A Rapamycin-Based GMP-Compatible Process for the Isolation and Expansion of Regulatory T Cells for Clinical Trials

TLDR

Regulatory T cell (Treg)-based immunotherapy holds promise for inducing tolerance in autoimmunity and transplantation, but clinical translation demands production processes that meet Good Manufacturing Practice standards. This study reports a robust GMP‑compliant process for ex vivo expansion of clinical‑grade Tregs, demonstrating its feasibility for producing a final product suitable for clinical use. The isolation protocol yields a highly pure Treg population that expands 300‑fold over 36 days while retaining >75 % CD4⁺CD25⁺FOXP3⁺ purity and >80 % suppressive function. The expanded Tregs can be cryopreserved without loss of phenotype or function, and the process has been successfully applied in the ONE phase I/IIa renal transplantation trial.

Abstract

The concept of regulatory T cell (Treg)-based immunotherapy has enormous potential for facilitating tolerance in autoimmunity and transplantation. Clinical translation of Treg cell therapy requires production processes that satisfy the rigors of Good Manufacturing Practice (GMP) standards. In this regard, we report our findings on the implementation of a robust GMP compliant process for the ex vivo expansion of clinical grade Tregs, demonstrating the feasibility of this developed process for the manufacture of a final product for clinical application. This Treg isolation procedure ensured the selection of a pure Treg population that underwent a 300-fold expansion after 36 days of culture, while maintaining a purity of more than 75% CD4+CD25+FOXP3+ cells and a suppressive function of above 80%. Furthermore, we report the successful cryopreservation of the final product, demonstrating the maintenance of phenotype and function. The process outlined in this manuscript has been implemented in the ONE study, a multicenter phase I/IIa clinical trial in which cellular therapy is investigated in renal transplantation.