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| Home > Publications database > Automated GMP-compatible production of universal CAR Tregs for organ-targeted tolerance induction. |
| Journal Article | DKFZ-2025-03007 |
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2025
BioMed Central
London
Abstract: Adoptive transfer of regulatory T cells (Tregs) has demonstrated safety, feasibility and early signs of efficacy in promoting immunological tolerance in inflammatory conditions such as graft-versus-host disease (GvHD). Chimeric antigen receptor (CAR)-engineered Tregs offer localized activation and suppression compared to polyclonal Tregs, but their clinical translation is limited by high manufacturing costs, lengthy developing times and fixed single-antigen specificity. To address these limitations, we employed the universal adapter Reverse CAR (RevCAR) system, which harbors a peptide epitope lacking intrinsic antigen specificity but provides flexibility in targeting through the use of an antigen-specific RevCAR Target Module (RevTM). As a proof-of-concept, we used a RevTM targeting carcinoembryonic antigen (CEA), which is highly expressed in the gastrointestinal (GI) tract, as a potential strategy to achieve localized immunosuppression in GI acute GvHD.To support clinical translation, we established an automated, GMP-compatible, clinical-scale manufacturing process. Tregs were magnetically enriched from leukapheresis using the CliniMACS® Plus, followed by high-purity sorting on the MACSQuant® Tyto®. The sorted cells were virally transduced and the RevCAR Tregs were expanded on the CliniMACS Prodigy® to obtain clinically relevant cell numbers. The harvested products were evaluated for phenotype, stability, antigen specificity and suppressive function.Across five manufacturing runs, Tregs (CD4+CD25highCD127lowFOXP3+) with a median initial purity of 94% were expanded to achieve a median therapeutic yield of 602 × 106 cells. The final product maintained a high purity (median: 91.9%) and exhibited high RevCAR expression (median: 60% RevCAR+). Mass cytometry analysis revealed that expanded RevCAR Tregs predominantly exhibited a central memory phenotype with high expression of functional and homing markers. Under experimental pro-inflammatory conditions, the cells maintained stable FOXP3 and Helios expression with minimal pro-inflammatory cytokine production. Importantly, RevCAR Tregs showed antigen-specific activation upon target engagement via the CEA-specific RevTM and robust, dose-dependent suppression.The study establishes a scalable, GMP-compatible process for manufacturing pure, stable and functional universal RevCAR Tregs for clinical applications. Furthermore, the RevCAR system offers a promising approach toward an allogenic, off-the-shelf Treg therapy capable of treating diverse immune-mediated diseases with spatial precision.
Keyword(s): T-Lymphocytes, Regulatory: immunology (MeSH) ; T-Lymphocytes, Regulatory: cytology (MeSH) ; Humans (MeSH) ; Receptors, Chimeric Antigen: metabolism (MeSH) ; Immune Tolerance: immunology (MeSH) ; Automation (MeSH) ; Organ Specificity (MeSH) ; Carcinoembryonic Antigen: metabolism (MeSH) ; Carcinoembryonic Antigen: immunology (MeSH) ; Graft vs Host Disease: immunology (MeSH) ; Automated expansion ; Closed-system manufacturing ; Good manufacturing practice ; Precision immunotherapy ; Regulatory T cells ; Treg cell therapy ; Universal adapter CAR ; Receptors, Chimeric Antigen ; Carcinoembryonic Antigen
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