehicle 30 100 300 1000 MBP dose (μg) MBP dose (μg) IL-17 IFN-γ c d 400 150 0 0 Vehicle 30 100 300 1000 Vehicle 30 100 300 1000 MBP dose (μg) MBP dose (μg) Fig. 5 Response of (DR2 9 Ob1)F1 mice to an acute *, ** and *** indicate p \ 0.05, \ 0.01 and \ 0.001, challenge with MBP. Dose-dependent secretion of cytoki- respectively, versus the vehicle (PBS)-treated group nes in the serum of (DR2 9 Ob1)F1 mice 2 h after (n = 4–5). ANOVA analysis of variance, IFN interferon, subcutaneous injection of MBP (a–d). Data were analyzed IL interleukin, MBP myelin basic protein, PBS phosphate- by ANOVA followed by Dunnett’s multi-comparison test. buffered saline or immediately after (dpi 8) the appearance of DISCUSSION the first signs of disease [14]. To study this effect in more detail, mice were treated with four Progressive axonal loss secondary to immune doses of ATX-MS-1467 at the onset and the peak cell infiltration into the CNS, inflammation- of disease. As shown here, therapeutic treat- mediated damage and demyelination, and ment with ATX-MS-1467, both early and late diminishing repair are widely considered to be after EAE induction in (DR2 9 Ob1)F1 mice, the pathological correlates of clinical disability reduced inflammation and T-cell and B-cell in MS [23]. In the preclinical study published by infiltration in the spinal cord, reduced Streeter et al., treatment with ATX-MS-1467 demyelination and reduced BBB disruption. prevented the increase of neurological disability These effects were more profound in the ani- when the drug was administered prior to (dpi 4) mals treated early in the course of EAE than in Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Concentration (pg/mL) Neurol Ther (2018) 7:103–128 119 those treated later, thus demonstrating the an anti-inflammatory cytokine profile was seen neuroprotective benefit of early intervention in treated mice in response to repeated doses of with ATX-MS-1467 in this animal model of MS. ATX-MS-1467 but not in mice treated with Although the exact association between the vehicle or in response to HLAbp, which binds to severity of disability and the extent of damage the MHC but contains none of the epitopes in the CNS is not fully understood [24], these involved in the autoimmune response to MBP. results show good agreement between the Consideration of ATX-MS-1467 as a tolero- abatement of inflammatory markers and genic approach presupposes that a rebalance of demyelination and improvement in clinical antigen-specific immunity occurs sometime score, particularly when treatment is started after the initial treatment. It also presupposes soon after the onset of EAE. that because the immune system is, at least Autoantigen T-cell responses to MBP are temporally, reprogramed against a particular involved in the pathogenesis of MS [25–28]. antigen, the effect of ATX-MS-1467 persists T-cell responses to MBP were initially identified when the drug is cleared from the circulation. in eight regions of MBP, of which five (MBP The induction of tolerance in type-1 helper 30–44, MBP 83–99, MBP 130–144, MBP 140–154 (Th1) cells after repeated peptide immunization and MBP 156–170) are the regions most fre- of MBP-specific-TCR transgenic mice led to a quently recognized by T cells. ATX-MS-1467 is a reduction in the capacity of these cells to pro- mixture of four of these peptides (MBP 30–44 liferate and a shift to an IL-10-secreting phe- [ATX-MS1]; MBP 131–145 [ATX-MS4]; 140–154 notype [7]. IL-10 suppresses dendritic cell [ATX-MS6]; MBP 83–99 [ATX-MS7]), which act maturation and prevents Th1-cell differentia- as apitopes and suppress EAE in a humanized tion, which is proposed as the negative arm of a mouse model [14]. feedback loop to limit Th1-driven Direct targeting of key regulators in the immunopathology [11]. As shown here, with development and maintenance of autoreactiv- repeated ATX-MS-1467 treatment, the concen- ity aims to silence or reprogram autoreactive T trations of the pro-inflammatory cytokines IL- cells in the periphery back to a regulatory phe- 17 and IFNc diminished relative to the con- notype, and thus promote a tolerogenic state to centration of IL-10, but the relation between IL- the targeted protein. Such targeted treatment 2 and IL-10 was less clear. IL-2 is secreted by may help to avoid many of the bystander effects effector cells but not by regulatory cells, seen with broad suppression of the immune whereas it is removed, via receptor-mediated system. An ex vivo antigen recall assay and internalization, by both subtypes [29]. IL-2 is a in vivo