Journal Article

Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System

Keßler, Jörg, Peter, Katja, Bonifacio, Ezio, Fuchs, Anke, Marín Morales, José Manuel, Flach, Anne-Christine, Freund, Daniel, Bornhäuser, Martin, Hölig, Kristina, Oelschlägel, Uta, Münch, Nadine, Böhm, Thea

2019

Overview

Adoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease. For the majority of applications, clinical administration of Treg requires laborious expansion and typically involves open handling for culture feeds and repetitive sampling. 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. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8 cell depletion and subsequent CD25 enrichment. 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. 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. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 10 cells with the CliniMACS Prodigy® and comparable product phenotype of >90% CD4 CD25 CD127 FOXP3 cells that had similar immunosuppressive function. 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 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).

• Publisher: Frontiers Media SA,Frontiers Media S.A
• Subject: Adoptive transfer / advanced therapy medicinal product / Anticoagulants / Autoimmune diseases / Automation / Batch Cell Culture Techniques / Biomarkers / CD25 antigen / CD4 antigen / CD8 antigen / Cell culture / Cell Separation / Cell- and Tissue-Based Therapy / Chimeric antigen receptors / Cloning / closed system / Good Manufacturing Practice / Graft-versus-host reaction / Humans / Immunology / Immunophenotyping / Immunoregulation / Immunotherapy, Adoptive - methods / Leukapheresis / Lymphocytes / Lymphocytes T / Manufacturers / Manufacturing / Phenotypes / Prodigies / Rapamycin / regulatory T cells / T cell receptors / T-Lymphocytes, Regulatory - cytology / T-Lymphocytes, Regulatory - immunology / T-Lymphocytes, Regulatory - metabolism / treg cell therapy / Treg expansion