Extended Data Fig. 4: Extended SMART logic and three-input systems.

a, SMART-SpyCatcher (i.e., SpyN and SpyC) was assigned to operate through AND logic involving combinations of EpCAM with HER2 and EGFR. Two combinations of K562 cell lines were used to test for the actuation of SMART-SpyCatcher and thereby recruitment of SpyTag003-AF594. Anticipated target cells are highlighted in red. Mixed-population 1 consisted of equal amounts of K562 (wildtype), K562^EGFR+, K562^HER2+, and K562^HER2+/EGFR+, whereas mixed-population 2 consisted of equal amounts of K562 (wildtype), K562^EGFR+, K562^{HER2+/EpCAMhigh}, and K562^{HER2+/EGFR+/EpCAMhigh}. The antigen profile of each cell line is indicated below each bar plot (L and H designates low endogenous and high ectopic levels respectively for EpCAM), whereas the antigens targeted by the added SpyN and SpyC pairs (i.e. the targeting DARPins employed in the constructs) are indicated at the bottom. Experiments were performed with SMART-SpyCatcher (100 nM, eNrdJ-1^cage), 100 nM SpyTag003-AF594. Data are presented as the mean of the AF594 median fluorescence intensities (MFI) from flow cytometry analysis with error bars signifying the standard error mean (n = 3 independent biological replicates; see Supplementary Table 13 for statistical one-way ANOVA followed by Dunnett’s test). b, Target cells were profiled for their relative surface levels of HER2, EGFR, and EpCAM. Cells were treated with DARPins labeled with AF594 followed by flow cytometry analysis. In parallel, combinations of SpyN and SpyC linked to targeting DARPins were used to solve the AND logic matrix for a given cell line. c, Top: Single cells lines were profiled for their relative surface levels of HER2, EGFR, and EpCAM using the three DARPin Alexa Fluor 594 conjugates αHER2-AF594, αEGFR-AF594, and αEpCAM-AF594. Cells were treated with the indicated DARPin-AF594 conjugate and analyzed by flow cytometry analysis. Bottom: Single cell lines were also treated with variations of SMART-SpyCatcher003 (100 nM, eNrdJ-1^cage) operating through different combinations of AND logic targeting HER2, EGFR, and EpCAM. The heatmaps represents the mean MFI of the AF549 signal from three independent replicates (see Supplementary Tables 3 and 4 for individual values). The cell lines are categorized as displaying low (MFI < 1000) or high (MFI ≥ 1000) levels of the three antigens. d, The quantity of the lesser-expressed antigen used in each AND gate and the resulting recruitment of SpyTag003-AF594 is plotted for the data from panel c. Errors = standard error mean (n = 3 independent biological replicates). e, Schematic illustrating SMART-SpyCatcher operating through [(HER2 OR EGFR) AND (HER2 OR EGFR)] logic on a mixed K562 population; sets of SpyN and SpyC are used to achieve [HER2 AND HER2], [EGFR AND EGFR], [HER2 AND EGFR], and [EGFR AND HER2] gating, essentially resulting in OR logic. f, The SMART-SpyCatcher [HER2 OR EGFR] logic operation was tested on mixed K562 cell population 1. Cells were treated with SMART-SpyCatcher (i.e. αHER2-SpyN/αEGFR-SpyN/SpyC-αHER2/SpyC-αEGFR, each at 100 nM, employing eNrdJ-1^cage), and SpyTag003-AF594 (100 nM) and analyzed by flow cytometry. Error bars signify the standard error mean (n = 3 independent biological replicates; see Supplementary Table 14 for statistical one-way ANOVA followed by Dunnett’s test). g, Schematic illustrating a 3-input logic operation, where SMART-SpyCatcher acts through the use of αAg₁-SpyN/SpyC-αAg₂/SpyC-αAg₂ to achieve [Ag₁ AND either Ag₂ OR Ag₃] cell targeting strategy. h, SMART-SpyCatcher (i.e. αAg₁-SpyN/SpyC-αAg₂/SpyC-αAg₂) was used in mixed K562 cell experiments to evaluate its AND/OR logic function involving combinations of HER2, EGFR and EpCAM. The two mixed populations of K562 cell lines described above were used to test for the actuation of SMART-SpyCatcher and thereby recruitment of SpyTag003-AF594. Experiments were performed with SMART-SpyCatcher (100 nM, employing eNrdJ-1^cage), and SpyTag003-AF594 (100 nM). Data are presented as AF594 MFI from flow cytometry analysis with error bars signifying the standard error mean (n = 3 independent biological replicates; see Supplementary Table 15 for statistical one-way ANOVA followed by Dunnett’s test).