Abstract
Background
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.
Methods
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.
Results
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.
Conclusion
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.