CD206-Targeted Liposomal Myelin Basic Protein Peptides in Patients with Multiple Sclerosis Resistant to First-Line Disease-Modifying Therapies: A First-in-Human, Proof-of-Concept Dose-Escalation Study

Previously, we showed that CD206-targeted liposomal delivery of co-encapsulated immunodominant myelin basic protein (MBP) sequences MBP46–62, MBP124–139 and MBP147–170 (Xemys) suppressed experimental autoimmune encephalomyelitis in dark Agouti rats. The objective of this study was to assess the safety of Xemys in the treatment of patients with relapsing-remitting multiple sclerosis (MS) and secondary progressive MS, who failed to achieve a sustained response to first-line disease-modifying therapies. In this phase I, open-label, dose-escalating, proof-of-concept study, 20 patients with relapsing-remitting or secondary progressive MS received weekly subcutaneously injections with ascending doses of Xemys up to a total dose of 2.675 mg. Clinical examinations, including Expanded Disability Status Scale score, magnetic resonance imaging results, and serum cytokine concentrations, were assessed before the first injection and for up to 17 weeks after the final injection. Xemys was safe and well tolerated when administered for 6 weeks to a maximum single dose of 900 μg. Expanded Disability Status Scale scores and numbers of T2-weighted and new gadolinium-enhancing lesions on magnetic resonance imaging were statistically unchanged at study exit compared with baseline; nonetheless, the increase of number of active gadolinium-enhancing lesions on weeks 7 and 10 in comparison with baseline was statistically significant. During treatment, the serum concentrations of the cytokines monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-7 decreased, whereas the level of tumor necrosis factor-α increased. These results provide evidence for the further development of Xemys as an antigen-specific, disease-modifying therapy for patients with MS.


Introduction
Multiple sclerosis (MS) is a chronic neurodegenerative disease with an evident autoimmune background resulting in inflammatory demyelination and axonal and neuronal injury Electronic supplementary material The online version of this article (doi:10.1007/s13311-016-0448-0) contains supplementary material, which is available to authorized users. [1]. MS, which was first described in 1868 [2], is one of the most common diseases of the nervous system. It affects people aged 20-40 years worldwide, although it has higher occurrence in women than in men and in those residing in northern than in southern latitudes. Despite its long history and the finding that immune cells rather than exogenous pathogens are responsible for MS development [3], the etiology of MS remains unclear.
Several treatment strategies for MS have been found to be moderately successful [4]. For example, the β-interferons and glatiramer acetate (GA) are disease-modifying therapies with an established history of efficacy and safety in clinical practice [5][6][7]. In addition, monoclonal antibodies binding to specific ligands have been found effective; these include natalizumab, which binds to α4 integrins [8]; daclizumab, which binds to CD25 [9]; and alemtuzumab, which binds to CD52 [10]. Natalizumab and alemtuzumab have been approved by the US Food and Drug Administration for the treatment of refractory MS, while daclizumab approval is very likely in the near future. Despite their effectiveness, however, these agents have been associated with serious adverse events (SAEs), significantly restricting their further application [4]. Novel, convenient oral therapies, including fingolimod [11], teriflunomide [12], and dimethyl fumarate [13], have shown efficacy and tolerability and have been approved for the treatment of patients with MS.
However, some patients remain refractory to these agents. This may be due to as yet unknown triggers of MS, together with high heterogeneity of this disease. Therefore, searching for novel, antigen-specific immunotherapeutic treatment options for MS is highly feasible [14]. For example, myelin basic protein (MBP), the structural component of the myelin membrane, is thought to be a primary target of the immune system during MS development [15]. Attempts have been made to induce tolerance toward MBP and its structural constituents [16][17][18][19][20], including MBP pulsing of dendritic cells [21]. In our previous studies using a newly designed MBP epitope library, we determined that MBP peptides 46-62, 124-139 and 147-170, but not 83-99, were the most immunodominant in terms of autoantibody responses in patients with MS when compared with healthy individuals and patients with other neurological diseases lacking an autoimmune background [22,23]. Nasal administration of these MBP peptides suppressed protracted relapsing experimental allergic encephalomyelitis (EAE) in dark Agouti rats [24]. Further, selected immunodominant MBP peptides encapsulated into mannosylated liposomes were reported effective in the treatment of EAE [25]. Mannosylation of these liposomes was found critical for their therapeutic efficiency, as animals that received nonmannosylated peptide-loaded liposomes were unable to recover from the first EAE attack. The most reasonable explanation that was confirmed experimentally suggests that mannosylation of liposomes significantly enhances their uptake by dendritic cells via the CD206 receptor [25], resulting in immune system tolerance towards myelin antigens. The synergistic liposome-mediated effects of coencapsulated MBP peptides reduced overall disease course, resulting in moderate severity of attacks and faster recovery from exacerbations [25]. Preclinical studies showed that administration of the designed formulation, at doses largely exceeding those proposed for humans, did not induce significant AEs in animals. The aim of the present study was to explore the AE profile and tolerability of encapsulated MBP peptides in a cohort of patients with MS. Secondary outcomes were to evaluate the effects of these peptides on the clinical course of MS.

