An Assay to Monitor the Engagement and Modulation of CD6 on T cells as a Clinical Biomarker of Treatment with Itolizumab

Abstract

Itolizumab is a novel first-in-class monoclonal antibody that selectively targets the co-stimulatory molecule CD6, a receptor that is highly expressed on CD4 and CD8 T cells and plays an important role in activation and migration. Monitoring target engagement and changes in receptor levels is critically important to interpreting clinical data. To evaluate the pharmacodynamic properties of itolizumab treatment on T cells in patients including those with graft versus host disease (GvHD), Precision for Medicine has developed and validated a 10-color flow cytometry assay to assess the engagement and modulation of cell-surface CD6. The assay was validated using whole blood from healthy donors. Blood was spiked with five concentrations of drug selected based on expected PK in the clinical study. The assay conditions were optimized for sensitivity, signal: noise ratio, detection of free receptor, receptor-bound itolizumab and total surface CD6. Itolizumab was detected using a fluorochrome conjugated α-human IgG1 antibody while total surface CD6 expression was assessed using an antibody to a non-competing site on CD6. For validation, pre-set criteria were used to assess inter-assay, intra-assay, inter-operator precision and post-staining stability. Technical validation was successfully met; and the assay performs within acceptable precision parameters. Measuring cell-based receptor engagement and fate in patients on immuno-modulatory therapies is very challenging. This assay was designed and validated to be both sensitive and selective in the quantification of CD6 receptor occupancy and modulation to facilitate the determination of an optimal therapeutic dose in autoimmune and inflammatory diseases.