Study design

This was a Phase 1, multicenter, open-label, dose-escalation study of single and multiple IV doses of MEDI-546 in adult subjects with SSc. The study is registered on ClinicalTrials.gov (Study MI-CP180; NCT00930683). The study protocol, protocol amendments, and subject informed consent documents were approved by Institutional Review Boards (IRBs). A list of the IRBs is provided below. Written informed consent was obtained from all subjects before study entry or any study-specific activities were carried out. An electronic data capture system was utilized for this study.

The primary objective was to evaluate the safety and tolerability of single and multiple IV escalating doses of MEDI-546. Secondary objectives of the study were to assess the PK, immunogenicity potential, and PD of MEDI-546.

Following a 28-day screening period, eligible subjects were randomized into 9 groups: 6 groups received 1 of 6 single MEDI-546 doses (0.1, 0.3, 1.0, 3.0, 10.0, or 20.0 mg/kg) sequentially, and 3 groups received 1 of 3 multiple MEDI-546 doses (0.3, 1.0, or 5.0 mg/kg/week, 4 doses in total) (Figure 1). The single doses were started at 0.1 mg/kg. The single doses were escalated sequentially. For single-dose groups ≤10.0 mg/kg, dose escalation decisions were made based on the review of safety data up to day 7 in the preceding lower dose group. The 20.0 mg/kg single-dose group and all multiple-dose groups were enrolled simultaneously, after the cumulative safety data for the lower single doses were approved. MEDI-546 was administered as IV infusion over ≥60 minutes. Administration occurred on day 0 for single-dose groups, with follow-up until day 84, and on days 0, 7, 14, and 21 for multiple-dose groups, with follow-up until day 105. Evaluations were scheduled on study days 0, 1, 7, 14, 21, 28, 56, and 84 for single-dose groups and, additionally, on days 22 and 105 for multiple-dose groups.

Subjects

Adults aged ≥18 years who met the American College of Rheumatology preliminary criteria for SSc [22] were enrolled in the study. All study participants were required to have at least moderate skin thickening (≥2 by modified Rodnan Skin Score [mRSS]) in an area suitable for repeat biopsy. Stable concomitant medication to treat SSc or other medical conditions was allowed, unless the treatment met the following exclusion criteria. Subjects were excluded if they had received any of the following medications within 28 days before study day 0: cyclophosphamide, systemic cyclosporine, or thalidomide. Subjects were excluded if they had received fluctuating doses or in excess of permitted doses of antimalarials (hydroxychlorquine >600 mg/day), mycophenolate mofetil (>3 g/day), methotrexate (>25 mg/week), leflunomide (>20 mg/day within 6 months of study entry), or azathioprine (>3 mg/kg/week); any antiviral treatment; or an investigational treatment within 28 days of study day 0. Also excluded were subjects who had received prednisone (>20 mg/day or fluctuating doses) or nonsteroidal anti-inflammatory drugs (fluctuating doses) within 14 days of study day 0. Other exclusions included history of primary immunodeficiency, severe viral infection, herpes zoster infection, cancer, and alcohol or drug abuse.

Safety and tolerability

Adverse events (AEs) and serious AEs (SAEs) were monitored at screening and at each scheduled evaluation visit. AEs and SAEs were graded on a scale of 1–5 using the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03 [23] and by relationship to study drug. Grade refers to the severity of the AE: grade 1: mild AE; grade 2: moderate AE; grade 3: severe AE; grade 4: life-threatening or disabling AE; grade 5: death related to AE.

Hematology, serum chemistry, and urinalysis were assessed at screening and at each scheduled evaluation visit, except for study days 1 and 22 (multiple doses only). Viral monitoring was performed on days 0 (before dosing), 28, 56, 84, and 105. Surveillance was performed for cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV-1 and HSV-2), and human papilloma virus (HPV). Oropharyngeal and vaginal swabs were cultured for HSV-1 and HSV-2, vaginal swabs were tested for HPV, and blood samples were tested for CMV and EBV. HPV DNA testing was performed using the Hybrid Capture 2 High-Risk assay (HC2), with a sensitivity and specificity range of 81–93% and 80–94%, respectively [24]. CMV, EBV and HSV testing were performed at ACM Global Central Laboratory (Rochester, NY, USA). CMV and EBV were detected using quantitative real-time polymerase chain reaction assays. HSV was identified by its characteristic cytopathic effect; virus typing was accomplished using fluorescent antibodies against HSV-1 and HSV-2.