Study Design
This was a phase I, multicenter, open-label, dose-escalating safety, and proof-of-concept study of the oligopeptides MBP 46-62 , MBP 124-139, and MBP 147-170 coencapsulated in CD206-targeted small monolammelar liposomes (Xemys; Pharmsynthez, St. Petersburg, Russia [25]) in patients with relapsing-remitting (RRMS) or secondary progressive MS with superimposed relapses (SPMS) who failed to achieve sustained responses to first-line disease-modifying therapies (FASEMS). The FASEMS clinical trial schedule is summarized in Fig. 1A. Patients received 6 weekly subcutaneous injections, on the same day each week, of Xemys at doses ascending from 50 μg to 900 μg. After the last injection, patients were followed up for 12 weeks.
The primary endpoint was the safety of Xemys, as determined by the frequency and severity of adverse events (AEs) and SAEs. To ensure patient safety, the patients were divided into 2 cohorts. Dose-limiting toxicities (DLT) and dose adjustments were assessed in the first cohort; doses in the second cohort were limited to the highest first-cohort dose not associated with DLT. If there were no DLTs in the first cohort, the dosing regimen would remain unaltered in the second cohort.
Secondary clinical endpoints included the number of relapses during the study period and Expanded Disability Status Scale (EDSS) score at the end of the trial. Secondary magnetic resonance imaging (MRI) endpoints included the number of gadolinium-enhancing T1 lesions and the total number of lesions in T2 and fluidattenuated inversion recovery (FLAIR) sequences. Laboratory endpoints included the concentrations of proand anti-inflammatory cytokines. Reasons for exclusion -primary-progressive MS -clinically relevant infection or surgical intervention <30 d.

Subjects
Subjects were screened 2 weeks before enrollment into the treatment phase of the study (Fig. 1B). The trial involved patients with RRMS or SPMS with superimposed relapses, defined as previously described [26]. Subjects were included if they were aged 18-55 years, had an EDSS from 3 to 5.5 and a ≥ 1.0 increase during the previous 6 months, and had > 1 relapse during the previous year [27]. Subjects also had to be stable for > 30 days at the time of screening and to have not received treatment with GA or β-interferons for > 30 days at the time of visit 1. Female subjects had to have negative pregnancy tests.
Subjects were excluded if they had primary progressive MS; clinically relevant infection or surgical intervention < 30 days before the screening visit; contraindications to MRI scanning, including hypersensitivity toward gadolinium; a body mass index > 40 kg/m 2 ; or hypersensitivity toward components of a test item (egg phosphatidylcholine, monomannosyl dioleyl glycerol, α-tocopherol, or lactose) or GA. Subjects were also excluded if they had liver decompensation; heart diseases; tuberculosis in anamnesis; significantly abnormal hematological or biochemical parameters; oncological diseases; previous therapy with cladribine, alemtuzumab, rituximab, natalizumab, daclizumab, or intravenous immunoglobulin; treatment with any disease-modifying therapy during the previous 6 months, including cyclophosphamide, mitoxantrone, ciclosporin, mycophenolate mofetil, azathioprine, methotrexate, or plasmapheresis; or had been administered glucocorticoids within the last month at a daily dosage equivalent to > 60 mg of prednisolone.
The study was authorized by the Russian Public Health Ministry #930 (FASEMS-01/01) issued on 28 April 2012. All patients provided written informed consent at enrollment after discussion of the study with investigators including possible alternative treatment options. Details of full medical history and MS pathogenesis were collected.

Procedures
Lyophilized Xemys consists of equimolar amounts of lyophilized, chemically synthesized MBP peptides , a n d 1 4 7 -1 7 0 (QGTLSKIFKLGGRDSRSGSPMARR) (total 0.45 mg) encapsulated in small unilamellar liposomes prepared from egg phosphatidylcholine and monomannosyl dioleyl glycerol with the addition of α-tocopherol and lactose (total 125 mg). Each dose was rehydrated in 1.0 ml sterile water immediately before administration. The starting dose, consisting of 0.05mg peptides, was chosen as the minimum to detect any unpredictable AEs without significant risk to the patients. Patients received weekly subcutaneous injections of Xemys at escalating doses over 6 weeks of 50 μg, 150 μg, 225 μg, 450 μg, 900 μg, and 900 μg, yielding a total dose of 2.675 mg.
Patients were followed-up 1, 4, and 12 weeks after administration of the last dose, corresponding to study weeks 7, 10, and 18, respectively. AEs were monitored by 12-lead electrocardiography, hematology, and laboratory tests, and scored according to the Common Terminology Criteria for Adverse Events version 4.0. Any SAEs deemed "certainly" or "likely" due to the study drug were considered DLTs, with the next lowest dose level considered the maximum tolerated dose. Patients also underwent complete neurological examinations during each study visit. An MS exacerbation was defined as a new worsening of neurological function lasting for > 24 h that was unrelated to other comorbidities.

