Fig. 1. Design of T cells with ultrasensitive antigen-density sensing.

(A) Ideal therapeutic T cells will distinguish between tumor cells that express high antigen density and normal cells that express low antigen amounts. A CAR-T cell with a standard linear response curve distinguishes poorly between high- and low-density cells. Effective discrimination requires a sigmoidal ultrasensitive dose-response curve. (B) Design of two-step recognition circuit. A synNotch receptor detects an antigen (HER2) with low affinity. This synNotch receptor, when fully activated, induces expression of a high-affinity CAR. The low-affinity synNotch acts as a high–antigen-density filter, and the high-affinity CAR activates T cell killing and proliferation, acting as an amplifier. TF, transcription factor. (C) Densities of the tumor-associated antigen HER2 on engineered stable cell lines of human leukemia K562. Representative flow cytometry plots (n = 3) are shown. These cell lines can be compared to tumor cell lines (fig. S1A). The average HER2 molecules per cell was measured (n = 3) as shown in fig. S1A. To construct different HER2 sensing systems, we used a series of anti-HER2 scFvs with affinities spanning a 100-fold range. Ab, antibody; APC, allophycocyanin; AU, arbitrary units. (D) Binding affinities for anti-HER2 scFvs used in this study (for details of sequences and binding affinity measurements, see fig. S2). Biolayer interferometry sensograms show the binding kinetics for human HER2 and immobilized anti-HER2 scFvs. Data are shown as colored lines, and the best fit for data to a 1:1 binding model is shown in pink. HER2 concentrations used for binding affinity measurements are indicated. BLI, biolayer interferometry.