Pharmacokinetics

Blood samples for PK assessments were collected at each scheduled evaluation visit. Serum concentrations of MEDI-546 were measured using a validated electrochemiluminescent (ECL) assay. In this assay, biotinylated IFNAR1 and a sulfo-TAG labeled monoclonal antibody specific for MEDI-546 were utilized as the capturing and detecting reagents, respectively. The quantitation range of the assay was 20–1280 ng/mL, for 1:10 diluted human serum.

Immunogenicity

Blood samples for anti-drug antibody (ADA) assessments were taken on days 0 (pre-dose), 28, 56, 84, and 105. The presence of ADAs in human serum samples was determined using an ECL, solution-phase, bridging immunoassay. Positive titers, expressed as reciprocal dilutions, were defined as 1:30 or higher.

Auto-antibody levels, including antinuclear antibody and anti-topoisomerase I (anti-SCL-70), were measured on day 0, as part of the baseline SSc evaluation.

Pharmacodynamics

Five type I IFN-inducible genes (RSAD2, IFI44, IFI44L, IFI27, IFI6) were selected as pharmacodynamics markers for MEDI-546 based on their prevalence and magnitude of overexpression in patients with SSc as compared with healthy controls [25, 26]. Total RNA was extracted from blood and skin biopsies using the PAXgene Blood RNA kit and the Qiagen RNeasy Fibrous Tissue Mini kit (Hilden, Germany), respectively. RNA purity and concentration were determined spectrophotometrically (260/280 > 1.9). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip® (Santa Clara, CA, USA).

The expression level of the 5-gene marker in whole blood and skin was measured using Affymetrix Human Genome U133 Plus arrays (Santa Clara, CA, USA) [25]. Blood samples were procured at each evaluation visit, except for days 21 and 22, and skin biopsies were collected on days 0, 7 (single doses only), and 28 (multiple doses only). Transcript profiling was conducted according to standard methods [27]. The rationale for the definition of positive gene signatures as >2.9 for skin and >1.8 for blood and the details of score generation are included in the Wang publication [26].

Statistical methods

No formal sample size calculation was performed, as the primary endpoint was safety and tolerability. A sample size of 33 subjects was planned, with 53 being the maximum number of subjects allowed in the study. The safety population, comprising all subjects who received MEDI-546, was used for safety and tolerability evaluations and for measurements of PD; the evaluable population for PK included all subjects in the safety population with ≥1 valid MEDI-546 serum concentration assessment; the evaluable population for immunogenicity included all subjects in the safety population with ≥1 valid immunogenicity test result post dose.

No formal statistical hypothesis testing was planned for the primary endpoint. Noncompartmental PK data analysis was performed using WinNonlin Professional version 5.2 (Pharsight Corp, St Louis, MO, USA). The maximum serum concentration (C_max), area under the serum concentration-time curve from 0 to infinity (AUC_inf; single doses only), AUC from time 0 to 7 days post dose administration (AUC_0–7; multiple doses only), serum elimination half-life (t_1/2), and systemic clearance (CL) were calculated and reported. Incidences of positive ADA were summarized for each dose group and all groups combined.

Type I IFN gene signature scores were calculated as median-fold change in the expression of 5 IFN-inducible genes in SSc subjects, with respect to a pooled panel of healthy volunteers. Positive gene signature scores were defined as >2.9 in whole blood and >1.8 in skin, using blood and skin specimens from 54 and 30 healthy volunteers, respectively (data on file). Subjects were divided into signature positive and signature negative groups based on type I IFN signature scores at baseline.

Baseline characteristics

All 34 subjects were included in the safety population and the evaluable population for PK. The population for ADA comprised 32 subjects; 2 subjects in the 5.0 mg/kg/week multiple-dose group were excluded for discontinuing treatment.

Safety and tolerability assessments

Three subjects (8.8%) reported 4 treatment-emergent SAEs (TESAEs): 1 subject in the 3.0 mg/kg single-dose group (osteomyelitis and skin ulcer), 1 subject in the 5.0 mg/kg/week multiple-dose group (vertigo), and 1 subject in the 1.0 mg/kg/week multiple-dose group (chronic myelogenous leukemia [CML]). Of these, only CML, with onset 10 months after study completion, was considered possibly related to treatment. Two subjects in the 5.0 mg/kg/week multiple-dose group discontinued treatment: 1 treatment discontinuation was due to TEAEs of head injury and vertigo, and the other due to a non-TESAE of normochromic normocytic anemia, with onset prior to administration of the first MEDI-546 dose. Both events were considered unrelated to treatment. There were no deaths in this study.

