Decoding CAR T cell phenotype using combinatorial signaling motif libraries and machine learning.

Chimeric antigen receptor (CAR) costimulatory domains derived from native immune receptors steer the phenotypic output of therapeutic T cells. We constructed a library of CARs containing ~2300 synthetic costimulatory domains, built from combinations of 13 signaling motifs. These CARs promoted diverse human T cell fates, which were sensitive to motif combinations and configurations.

T cell circuits that sense antigen density with an ultrasensitive threshold.

Overexpressed tumor-associated antigens [for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2)] are attractive targets for therapeutic T cells, but toxic "off-tumor" cross-reaction with normal tissues that express low levels of target antigen can occur with chimeric antigen receptor (CAR)-T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive-feedback circuit that allows human cytotoxic T cells to discriminate targets on the basis of a sigmoidal antigen-density threshold. In this circuit, a low-affinity synthetic Notch receptor for HER2 controls the expression of a high-affinity CAR for HER2.