quantification of cytokine concentra- broad regulator of Th-cell differentiation, nota- tions in response to an acute challenge with bly in the differentiation of Th cells into both MBP and also in response to chronic dosing effector and cytolytic T cells, and is vital for the with ATX-MS-1467 were used to investigate the normal development of iTregs [29]. Therefore, effect of ATX-MS-1467 on the immune envi- IL-2 may have a gateway role in the early ronment. ATX-MS-1467 elicited a dose-depen- development of tolerance that diminishes with dent switch to an IL-10-dominant environment the development of a tolerogenic state follow- after chronic treatment. Following repeated ing repeated exposure. Such a role would be exposure to ATX-MS-1467, there was a positive consistent with the results reported here, as well and robust shift in the ratio of anti-inflamma- as with other reports in which single peptide tory to pro-inflammatory cytokines, which exposure led to an initial variable peripheral persisted for as long as the mice were exposed to T-cell death that was transient and incomplete. ATX-MS-1467. Some aspects of the effect Taken in isolation, the reduction in ex vivo induced by ATX-MS-1467 remained, at least secretion of IL-2 from splenocytes of mice trea- partially, beyond 21 days, such as the shift in IL- ted in vivo with ATX-MS-1467 does not allow us 10/IFNc ratio; however, this effect was not as to conclude whether the treatment led to an persistent as the effect on the ratio of other increase in MBP-specific Tregs, a reduction in cytokine pairs (e.g., IL-10-to-IL-17). The shift to MBP-specific effector Tregs or both, because, in 120 Neurol Ther (2018) 7:103–128 Week -3 Week -2 Week -1 Week 0 Legend: Vehicle vehicle ATX-MS-1467 (100μg three times/week) 1x 2x 2h 3x Cytokine 4x quantification 5x 7x 10x b c IL-2 IL-10 600 120 0 0 Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Number of treatments with ATX-MS-1467 Number of treatments with ATX-MS-1467 d e IL-17 IFN-γ 300 100 0 0 Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Number of treatments with ATX-MS-1467 Number of treatments with ATX-MS-1467 fg IL-10/IL-2 ratio IL-10/IL-17 ratioh IL-10/IFN-γ ratio 2.5 2.5 5 2.0 2.0 4 1.5 1.5 3 1.0 1.0 2 0.5 0.5 1 0.0 0.0 0 Number of treatments with Number of treatments with Number of treatments with ATX-MS-1467 ATX-MS-1467 ATX-MS-1467 **** **** *** ** **** **** *** ** ** *** ** *** Concentration (pg/mL) Concentration (pg/mL) Ratio Ratio Concentration (pg/mL) Concentration (pg/mL) Ratio Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Vehicle 1x 2x 3x 4x 5x 7x 10x Neurol Ther (2018) 7:103–128 121 bFig. 6 Effect of chronic dosing with ATX-MS-1467 on tolerogenic state and the recruitment of resting in vivo proinflammatory cytokine secretion. (DR2 9 Th cells from more than one compartment. This Ob1)F1 mice received from 1 to 10 treatments with is also in keeping with the more consistent 100 lg of ATX-MS-1467 or vehicle (PBS) on days 0, 2, 5, response seen following repeated exposure to 7, 9, 12, 14, 16, 18 and 20. The top panel shows the overall peptides, as compared with the response to dosing schedule (a). Serum cytokine concentrations were single exposure, which was transient, incom- assayed 2 h after the last injection. The bottom panel plete and resulted in only variable peripheral shows the absolute concentrations of each cytokine in T-cell apoptosis [11]. serum (b–e) used to generate ratios of interleukin 10 Analysis of the phenotype of the IL-10-se- versus the other cytokines, shown in the insert (f–h). Data creting cells showed that the splenic cell com- were analyzed by ANOVA followed by Dunnett’s multi- partment contained iTregs that were derived comparison test. *, **, *** and **** indicate p \ 0.05, from peripheral compartments. iTregs that co- \ 0.01, \ 0.001 and \ 0.0001, respectively, versus the ? ? express the LAG3 CD49 phenotype represent vehicle (PBS)-treated group. ANOVA analysis of variance, a population of cells that migrate to the bone IFN interferon, IL interleukin, PBS phosphate-buffered marrow following the effector stage of an saline 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 each case, the observed result (i.e., reduced IL-2 treatment also led to greater secretion of IL-10 concentration in culture supernatant) was to be from iTregs that lacked the transcription factor ? – ? expected. Nevertheless, any of these treatment Foxp3 (IL-10 Foxp3 ) but not from IL-10 Fox- effects would have a beneficial therapeutic p3 nTregs. This indicated that the main