Unlocking the potential of Tregs: innovations in CAR technology

Abstract

Regulatory T cells (Tregs) adoptive immunotherapy is emerging as a viable treatment option for both autoimmune and alloimmune diseases. However, numerous challenges remain, including limitations related to cell number, availability of target-specific cells, stability, purity, homing ability, and safety concerns. To address these challenges, cell engineering strategies have emerged as promising solutions. Indeed, it has become feasible to increase Treg numbers or enhance their stability through Foxp3 overexpression, post-translational modifications, or demethylation of the Treg-specific demethylated region (TSDR). Specificity can be engineered by the addition of chimeric antigen receptors (CARs), with new techniques designed to fine-tune specificity (tandem chimeric antigen receptors, universal chimeric antigen receptors, synNotch chimeric antigen receptors). The introduction of B-cell targeting antibody receptor (BAR) Tregs has paved the way for effective regulation of B cells and plasma cells. In addition, other constructs have emerged to enhance Tregs activation and function, such as optimized chimeric antigen receptors constructs and the use of armour proteins. Chimeric antigen receptor expression can also be better regulated to limit tonic signaling. Furthermore, various opportunities exist for enhancing the homing capabilities of CAR-Tregs to improve therapy outcomes. Many of these genetic modifications have already been explored for conventional CAR-T therapy but need to be further considered for CAR-Tregs therapies. This review highlights innovative CAR-engineering strategies that have the potential to precisely and efficiently manage immune responses in autoimmune diseases and improve transplant outcomes. As these strategies are further explored and optimized, CAR-Treg therapies may emerge as powerful tools for immune intervention.

Keywords: cell therapy; chimeric antigen receptor (CAR); genetic engineering; immunotherapy; regulatory T cells; tolerance.

FIGURE 1

CAR-Treg development workflow. For CAR-Treg development, peripheral blood mononuclear cells (PBMC)s can be retrieved from various sources, including the peripheral blood, umbilical cord blood, or even the thymus. Tregs are then isolated through magnetic enrichment or sorted as CD4⁺CD25^hiCD127l^ow cells. For naïve Tregs, CD45RA^hi cells are then selected. Given that obtaining Tregs fromPBMCs can yield limited quantities due to their low proportion, conventional CD4⁺ T cells overexpressing Foxp3 emerge as a possible substrate for CAR-Treg production. Foxp3 can be expressed in those cells after viral transduction, TSDR demethylation, HDR, or the use of HDAC inhibitors. Then, CAR constructs can be integrated into the cells by viral transduction, HDR, or CRISPR-Cas9 technology, among others. To date, four types of CAR constructs have been tested in Tregs: classical CAR, Foxp3-CAR, BAR, and UCAR. Other constructs have, until now, been exclusively tested in effector T cells but holds potential as CAR-Treg therapies: SynNotch-CAR, TanCAR, and Armour-CAR. Then, the desired CAR-Tregs can be infused into patients to treat and/or prevent graft-versus-host disease, transplant rejection, allergies, and autoimmune diseases. CAR, Chimeric Antigen Receptor; TSDR, Treg-specific demethylated region; HDR, Homology-Directed Repair; HDAC, Histone deacetylases; BAR, B cell-targeting Ab receptors; UCAR, Universal CAR; SynNotch, Synthetic Notch CAR; TanCAR, Tandem CAR. Created with BioRender.com.

FIGURE 2

Progress in CAR design and strategies. Basic CARs are divided usually into three generations based on the number of co-stimulatory domains associated with CD3z, CD28 and 4-1BB being the most common. In Tregs, BAR receptors replace the scFv portion for a target protein recognized by the BCR, enabling interaction between Tregs and B cells and inhibiting antibody production. Incorporating “armour proteins” into CAR-T cells improve their function and provides precise control over their activity, enhancing the safety and efficacy of CAR-T therapies in various diseases. These strategies can also be applied to CAR-Tregs to enhance their suppressive capacity and survival. SynNotch CAR strategy employs a positive feedback receptor to regulate CAR-T cell activity, releasing a transcription factor that induces the expression of a CAR targeting a second antigen following recognition of the initial antigen. Bispecific TanCAR cells can recognize two different antigens using distinct scFv portions, but only one of them is required for cell activation, thereby avoiding antigen escape. UCARs enable the binding to target cells through independent and antigen-specific switch modules, allowing for adaptability to different tissue antigens by exchanging adapters. Finally, Foxp3-CAR allows for an increase and stable expression of Foxp3 in Tregs, leading to significant improvements in the safety and efficacy of CAR-Treg products. BAR, B cell-targeting Ab receptors; BCR, B cell receptor; CAR, Chimeric antigen receptor; Foxp3, Forkhead box 3; scFv, Single-chain variable fragment; SynNotch CAR, Synthetic Notch CAR; TanCAR, Tandem CAR; Treg, Regulatory T cells; UCAR, Universal CAR. Created with BioRender.com.