Statistical Analysis
Demographic data, baseline characteristics, safety and tolerability variables, and other parameters under investigation were calculated using descriptive statistics. Safety and tolerability were assessed in patients who received at least 1 dose of the studied substance. AEs were grouped by dose and classified by MedDRA system organ classes and preferred terms, with severity classified by Common Terminology Criteria for Adverse Events version 4.0. Secondary endpoints were analyzed in patients who received at least 1 dose of the studied substance and underwent at least 1 assessment. The normality of the data was determined using Kolmogorov-Smirnov tests; all datasets were non-normally distributed. Changes from baseline in the number of MRI lesions were assessed by analysis of variance. Mann-Whitney t tests were used to compare between-group variables and the Wilcoxon signed rank test for within-group variables. All tests were two sided, and pvalues < 0.05 were considered significant. All statistical analyses were performed with SPSS (IBM, Armonk, NY, USA) and GraphPad Prism 6.0 (GraphPad Inc., La Jolla, CA, USA).

Results
Between April 2013 and July 2014, 20 patients with RRMS or SPMS matching all criteria were directly recruited into the trial at 4 clinical centers in the Russian Federation. Baseline characteristics of patients with MS are listed in Table 1.    (Fig. 1A). One patient who received all 6 doses of Xemys discontinued from the study after treatment period at week 6 (his own decision); 1 patient received only the first 50-μg dose and chose to discontinue after 1 week (his own decision). As no patient experienced a DLT during treatment, an maximum-tolerated dose was not reached, making it likely to be > 900 μg per week. Eight patients (40%) experienced 16 AEs (Table 3), with 11 events in 5 (25%) patients regarded as related to the Xemys injections. No SAEs, serious drug reactions, or deaths occurred during the study. Of the 16 AEs, 13, in 6 (30%) patients, were regarded as grade 1, and 3 AEs, in 2 (10%) patients, were regarded as grade 2 (Table 4). No AE met the seriousness criteria of International Conference on Harmonisation E6. All drugrelated AEs were grade 1 in severity, except for diarrhea, which was grade 2 ( Table 5). All AEs resolved without treatment and did not require interruption or discontinuation of the investigational drug.
The most common AE was local reaction at the site of injection, which was observed 8 times in 4 (20%)    Other AEs occurred only once in single patients. Xemys did not have any effect on laboratory safety tests, vital signs (body temperature, heart rate, respiration rate and blood pressure), results of physical examination, or electrocardiography parameters (heart rate, PR interval, QRS duration, and QT interval). In general, the administered doses of Xemys were considered safe and tolerable. Patients underwent MRI scans at baseline (week -2) and after treatment, at weeks 7, 10, and 18, using T1-weighted (with and without contrast), T2, and FLAIR regimens. Nineteen patients were evaluated at baseline and week 10, and 18 patients at weeks 7 and 18 ( Table 6, Fig. 2A). At baseline, 16 (84%) patients had no active gadoliniumenhancing lesions. By week 7, however, active lesions were detected in 10 (56%) patients, and at last follow-up, 8 (33.7%) patients had active lesions. Although a trend towards an increasing number of gadolinium-enhancing lesions was detected, per subject-specific analysis of MRI results at time of study exit showed that 83% of patients had 0±1 new lesions, with only 3 patients (17%) having > 1 new lesion (Fig. 2B). To assess changes in the number of MRI lesions in comparison with baseline, ANOVA was performed, with the dependent variable being the rank of the number of MRI lesions ( Table 7). All datasets were non-normally distributed.  Table 8.
To analyze the immunological consequences of Xemys administration, the concentrations of 17 serum cytokines and chemokines were analyzed at follow-up time points (Fig. 2C). Compared with baseline, MCP-1, MIP-1β, and IL-7 concentrations were significantly lower and TNF-α was significantly higher at study exit (week 18).  -0.5 ± 0.3 0.115 (-1.11 to 0.12) * Data are presented ± SE † Difference of means between baseline and weeks 7, 10, and 18 ‡ For "visit" factor assessment CI = confidence interval