Credit: Macmillan Publishers Limited

T cells engineered to express a chimeric antigen receptor (CAR) for the B cell antigen CD19 have had impressive clinical success in treating B cell malignancies, but CAR T cells have yet to show therapeutic efficacy for other types of tumour. Kagoya et al. describe a new CAR construct that mimics cytokine-induced signalling, resulting in superior antitumour effects both in vitro and in vivo.

a new CAR construct that mimics cytokine-induced signalling, resulting in superior antitumour effects

Full T cell activation requires antigen-dependent signals through the T cell receptor (TCR; signal 1), co-stimulatory signals for example through CD28 (signal 2) and cytokine-induced signals (signal 3). Current CAR constructs provide signals 1 and 2, but not signal 3. Previous studies have shown that forced expression of cytokine genes in these CAR T cells improves their antitumour efficacy but is associated with severe side effects. Therefore, the authors sought to design a CAR construct that provides signal 3 only when the CAR T cell sees its specific antigen.

To achieve this, they engineered a CD19-specific CAR to express the truncated cytoplasmic domain of IL-2 receptor β-chain (ΔIL-2Rβ) between the cytoplasmic domains of CD28 (signal 2) and CD3ζ (signal 1), and added a YXXQ motif to CD3ζ. The resulting 28-ΔIL2RB-z(YXXQ) CAR construct significantly upregulated levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) through the YXXQ motif and of pSTAT5 through ΔIL-2Rβ, in an antigen-dependent manner. This STAT activation mimics a cytokine-induced signal 3.

After in vitro antigen stimulation, 28-ΔIL2RB-z(YXXQ) CAR T cells had greater proliferation than control CAR T cells lacking signal 3, and increased IL-2 secretion and cytokine polyfunctionality. Using specific inhibitors of STAT3 and STAT5, the authors showed that STAT3 signalling prevented the terminal differentiation of T cells and maintained a CD45RA+CD62L+CCR7+ population of stem-cell-like memory CAR T cells. Gene expression profiling following antigen stimulation in vitro showed that the 28-ΔIL2RB-z(YXXQ) CAR T cells were enriched for the expression of STAT3 target genes, including those induced by IL-21, and of multiple genes encoding cytolytic effector molecules, such as granzymes. Indeed, the 28-ΔIL2RB-z(YXXQ) CAR T cells had greater cytolytic activity than control CAR T cells after repeated antigen exposure, which attenuated the effector function of the control cells.

In an in vivo model, CAR T cells were transplanted into immunodeficient mice bearing human leukaemia cell-derived tumours. In line with the in vitro results, 28-ΔIL2RB-z(YXXQ) CAR T cells persisted for longer in the peripheral blood and had increased proliferation and cytokine polyfunctionality compared with control CAR T cells. They efficiently suppressed leukaemia progression without increasing graft-versus-host disease, resulting in significantly longer overall survival of the mice treated with 28-ΔIL2RB-z(YXXQ) CAR T cells. Similar results were observed for a solid tumour model in vivo. In light of these findings, the authors believe that clinical translation of their novel CAR is warranted, although they caution about the possible increased risk of cytokine release syndrome.