Clinical grade expansion protocol for the manufacture of thymus-derived Treg cells for clinical application

Adoptive transfer of regulatory T cells (Tregs) has provided promising results in treating autoimmune disorders, transplant rejection and graft versus-host disease in early clinical trials. However, major challenges remain for developing a standardized and robust good manufacturing practice (GMP)-compliant cell product which is severely hampered by low frequency of Tregs in circulation and laborious ex vivo expansion.

Paediatric thymuses routinely obtained during heart surgery have been shown by us and others to be a valuable source of large numbers of pure Tregs (Thy-Tregs). Here we show results from our process development approach including systematic laboratory-scale testing of activation reagents, restimulation timing, and cryopreservation to translate our expansion protocol of Thy-Tregs into a clinical grade cell product.

Thy-Tregs obtained through CD8⁺ cell depletion and subsequent CD25⁺ enrichment were expanded with αCD3/αCD28 beads in the presence of Rapamycin and IL-2 for 10-23 days using G-Rex bioreactors. We successfully embedded bead removal and final formulation of a cryopreserved cell product ready to be used at bedside transfusion.

This process has proved the capability of efficiently producing high number of functional Thy-Tregs, which will be administered as cell therapy in children undergoing heart transplantation (ATT-Heart, ISRCTN15374803), and enhancing the potential of using expanded Thy-Tregs for broad-ranging therapeutic applications.