The costimulatory domain in CAR T cells determines the resistance to immunosuppression by regulatory T cells

Chimeric antigen receptor (CAR)-modified T cells are intensively studied for their application in cancer patients and already proved incredible success in clinical trials. However, the choice of the intracellular signaling domain integrated into the CAR architecture can largely influence T cell function and fate, as already shown in vitro and in vivo. Moreover, especially within solid tumors regulatory T cells (Tregs) play an important role in establishing an anti-inflammatory milieu and suppressing effector cells. Consequently, endogenous Tregs might impair CAR-engrafted T cells and thereby affect treatment outcome of cancer patients. Therefore, it is of large interest to investigate the responsiveness of T cells comprising CARs with different intracellular signaling domains on Treg suppression.
To address this question, we isolated CD4+CD25- conventional T cells (Tconv) and genetically modified them to express a universal CAR (UniCAR) construct as part of our previously developed UniCAR platform technology. In contrast to conventional CARs, UniCARs are indirectly linked to their target cells via a separate antigen-specificity providing target module (TM), which allows a flexible application of UniCAR-engrafted T cells against a wide range of tumor-associated antigens. It also enables a modulation of T cell activity between an “on” and “off” status. To compare the influence of different intracellular costimulatory signals, we designed UniCARs containing either a CD3ζ, CD28-CD3ζ or CD137-CD3ζ domain.
By using a lentiviral gene transfer system for genetic modification, transduction rates of more than 80 % were achieved. Upon TM-mediated activation via the UniCAR, Tconvs containing UniCAR28/ζ produced significantly higher amounts of the pro-inflammatory cytokine TNF and the growth-related cytokine IL 2 than UniCAR137/ζ- or UniCARζ-engrafted cells. To investigate the impact of Tregs, Tconvs containing the individual UniCAR constructs were cultured in the presence of expanded, autologous CD4+CD25+CD127lowCD45RA+ Tregs for 96h. On the one hand, Tregs were pre-stimulated with anti-CD3/CD28 beads to mimic polyclonal activation via the endogenous TCR. On the other hand, an antigen-specific stimulation was achieved by engrafting Tregs with UniCARs. In both cases, UniCAR-armed Tconvs showed a distinct responsiveness on Treg suppression in dependence on the intracellular signaling domain. We observed, that in contrast to UniCAR28/ζ-armed Tconvs, UniCAR137/ζ- and UniCARζ-engrafted cells could be substantially repressed by Tregs.
In summary, we could demonstrate that Tconvs containing UniCARs with different intracellular signaling domains display not only a distinct cytokine secretion profile but also a disparate resistance against Treg suppression. These data indicate, that the chosen costimulatory signal has an impact on both the efficacy and the safety of a cancer treatment conducted with genetically modified CAR T cells.