Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.

IL-17A secreting T cells represent a distinct lineage of CD4+ T effector cells (Th17) that have been suggested to play a role in the pathogenesis of inflammatory/autoimmune diseases such as Crohn’s disease. Precision for Medicine cryopreserves primary Th17 cells (AccuCell®) to provide an easy alternative to isolating fresh cells, for use in screening assays and functional immunological studies. The human MDR1 gene encodes a membrane protein, P glycoprotein (Pgp), whose function is to export toxic substances or metabolites from the cell. MDR1 expresssion has been demonstrated in tissues of Crohn’s disease patients and on their circulating Th17 cells. Using AccuCell® Th17 cells from healthy and diseased patients we investigate how MDR1 expression differs in the disease state and highlight the challenges of screening drugs using healthy cells as opposed to the targeted disease state. First we evaluated our cryopreserved Th17 cells to fresh isolations for surface marker, chemokines, intracellular cytokines (IL-17A, IFNγ, IL-22), and RORγt. Full retention of the tested markers were observed in the recovered cryopreserved cells. On our disease state PBMCs phenotypic and functional evaluation of MDR1+ Th17/17.1 cells included activation markers CD5, CD69, CD2, human leukocyte antigen-DR (HLA-DR) and co-stimulatory receptor CD28, compared to normal healthy donors. T-cell receptor dependent proliferation in MDR1+ Th17 cells was compared to healthy donor demographic matched Th17 cells. Utilizing AccuCell® cryopreserved Th17 cells which are fully functional empowered these studies to elucidate mechanisms as discussed above; thus eliminating fresh isolations so researchers can focus on studying the pathogenesis of these cells.

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.