Study design and conduct
This open-label, single arm multi-institutional study was planned for approximately 110 subjects. The original study protocol stated that enrollment should terminate if 3 or more subjects experienced tremelimumab-related adverse events (AEs) during or immediately after the infusion. All subjects signed a written informed consent approved by the Institutional Review Board (IRB) at each study site. The study was conducted in accordance with local regulations, the guidelines for Good Clinical Practice (GCP), and the principles of the current version of the Declaration of Helsinki. The study opened to accrual at 7 US centers, was sponsored by Pfizer Inc. (New York, NY), and had the clinical trial registration number NCT00585000.
Study objectives and assessments
The primary objective was to assess the safety and tolerability of tremelimumab at 15 mg/kg as a one-hour infusion. Secondary objectives included characterizing the pharmacokinetics (PK), monitoring for human anti-human antibody (HAHA), and assessing anti-tumor activity by best overall tumor response rate using Response Evaluation Criteria in Solid Tumors (RECIST) criteria.
Eligible subjects were aged 18 years or older with histologically confirmed stage III or IV melanoma considered to be surgically incurable, with evidence of at least 1 measurable or non-measurable lesion according to RECIST criteria, with an Eastern Cooperative Oncology Group (ECOG) performance status of 1 or lower, with adequate bone marrow, hepatic, and renal function. Subjects were excluded if they had had a potential requirement for systemic corticosteroids or concurrent immunosuppressive drugs, had an inherited or acquired immunodeficiency, had a history of chronic autoimmune disease, inflammatory bowel disease, active or chronic viral hepatitis, uveitis or melanoma-associated retinopathy. Patients with a history of brain metastases were eligible if they had been adequately treated with surgery or stereotactic radiosurgery and were stable for at least 3 months prior to enrollment.
Subjects received an intravenous administration of tremelimumab at a dose of 15 mg/kg via infusion pump as a 1-hour infusion, repeated every 90 days up to a maximum of 4 doses.
Safety, efficacy, and pharmacokinetic evaluations
Adverse events (AEs) were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE Version 3.0). Radiologic and clinical evaluations were performed within 28 days prior to dosing and within 10 days prior to the planned start of each 90-day cycle. Blood samples for pharmacokinetic analyses were obtained just prior to administration of tremelimumab for every treatment cycle (cycles 1–4), with multiple additional samples taken in cycle 1 (1 and 6 hours after the end of infusion, on days 2, 3, 8, 15, 30, 45, 60 and 75 with ± 2 days to accommodate scheduling problems) to fully characterize the pharmacokinetic profile of the infusion regimen.
Plasma samples were analyzed for tremelimumab concentrations using a validated, sensitive and specific enzyme-linked immunosorbent assay (ELISA) (ALTA Analytical Laboratory, San Diego, CA) as previously described
[5, 6]. The PK assay underwent formal methodology optimization, with linear calibration of standard responses over the range of 7.8 to 250 ng/mL using a 5 parameter curve-fit weighted model. Those samples with concentrations above the upper limits of quantification were adequately diluted into the calibration range. The lower limit of quantification (LLOQ) for tremelimumab was 7.8 ng/mL. Assay precision, expressed as the between-day coefficients of variation (CV [%]) of the mean estimated concentrations of quality control samples ranged from 9.34% to 11.5% for low (25.0 ng/mL), medium (50.0 ng/mL), high (100 ng/mL), and diluted (25.0 ng/mL) concentrations. Samples below the LLOQ were set to 0 ng/mL for analysis. Nominal sample collection times were used for the PK analysis. The concentration-time data were analyzed by non-compartmental analysis. Parameters of tremelimumab disposition included clearance (CL), volume of distribution at steady state (Vss), and terminal phase half-life (t1/2). Parameters of tremelimumab exposure included plasma concentration of tremelimumab one hour after the end of infusion (Cmax) and area under the concentration curve from time zero to infinity (AUCinf). Comparison PK data is presented from study A3671008, a pivotal phase II study in which tremelimumab was administered as a 5-hour infusion (11).
Human anti-human antibody (HAHA) assessment
A blood specimen for HAHA was obtained just prior to administration of tremelimumab every treatment cycle. In cycle 1, blood specimens were also obtained on days 30 and 60. HAHA samples were analyzed for the presence or absence of anti-tremelimumab antibodies following a tiered approach using screening, confirmation and titer/quantitation as previously described
. The semi-quantitative ELISA with electrochemiluminescence detection (ECL) included a positive control (cynomolgus anti-CP675,206 antibody sera) and negative control (pooled normal human plasma). Assay precision, expressed as the between-day CV%, was less than 16.7% for the positive control (used to calculate assay cut point) and 13.1% for the negative control.
The primary goal of the study was to demonstrate that the infusion-related reaction rate for one-hour infusion did not exceed 5% with a one-sided exact binomial test at 10% significance level (alpha=0.10). 110 subjects would provide 90% power to reject the null hypothesis (infusion-related reaction rate ≥5%) when the true infusion-related reaction rate did not exceed 1%.