Possible alternative strategies to implement basophil activation testing in multicentric studies.

The Basophil Activation Test (BAT) enables flow cytometry characterization of basophil reactivity against specific allergenic molecules. The focus now revolves around democratizing this tool, but, as blood sample stability could be challenging, after having developed a simplified approach, herein, we aimed to characterize two strategies for implementing BAT in multicentric studies: store and ship blood before or after sample processing. Fresh heparin- and EDTA-anticoagulated whole blood samples followed both BAT workflows: "collect, store, process & analyze" or "collect, process, store & analyze". Storage temperatures of 18-25 °C or 2-8 °C and preservation times from 0 to 7 days were considered. Interleukin-3 was also evaluated. With the "collect, store, process & analyze" workflow, heparin-anticoagulated blood and 18-25 °C storage were better than other conditions. While remaining possible, basophil activation exhibited a possible reactivity decay after 24 h. Under the conditions tested, interleukin-3 had no role in enhancing basophil reactivity after storage. Conversely, the "collect, process, store & analyze" workflow demonstrated that either heparin- or EDTA-anticoagulated blood can be processed and kept up to 7 days at 18-25 °C or 2-8 °C before being analyzed. Various strategies can be implemented to integrate BAT in multicentric studies. The "collect, store, process & analyze" workflow remains a simplified logistical approach, but depending on time required to ship from the clinical centers to the reference laboratories, it might not be applicable, or should be used with caution. The "collect, process, store & analyze" workflow may constitute a workflow improvement to provide significant flexibility without impact on basophil reactivity.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n = 56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3 mg/kg), or subcutaneous iscalimab (3 mg/kg), or placebo. Rheumatoid arthritis patients (n = 20) received single doses of intravenous iscalimab (10 or 30 mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4 µg/mL. In subjects receiving 3 mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Gene expression profiling of immunomagnetically separated cells directly from stabilized whole blood for multicenter clinical trials.

BACKGROUND: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. METHODS: Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). RESULTS: Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. CONCLUSIONS: The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.