Six subjects had abnormal laboratory results, with 3 abnormalities reported as TEAEs (Table 2). Of the 6 subjects, 4 also had abnormal values at baseline and 4 had a history of similar findings. Most events were transient and resolved by the following evaluation visit. One laboratory abnormality of low red blood cell count, reported as a grade 1 TEAE of anemia, was considered possibly related to treatment.

Infections occurred in 14 subjects (41.2%). All infections were grade 1 or 2 in severity, except for 1 grade 3 osteomyelitis in the 3.0 mg/kg single-dose group that lasted 8 days and was considered remotely related to treatment. One subject in the 5.0 mg/kg/week multiple-dose group reported a grade 1 infusion-related reaction (pruritus and urticaria) that required temporary (48 minutes) interruption of dosing and was judged as probably related to MEDI-546.

Viral surveillance showed a low percentage of subjects converting from normal test results at baseline to abnormal results post MEDI-546 administration: 4/31 subjects (12.9%) for EBV and 2/24 subjects (8.3%) for HPV. There were no abnormal CMV, HSV-1, or HSV-2 test results throughout the study. All 7 subjects with a history of viral reactivation with HSV tested negative after receiving MEDI-546. One subject in the 20.0 mg/kg single-dose group with a history of viral reactivation developed oral herpes (grade 1) 52 days after dosing. The infection lasted 15 days and the investigator determined it to be unrelated to treatment.

Pharmacokinetics

After single dose administration, MEDI-546 exhibited non-linear PK at lower dose levels (<10.0 mg/kg) (Figure 2A, Table 3). Mean AUC_inf increased more than proportionally with MEDI-546 dose from 0.1 to 10.0 mg/kg, and proportionally from 10.0 to 20.0 mg/kg. Systemic clearance decreased from 40.8 to 4.67 mL/kg/day when the dose was increased from 0.1 to 20.0 mg/kg. MEDI-546 terminal t_1/2 was more prolonged at higher doses. At the highest dose level investigated (20.0 mg/kg) the terminal t_1/2 was 11.8 days.

In the multiple-dose groups, non-linear PK was also observed at lower doses (<1.0 mg/kg/week). AUC_0–7 was more than dose proportional to MEDI-546 dose from 0.3 to 1.0 mg/kg/week, and proportional from 1.0 to 5.0 mg/kg/week (Figure 2B, Table 3). Mean t_1/2 ranged from 1.1 to 6.3 days across the multiple dose range 0.3 to 5.0 mg/kg/week. After 4 weekly doses, steady-state was not reached, as shown by increasing trough (pre-dose) concentrations.

Immunogenicity

Two of 21 subjects (9.5%) in the single-dose groups and 3 of 11 subjects (27.3%) in the multiple-dose groups tested positive for ADAs following MEDI-546 administration. Most positive titers were low (≤1:30) and transient, detectable only at 1 or 2 study visits. One subject in the 0.3 mg/kg/week multiple-dose group had persistent positive titers of 1:240–1:960 between study days 28 and 105. Serum MEDI-546 levels for subjects who were positive for ADA did not appear to differ from those of subjects who were negative for ADA, in the same dose groups.

Pharmacodynamics

At baseline, 22 and 15 subjects had positive type I IFN gene signature score in whole blood and skin, respectively. Subjects with elevated baseline signature in whole blood who received MEDI-546 had suppressed gene signature; the effect seemed to be greater when the amount of drug was increased. All groups reached or approached gene signature peak inhibition at day 1 post-dose (Figures 3A and 3B).

Of the 22 subjects with a positive baseline gene signature in whole blood, 15 were in the single-dose groups and 7 in the multiple-dose groups. In single-dose groups ≥1.0 mg/kg, 11 subjects with positive baseline signature score in whole blood had ≥70% inhibition of the signature at day 14. Persistent decreases until day 84 were observed in 2 of 3 subjects in the 20.0 mg/kg group (Figure 3A). In multiple-dose groups, ≥1.0 mg/kg/week, 5 of 6 subjects had ≥70% inhibition at day 14. The inhibition persisted until day 84 in 2 of 3 subjects in the 1.0 mg/kg/week group and both subjects in the 5 mg/kg/week group (Figure 3B).

Of the 15 patients with a positive baseline skin signature, 11 were in the single-dose groups and 4 in the multiple-dose groups. In single-dose groups ≥1.0 mg/kg, MEDI-546 decreased the expression of type I IFN-inducible genes in skin at day 7 (Figure 3C). In multiple-dose groups ≥1.0 mg/kg/week, expression was decreased at day 28 (Figure 3D). Five of 11 patients in the ≥1.0 mg/kg single-dose cohorts and all 3 patients in the 1.0 mg/kg/week multiple-dose cohort had ≥70% inhibition.