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 (Cmax), area under the serum concentration-time curve from 0 to infinity (AUCinf; single doses only), AUC from time 0 to 7 days post dose administration (AUC0–7; multiple doses only), serum elimination half-life (t1/2), 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.