Toll-like receptor 4 signaling activation domains promote CAR T cell function against solid tumors.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses, there is an exciting prospect of further enhancing CAR functionality. Here, we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-ß (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human T cells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and T cell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model, MyD88 and TLR4 CAR T cells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors.

Regulation of CD19 CAR-T cell activation based on an engineered downstream transcription factor.

CAR-T cells present a highly effective therapeutic option for several malignant diseases, based on their ability to recognize the selected tumor surface marker in an MHC-independent manner. This triggers cell activation and cytokine production, resulting in the killing of the cancerous cell presenting markers recognized by the chimeric antigen receptor. CAR-T cells are highly potent serial killers that may cause serious side effects, so their activity needs to be carefully controlled. Here we designed a system to control the proliferation and activation state of CARs based on downstream NFAT transcription factors, whose activity can be regulated via chemically induced heterodimerization systems. Chemical regulators were used to either transiently trigger engineered T cell proliferation or suppress CAR-mediated activation when desired or to enhance activation of CAR-T cells upon engagement of cancer cells, shown also in vivo. Additionally, an efficient sensor to monitor activated CD19 CAR-T cells in vivo was introduced. This implementation in CAR-T cell regulation offers an efficient way for on-demand external control of CAR-T cell activity to improve their safety.