source impact. By comparison, multiple doses of Ac of IL-10 is from iTregs that originated in the 1–9 of MBP completely protected TCR-trans- periphery during the effector phase of the initial genic mice inoculated with myelin, leading to immune response to ATX-MS-1467, as opposed down-regulation of the capacity of CD4 T cells to nTregs of thymic origin [30]. The presence of to proliferate or to produce IL-2, IFNc and IL-4, ? ? ? – iTregs with LAG3 CD49b and IL-10 Foxp3 but increased the production of IL-10. This phenotypes in the spleens of ATX-MS-1467- effect was completely reversed by neutralization treated mice might indicate an overlapping cell of IL-10 [7]. population that express IL-10 transiently and at Treg cells are a crucial subpopulation of T low concentrations during differentiation [31]. cells that suppress the activity of several The results presented here raise some inter- immune-cell lineages and are thought to have esting questions for further investigation in diverse and overlapping roles in the suppression preclinical studies and clinical trials in humans of foreign-stimulated and autoantigen-stimu- that were outside the scope of this manuscript. lated immune reactions. Pharmacological Future investigators should look at performing induction of tolerance may, theoretically, occur the ex vivo assay in co-cultures of purified by expansion of the nTreg compartment, by dendritic cells and T cells, instead of whole- way of differentiation, expansion of induced spleen cultures. Furthermore, ex vivo data could Tregs (iTregs, also called type 1 regulatory T also be obtained only from leukocytes that cells), or by both mechanisms. Chronic treat- infiltrated the CNS, which is more relevant for ment with ATX-MS-1467 preferentially induced diseases such as EAE, and a wider complement differentiation of naıve T cells into iTregs and of cytokines (such as IL-6 and transforming there is also evidence of re-education of the growth factor beta) could be measured. The immune system, with sparing of normal cellular regulatory effect of IL-10 could be further con- immune surveillance. These findings are con- firmed in the EAE mouse model by the use of IL- sistent with the development of a devolved 10-neutralizing antibodies or by adoptive- 122 Neurol Ther (2018) 7:103–128 Legend: Vehicle HLAbp (100 μg, 3x/weekly) ATX-MS-1467 (100μg, 3x/weekly) MBP (300μg) Chronic Wash out Challenge Week -9 Week -8 Week -7 Week -6 Week -5 Week -4 Week -3 Week -2 Week -1 Week 0 treatment period (days) None n.a. PBS None n.a. MBP Variable wash-out between 2-42 days HLAbp Variable MBP 2h Cytokine ATX-MS-1467 2 MBP quantification 7 days ATX-MS-1467 7 MBP 21 days ATX-MS-1467 21 MBP 42 days ATX-MS-1467 42 MBP b IL-2 c IL-10 4000 160 **** 3000 **** ** *** * ## ## ## ## ## ## ### 0 0 Chronic treatment none none H LAbp ATX-MS-1467 Chronic treatment none none H LAbp ATX-MS-1467 † † Wash-out (days) none none 2-42 2 7 21 42 Wash-out (days) none none 2-42 2 7 21 42 Challenge PBS MBP Challenge PBS MBP d IL-17 e IFN-γ 500 500 ** ** 400 400 300 300 200 200 100 100 # # # # 0 0 Chronic treatment none none H LAbp ATX-MS-1467 Chronic treatment none none H LAbp ATX-MS-1467 † † Wash-out (days) none none 2-42 2 7 21 42 Wash-out (days) none none 2-42 2 7 21 42 Challenge PBS MBP Challenge PBS MBP f IL-10/IL-2 ratio g IL-10/IL-17 ratio h IL-10/IFN-γ ratio 1.4 8 8 #### #### **** 1.2 #### 6 6 **** 1.0 0.8 #### 4 4 ## 0.6 *** 0.4 2 2 *** *** 0.2 *** *** *** 0.0 0 0 Chronic none none H LAbp ATX-MS-1467 Chronic none none H LAbp ATX-MS-1467 Chronic none none H LAbp ATX-MS-1467 treatment treatment treatment Wash-out none none 2-42 2 7 21 42 Wash-out none none 2-42 2 7 21 42 Wash-out none none 2-42 2 7 21 42 † † † (days) (days) (days) Challenge PBS MBP Challenge PBS MBP Challenge PBS MBP Ratio Concentration (pg/mL) Concentration (pg/mL) Ratio Concentration (pg/mL) Concentration (pg/mL) Ratio Neurol Ther (2018) 7:103–128 123 bFig. 7 Effect of chronic treatment with ATX-MS-1467 ATX-MS-1467 has so far been restricted to ani- on the ratio of interleukin 10-to-proinflammatory cytoki- mals designed to model the MS-associated HLA- nes after an acute challenge with myelin basic protein. The DR2b allele; therefore, the generalizability of length of the wash-out period is indicated by the arrows in the treatment for patients who do not have the schematic representation of the study design (a). The genetic susceptibility and the efficacy in absolute serum cytokine concentrations (b–e) were used to patients with different forms of MS will need to generate