Plates were washed and an alkaline phosphatase-conjugated goat anti–human IgG (Jackson Immunoresearch Laboratories, West Grove, USA) antibody was added. Following 1 h incubation at 37 °C, plates were washed again and 1 mg/ml paranitro-phenyl phosphate in diethanolamine buffer was added to each well. After 30 min at 25 °C in dark place, the reactions were stopped with 3 N NaOH,

and the absorbance (405 nm) was recorded. A post-treatment OD/pre-treatment OD ratio = 2 was defined as cutoff value for positive responses. Safety was evaluated in the population who received at least one dose of itolizumab, while clinical effect was evaluated selleckchem in the evaluable population defined as patients who received at least six doses of the mAb. Patients who did not achieve an ACR20 were considered as non-responders. Patients who dropped out the study or did not attend ZD1839 ic50 physician evaluation at the time point to assess clinical effect were considered as not available. The incidence of adverse events and the proportion of patients with a clinical benefit expressed in a 20% improvement of signs and symptoms (ACR20) or superior (ACR50 and ACR 70) were reported as counts and percentages. The ACR core data set consists of seven components: swollen joint count (66 joints), tender joint count (68 joints), subject global

assessment of pain (VAS 100 mm), subject global assessment of disease activity (VAS 100 mm), physician global assessment of disease activity (AS 100 mm), and subject assessment of physical function using HAQ and eritrosedimentation rate (ESR). A total of 15 patients were enrolled in the study. Three patients were included into the three dose levels groups previously defined (0.2 mg/kg, 0.4 mg/kg and 0.8 mg/kg). Two patients were additionally included in the 0.4 mg/kg group since two patients dropped-out the study before the clinical assessment was completed (week 7). A protocol amendment to include a 0.1 and 0.6 mg/kg dose cohorts was made after initiation of the trial, with two patients accrued in each one (Table 1). Data on patient disposition, Lck demographics and other characteristics at baseline are summarized in Tables 1 and 2. The patients were

predominantly women (73%) with moderate disease activity (80%) and a median duration of the disease of 10 years across the five dose groups. Patients showed active disease at recruiting despite previous DMARD therapy, evidenced by more than four swollen and tender joints at baseline (data not shown). All patients had received two or more DMARDs before enrolment (Table 1). Since the washout period accounted for a high baseline disease activity, the clinical status immediately before the first itolizumab dose was considered as baseline (W0) (Table 3A). Fourteen patients, out of 15 that participated in the study, received the scheduled six-infusions of itolizumab. Thirteen patients reached the first assessment point of the follow-up period (week 7); while nine patients completed all the scheduled follow-up visits.