Antigen-specific redirection of human regulatory T cells via a universally applicable chimeric antigen receptor technology

Regulatory T cells (Tregs) play a fundamental role in preventing inflammatory diseases and, therefore, their adoptive transfer emerged as a promising therapeutic strategy for the treatment of autoimmunity, graft rejection and Graft-versus-Host disease. However, preclinical animal models have already substantiated that the application of antigen-specific instead of polyclonal Tregs results in a far more efficient suppression of pathogenic immune reactions. Due to their low frequency in human peripheral blood, the isolation of Tregs with defined antigen specificity is a highly time-consuming and labour-intensive process that does not yet provide therapeutically relevant cell numbers. To overcome this obstacle, we equipped polyclonal Tregs with a novel modular chimeric antigen receptor (CAR) technology called UniCAR.
Unlike conventional CARs, the UniCAR binding domain does not directly recognize a target cell antigen but a small peptide epitope, which is a subunit of a separate target module (TM) providing antigen specificity. Hence, UniCAR-armed Tregs are silenced until they encounter the TM that mediates their cross-linkage with target cells. Therefore, this novel CAR technology not only allows for precise regulation of Treg activity between an “on” and “off” status but also enables their specific retargeting towards any desired antigen simply by replacing the TM.
To additionally compare the influence of different costimulatory signals on Treg properties and functionality, UniCARs were generated and introduced comprising either a CD3ζ, CD28-CD3ζ or CD137-CD3ζ signaling domain. For generation of UniCAR-expressing Tregs the CD45RA+ subpopulation was used, as these cells show the highest capacity in preserving Treg phenotype and functionality ex vivo.
Thus, highly pure, sorted CD4+CD25+CD127lowCD45RA+ Tregs were genetically modified by using a lentiviral gene transfer system resulting in an average of 80 % UniCAR+ Tregs. These UniCAR-armed Tregs maintain their phenotype (≥ 93 % FOXP3+) and expand approximately 150- to 200-fold already after 8 days of in vitro culture. In addition, in the presence of target cells and a respective TM the genetically modified Tregs are activated antigen-specifically as shown by CD69 and LAP upregulation as well as an increased FOXP3 expression level. Most importantly, upon TM-mediated restimulation via the UniCAR, Tregs efficiently suppress proliferation and overall expansion of bead-activated autologous T effector cells.
Taken together, our results underline the enormous clinical potential of UniCAR-armed Tregs as this technology facilitates an antigen-specific activation of polyclonal Tregs at the side of inflammation where they subsequently exert their suppressive capacity. In addition, the UniCAR system enables a precise control over Treg activity. Moreover, the UniCAR-equipped Tregs can be applied for treatment of a wide range of inflammation-related diseases as their antigen specificity can be easily modified just by exchanging the TM.