ratios of interleukin 10 versus the other cytokines be established. shown in the insert (f–h). Data were analyzed by ANOVA Furthermore, in the absence of a model that followed by Dunnett’s multicomparison test. *, **, *** and responds to all the peptides in the ATX-MS- **** indicates p \ 0.05, \ 0.01, \ 0.001 and \ 0.0001, 1467 sequence, the model used in this study # ## ### respectively, versus naı¨ve mice (first column). , , and was constructed around only a small region of #### indicate p \ 0.05, \ 0.01, \ 0.001 and \ 0.0001, MBP: MS7 overlapping the MBP amino acids respectively, versus mice that were challenged with MBP 81–102 [14]. This means that the efficacy of the without any prior tolerization (HLAbp column only), drug in this model is restricted to this region, n = 6–8. After 10 doses of ATX-MS-1467. ANOVA rather than the whole mix of peptides in ATX- analysis of variance, HLAbp human leukocyte antigen- MS-1467. It would be interesting to fully assess binding peptide, IFN interferon, IL interleukin, MBP the efficacy of ATX-MS-1467 in a non-trans- myelin basic protein, na not applicable, PBS phosphate- genic model, in which the MBP sequences in buffered saline the drug are part of the encephalitic T-cell repertoire of the animal. We have already reported preliminary findings that ATX-MS- transfer experiments using purified iTregs, to 1467, given before the onset of EAE, delayed gain further validation of the regulatory the onset (p \ 0.04) and severity (p \ 0.002) in potential of the iTregs. non-transgenic Lewis rats [32]. A phase 1 first- Although these current results show that in-human study in six patients with secondary ATX-MS-1467 can induce tolerance in (DR2 9 progressive MS showed that ATX-MS-1467 was Ob1)F1 mice in which EAE has been induced, safe and well tolerated [14]. This study also this effect diminished with a 7-day wash-out showed a trend towards a transient induction period in the case of IL-10/IL-17. However, the of immunotolerance 1 month after chronic effect on the IL-10-to-IFNc ratio was more per- dosing with ATX-MS-1467. A phase 2 trial of sistent. This observation is consistent with the ATX-MS-1467 in patients with relapsing/ inhibition of antigen-specific IFNc release, but remitting MS to evaluate the clinical, biologi- not of IL-17 release, in vitro from the spleno- cal and radiological effects and the safety pro- cytes of mice treated with ATX-MS-1467. These file of ATX-MS-1467 for up to 36 weeks has also data suggest that, in this model, ATX-MS-1467 been completed [33]. is a more robust treatment to curb Th1-type responses than Th17, although this falling off may also be indicative of improved efficacy and CONCLUSIONS a reduction in the need for high levels of cytokines over time. This finding could have a ATX-MS-1467 improved clinical disability and substantial effect on the regularity of dosing histological markers of inflammation and required in patients to achieve and maintain a demyelination. It also restored the integrity of state of tolerance to MBP. Studies in patients the BBB following induction of EAE in the with MS will also determine whether the humanized (DR2 9 Ob1)F1 mouse. The greatest improvements in disability can be reproduced effectiveness was seen when treatment was and whether the magnitude of the treatment given at the onset of EAE, but the effect is still effect will be sufficient to make this viable for significant and reproducible with dosing at the the treatment of patients beyond the early peak of disease. Chronic treatment with ATX- stages of the disease. Experimental testing of MS-1467 led to the development of tolerance to 124 Neurol Ther (2018) 7:103–128 a c HLAbp ATX-MS-1467 1.2 1.0 0.8 0.6 0.4 0.2 0.0 de f g 5 3 HLAbp HLAbp ** ATX-MS-1467 ATX-MS-1467 0 0 + - % IL-10 from Foxp3 CD4 lymphocytes + + % IL-10 from Foxp3 CD4 lymphocytes + + + % LAG3 CD49b from CD4 lymphocytes Neurol Ther (2018) 7:103–128 125 bFig. 8 Effect of chronic treatment with ATX-MS-1467 involved in previous project management activ- on the splenic iTreg population of (DR2 9 Ob1)F1 mice. ities and model validation relevant to this work. For a–c, the spleens were harvested and fresh cells stained as described in the methods section. a, b are representative Funding. This study was funded by EMD ? ? plots showing the populations of LAG3 CD49b cells Serono, Inc., a business of Merck KGaA, Darm- ? - - ? (CD3 CD8 B220 ) from total CD4 lymphocytes, for stadt, Germany. At the time of the study, most which the averages are shown in c. For d–g, the harvested of the