A novel assay to measure B cell responses to keyhole limpet haemocyanin vaccination in healthy volunteers and subjects with systemic lupus erythematosus.

The aim of the study was to characterize performance of a complementary set of assays to measure antigen-specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre-existing anti-tetanus antibodies. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti-KLH IgG responses rose to a mean of 65-93-fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260-170-fold above baseline. High levels of anti-tetanus IgG were detected in pre-immunization samples and their levels did not change over the course of study. Anti-KLH IgG1-4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti-KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti-KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune-therapeutics.

Best practice recommendations for the transfer of cell-based assays for the measurement of neutralizing anti-drug antibodies.

We recommend the application of a strategically designed step-wise approach to transfer cell-based assays that includes assessing analytical performance (through a fit for purpose validation and/or design of experiment robustness characterization), clinical performance (i.e., concordance) and performance or proficiency testing for long-term method monitoring. Here we focus on the application of this strategy to cell-based assays for the measurement of neutralizing anti-drug antibodies. This application is unique in that it requires a custom cell-based assay to be used over a long period of time (potentially phase 1a through the life of a marketed product) with the confidence of consistent method performance and result reporting. But, the process is adaptable to a variety of assay types and applications. We present lessons learned from two cell-based assay transfers that met relevant challenges while implementing alternative permutations of the recommended method transfer process.

Treatment of systemic lupus erythematosus patients with the BAFF antagonist "peptibody" blisibimod (AMG 623/A-623): results from randomized, double-blind phase 1a and phase 1b trials.

INTRODUCTION: Blisibimod is a potent B cell-activating factor (BAFF) antagonist that binds to both cell membrane-expressed and soluble BAFF. The goal of these first-in-human studies was to characterize the safety, tolerability, and pharmacokinetic and pharmacodynamic profiles of blisibimod in subjects with systemic lupus erythematosus (SLE). METHODS: SLE subjects with mild disease that was stable/inactive at baseline received either a single dose of blisibimod (0.1, 0.3, 1, or 3 mg/kg subcutaneous [SC] or 1, 3, or 6 mg/kg intravenous [IV]) or placebo (phase 1a; N = 54), or four weekly doses of blisibimod (0.3, 1, or 3 mg/kg SC or 6 mg/kg IV) or placebo (phase 1b; N = 63). Safety and tolerability measures were collected, and B cell subset measurements and pharmacokinetic analyses were performed. RESULTS: All subjects (93 % female; mean age 43.7 years) carried the diagnosis of SLE for ≥ 1 year. Single- and multiple-dose treatment with blisibimod produced a decrease in the number of naïve B cells (24-76 %) and a transient relative increase in the memory B cell compartment, with the greatest effect on IgD(-)CD27+; there were no notable changes in T cells or natural killer cells. With time, memory B cells reverted to baseline, leading to a calculated 30 % reduction in total B cells by approximately 160 days after the first dose. In both the single- and multiple-dosing SC cohorts, the pharmacokinetic profile indicated slow absorption, dose-proportional exposure from 0.3 through 3.0 mg/kg SC and 1 through 6 mg/kg IV, linear pharmacokinetics across the dose range of 1.0-6.0 mg/kg, and accumulation ratios ranging from 2.21 to 2.76. The relative increase in memory B cells was not associated with safety signals, and the incidence of adverse events, anti-blisibimod antibodies, and clinical laboratory abnormalities were comparable between blisibimod- and placebo-treated subjects. CONCLUSIONS: Blisibimod changed the constituency of the B cell pool and single and multiple doses of blisibimod exhibited approximate dose-proportional pharmacokinetics across the dose range 1.0-6.0 mg/kg. The safety and tolerability profile of blisibimod in SLE was comparable with that of placebo. These findings support further studies of blisibimod in SLE and other B cell-mediated diseases. TRIAL REGISTRATION: Clinicaltrials.gov NCT02443506 . Registered 11 May 2015. NCT02411136 Registered 7 April 2015.