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

Abstract

Chimeric antigen receptor (CAR) Tcell 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)-β (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 Tcells 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 Tcell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model, MyD88 and TLR4 CAR Tcells 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.