authors were employees of EMD Serono, splenocytes were stimulated in vitro for 3 h in the presence Inc.; therefore, the sponsor was involved in the of PMA, ionomycin and monensin, to allow intracellular study design, the collection, analysis and accumulation and subsequent staining of IL-10. The - ? interpretation of the data and the decision to contributions of Foxp3 and Foxp3 cells to the total IL ? submit the study for publication. All authors 10-producing CD4 lymphocytes is shown in d and e, had full access to all of the data in this study which are representative dot plots of the groups of mice and take complete responsibility for the integ- treated with HLAbp (n = 8) or ATX-MS-1467 (n = 8), rity of the data and accuracy of the data respectively. The averaged data are shown in f and g. * and analysis. ** indicate p \ 0.05 and\ 0.01, respectively, by unpaired t test with Welch’s correction versus the HLAbp group Medical Writing and/or Editorial Assis- (n = 8). Foxp3 forkhead box p3, HLAbp human leukocyte tance. Medical writing support was provided by antigen-binding peptide, IL interleukin, iTreg induced Steven Goodrick, inScience Communications, regulatory T cells, PMA phorbol myristate acetate UK, and funded by Merck KGaA, Darmstadt, Germany. MBP in this model, accompanied by a shift to Authorship. All named authors meet the an immunotolerant state. The results of this International Committee of Medical Journal study indicate that ATX-MS-1467 is a potential Editors (ICMJE) criteria for authorship for this candidate in the management of MS. article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. ACKNOWLEDGEMENTS Author Contributions. Adriano Luı´s Soares de Souza wrote the manuscript, designed and ATX-MS-1467 is owned and being developed by performed the research and analyzed the data. Apitope Technology (Bristol) Limited. All of the Shuning Huang performed the research, MRI data presented here was generated under a scans and analysis. Ji-Kyung Choi performed license from Apitope Technology (Bristol) Lim- the MRI scans and analysis. Timothy Crandall ited. The authors are grateful to David Wraith performed the experiments. Stefan Rudin (University of Bristol) and Keith F. Martin (Api- designed and performed the research and ana- tope, Chepstow, Monmouthshire, UK) for sci- lyzed the data. Blake Tomkinson devised the entific exchange. The authors would like to strategy for the experiments and contributed to thank Professor Joseph Mandeville (Athinoula A. the research design. Rui Chang contributed to Martinos Center for Biomedical Imaging, Mas- the histological analysis and analyzed the data. sachusetts General Hospital, Charlestown, MA, Keith Mitchell contributed to antigen-specific USA) who helped to design the study and pro- proliferation assays and analyzed the data. vided guidance on the data analysis, and Fre´deric Shinji Okitsu contributed to the flow-cytometry Bernard (EMD Serono, Billerica, MA, USA), experiments and analyzed the data. Danielle Anneli Savinainen (EMD Serono, Billerica, MA, Graham designed the research and analyzed the USA) and Dongzi Yu (EMD Serono, Billerica, MA data. Tammy Dellovade wrote the manuscript, USA) for supporting the in vivo studies. The designed the research and analyzed the data. authors would also like to thank Paul Smith Images were acquired by Shuning Huang and Ji- (Merck, Geneva, Switzerland) and Alla Zozoulya Kyung Choi. Gadolinium leakage was evaluated (EMD Serono, Billerica, MA, USA) for being 126 Neurol Ther (2018) 7:103–128 independently by Shuning Huang, Ji-Kyung Attribution-NonCommercial 4.0 International Choi and Joe Mandeville. All authors critically License (http://creativecommons.org/licenses/ reviewed the content of the manuscript and by-nc/4.0/), which permits any noncommer- approved the final version for publication. cial use, distribution, and reproduction in any medium, provided you give appropriate credit Disclosures. Adriano Luı´s Soares de Souza is to the original author(s) and the source, provide a former employee of EMD Serono, Inc, a busi- a link to the Creative Commons license, and ness of Merck KGaA, Darmstadt, Germany. Ste- indicate if changes were made. fan Rudin is a former employee of EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. Rui Chang is a former employee of REFERENCES EMD Serono, Inc, a business of Merck KGaA, Darmstadt, Germany. 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Neurology and TherapySpringer Journals

Published: Mar 14, 2018

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