Thymus-derived regulatory t cells as a universal off-the-shelf anti-inflammatory therapy: technology transfer in preparation for a phase i clinical trial

Background & Aim The ability of CD4+ T regulatory cells (Tregs) to suppress immune-based pathology and promote tissue repair makes them a promising anti-inflammatory cell therapy. To date, most Treg cell therapies are autologous. An abundant source of pure, allogeneic Tregs enabling an “off-the-shelf” therapy would facilitate more widespread clinical translation. Thymus-derived tissue, routinely discarded during pediatric heart surgery, is a rich source of naïve Tregs that are potently suppressive and resistant to inflammation-induced lineage instability, a concern with blood-derived Tregs. Our group's allogeneic Tregs will be piloted as on off-the-shelf therapy in a phase 1 trial for prevention of graft vs. host disease (GVHD) post allogeneic hematopoietic stem cell transplant (HSCT). Here, we present the results of the GMP facility-based tTreg manufacturing runs to illustrate feasibility and quality in preparation for the clinical trial. Methods, Results & Conclusion: Methods CD25+CD8- thymocytes are isolated using magnetic beads (STEMCELL Technologies), cryopreserved and shipped to the Alberta Cell Therapy Manufacturing GMP facility for expansion using DynaBead Treg Expanders (Thermo Fisher Scientific, TFS) before harvest with the Lovo (ScaleReady), bead removal with the DynaCellect (TFS) and cryopreservation. Thawed cells are subjected to release and reference testing, including phenotyping for identity, purity and cytokine production, suppression assays, epigenetic testing and a flow cytometry-based residual bead assay. Product efficacy and toxicology will be tested using a xenogeneic (xeno) model of GVHD. Results The current manufacturing process yields 3-5x10E9 tTregs from 10-30x10E6 CD25+CD8- thymocytes in a 14-day expansion protocol. Phenotyping shows that the thawed tTregs are >70% apoptosis negative, >80% FoxP3+ and contain <1% CD8+ CD4- cells. tTreg identity is confirmed using a GMP-compatible Treg-specific demethylated region assay (PureQuant, TFS) and a suppression assay standardized in our lab that enables quantification of both costimulatory molecule removal and inhibition of proliferation. The automated DynaCellect system (TFS) allows rapid and thorough removal of magnetic beads as quantified by a flow cytometry-based assay developed in our lab. XenoGVHD and toxicology studies are ongoing. Conclusion These data will be presented to Health Canada in support of a clinical trial application for a phase 1 trial to reduce GVHD after allogeneic HSCT.