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We reported manual Treg expansion for cGvHD treatment using cell differentiation bags (Miltenyi Biotec) ( 18, 23) and since then have changed to G-Rex100 cell culture devices (Wilson Wolf manufacturing) due to enhanced growth rates, likely related to optimized gas exchange through the permeable membrane bottom, and convenient handling.

Efficient good manufacturing practice (GMP) compliant protocols for Treg expansion have been developed by us and others ( 6– 18) and in the case of CliniMACS isolated Treg, typically include rapamycin as cell culture medium supplement to prevent T effector cell outgrowth ( 11, 15, 17, 19– 22). Treg expansion requires activation through the T cell receptor (TCR) in the presence of high doses of IL-2 ( 3– 5). Most clinical applications require ex vivo expansion of Treg, classifying the cell product as advanced therapy medicinal product (ATMP). Nine years after the first in-man report, there are currently close to 30 recruiting or ongoing clinical trials administering Treg in autoimmune diseases, solid organ transplantation, pro-inflammatory diseases and graft-versus-host disease (GvHD) ( 1, 2). We anticipate this newly developed closed system expansion approach to be a starting point for the development of enhanced throughput clinical scale Treg manufacture, and for safe automated generation of antigen-specific Treg grafted with a chimeric antigen receptor (CAR Treg).

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Efficiency of expansion bead depletion was comparable to the CliniMACS ® Plus system and the final ready-to-infuse product had phenotype stability and high vitality after overnight storage. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 10 9 cells with the CliniMACS Prodigy ® and comparable product phenotype of >90% CD4 +CD25 highCD127 lowFOXP3 + cells that had similar in vitro immunosuppressive function. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We successfully integrated expansion bead removal and final formulation into the automated procedure, finalizing the process with a ready to use product for bedside transfusion. Treg were expanded with the CliniMACS Prodigy ® device using clinical-grade cell culture medium, rapamycin, IL-2, and αCD3/αCD28 beads for 13–14 days. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8 + cell depletion and subsequent CD25 high enrichment. Here we show results from our approach to translate manual Treg manufacturing to the fully closed automated CliniMACS Prodigy ® system reducing contamination risk, hands-on time, and quality variation from human intervention. For the majority of applications, clinical administration of Treg requires laborious ex vivo expansion and typically involves open handling for culture feeds and repetitive sampling. 5National Center for Tumor Diseases, Dresden, GermanyĪdoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease.

4DFG-Center for Regenerative Therapies Dresden, Center for Molecular and Cellular Bioengeneering, Technische Universität Dresden, Dresden, Germany.3Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.2Department of Hematology, Medical Clinic I, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.1GMP Facility, DFG-Center for Regenerative Therapies Dresden, Center for Molecular and Cellular Bioengeneering, Technische Universität Dresden, Dresden, Germany.José Manuel Marín Morales 1, Nadine Münch 1, Katja Peter 1, Daniel Freund 1, Uta Oelschlägel 2, Kristina Hölig 2, Thea Böhm 3, Anne-Christine Flach 3, Jörg Keßler 3, Ezio Bonifacio 4, Martin Bornhäuser 2,5 and Anke Fuchs 1,2,4 *