T cell circuits that sense antigen density with an ultrasensitive threshold

Abstract

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. Increasing HER2 density thus has cooperative effects on T cells-it increases both CAR expression and activation-leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100 times as much HER2, both in vitro and in vivo.

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.

Fig. 2.. A two-step low- to high-affinity recognition circuit yields ultrasensitive antigen-density sensing.

(A) In vitro cell killing curves as a function of target cell antigen density, using human primary CD8+ T cells expressing a constitutive CAR of high (scFv K_d = 17.6 nM) or low affinity (scFv K_d = 210 nM) (top) or a two-step circuit in which the low-affinity synNotch receptor induces expression of either a low- or a high-affinity CAR (scFv K_d = 210 nM synNotch, 17.6 nM CAR) (bottom). For the circuits, lines are fitted to a Hill equation (Hill coefficient for each curve is indicated) (fig. S4A). For constitutive CARs, the lines are drawn on the basis of inspection. The percentage of specific lysis was determined by using flow cytometry to count the number of target cells after 3 days relative to a coculture of targets in the presence of untransduced T cells (see fig. S3C for gating details). Data points denote means, and error bars represent SEM (n = 3). (B) Representative fluorescence-activated cell sorting (FACS) distributions (n = 3) for CAR expression and T cell proliferation measured as a function of target cell HER2 density (at 3 days) for T cells expressing a low- to high-affinity recognition circuit. T cell proliferation was only observed at HER2 densities of >10⁵ (fig. S4C). CFSE, carboxyfluorescein diacetate succinimidyl ester. (C) Model for the mechanism of a two-step circuit expressing a low-affinity synNotch to a high-affinity CAR. In principle, cells with this circuit display two very different responses; in the presence of a low–antigen-density target (left), the T cell activity is dominated by the low-affinity synNotch and low amounts of a CAR. In the presence of a high–antigen-density target (right), the expression of a CAR is increased, and the T cell activity is dominated by the high-affinity CAR response that activates proliferation and killing. T cell activity is predicted to show a sigmoidal response curve (shown in red) because as antigen density increases, CAR expression also gradually increases, transiting between the series of linear response curves shown in purple.

Fig. 3.. Low-to-high synNotch-to-CAR circuit: Discrimination between high- and low-density tumor cancer cell lines and 3D spheroids.

(A) Representative FACS distributions (n = 3) showing the HER2 expression of low- and high-HER2 cell lines. The HER2 score (as defined by ASCO-CAP scoring guidelines) is shown to the left, and the average HER2 density is shown to the right. (B) Area occupied by target cells as a function of time, normalized by the area occupied by target cells at time 0 (left). Low–HER2-density cancer cells (top plot), PC3 (1+ tumor line), or high–HER2-density cancer cells (bottom plot), SKOV3 (3+ tumor line), were cultured with human primary CD8+ T cells expressing either a two-step circuit low-affinity to high-affinity CAR (scFv K_d = 210 nM synNotch, 17.6 nM CAR) (purple lines) or a high-affinity CAR (scFv K_d = 17.6 nM) (blue lines). Gray lines correspond to the target area in the presence of untransduced T cells. Solid lines show the average normalized target area, and the shaded areas depict the SEM (n = 3 wells, three fields of view per well). To the right, representative images of the in vitro cell killing experiment are shown. T cells are shown in blue, the low–HER2-density cells in green, and the high–HER2-density cells in red (for data from additional cell lines, see fig. S6; see also movies S1 and S2). (C) Schematics of T cell killing assay of spheroids made of MCF10A cells expressing high or low HER2. A caspase dye (shown in green) was used to track cell death. The FACS distributions show the HER2 expression on MCF10A lines used to make the 3D spheroids. The MCF10A line engineered to express high HER2 is shown in blue, and wild-type MCF10As that express low levels of HER2 are shown in red. (D) Representative images of spheroids expressing low (shown in red) or high (shown in blue) HER2 in the presence of untransduced T cells (left) or T cell expressing either a low-affinity CAR (middle) or a two-step low-to-high recognition circuit (right). The caspase 3/7 signal is in green, and the T cells are labeled in yellow. (E) Violin plots showing the distributions of mean caspase 3/7 signal per spheroid. The distributions for the low–HER2-density spheroids are shown to the left in red and the ones for the high–HER2-density spheroids to the right in blue. The mean of the distribution is shown as a white circle, and the number of analyzed spheroids in each case is shown at the bottom. The statistical significance of differences in mean caspase 3/7 signal in each coculture condition was determined by a Kruskal-Wallis test with Bonferroni’s post hoc for multiple comparisons [not significant (ns) > 0.05, *P < 0.05, ****P < 0.0001]. (F) Schematics of experiment to study the distance dependence of killing activity of low-to-high–circuit T cells in a 3D culture system. High–HER2-density spheroids were mixed with a large excess of low–HER2-density cells and engineered low-to-high–circuit T cells. A caspase 3/7 dye was used as a reporter for cell killing. The spheroids and cells were embedded in a thin slab of cell-laden Matrigel to constrain their position along the z axis. A representative image of a high-HER2 spheroid is shown in blue surrounded by low-HER2 spheroids, highlighted in red circles, after 3 days of coculture. The corresponding image for the caspase 3/7 activity is shown below. (G) Caspase 3/7 activity (fluorescence per pixel within spheroids) is plotted as a function of distance from a high-HER2 spheroid, located at the origin. The distances for each low-density spheroid to the closest high-density spheroid were binned in 50-μm bins, and the means of the caspase signal of all spheroids within the bin were computed (on a per-pixel basis to account for differences in spheroid size). The gray bars show the mean values of caspase signal for spheroids cocultured with untransduced T cells; the purple bars show the mean values of caspase signal for spheroids cocultured with low-to-high–circuit T cells. The error bars indicate the SEM. A two-sample Kolmogorov-Smirnov test was used to determine the significance of distributions differences (ns > 0.05, *P < 0.05, ***P < 0.001). For representative images for each channel showing high-density spheroids (blue) surrounded by low-HER2 spheroids, see fig. S7. T cells were labeled with a yellow cell trace dye.

Fig. 4.. Low-o-high synNotch-to-CAR circuit: Antigen density discrimination in mouse models.

(A) Schematics of a two-tumor mouse model experiment to test the efficacy and safety of ultrasensitive antigen density–sensing T cells: low-affinity synNotch to high-affinity CAR circuit (scFv K_d = 210 nM synNotch, 17.6 nM CAR). Low- and high-HER2 tumor cells were injected subcutaneously in the flanks of NSG mice. Engineered primary human CD4+ and CD8+ T cells were injected intravenously at the indicated times after tumor injection. Tumor volume was monitored through caliper measurement over several days after tumor injection. (B) FACS distributions showing the HER2 expression of cell lines used in the experiment. The doses and injection times for tumors and T cells are indicated in the gray box. Tumor volumes of cells with high and low K562 HER2 density after treatment with T cells expressing a two-step circuit (low-affinity synNotch to high-affinity CAR) are shown. The high-density tumor is shown in dark purple and the low-density tumor in pink. The solid lines connect the means, and the error bars indicate the SEM (n = 7). The gray and black dotted lines show the low-density and high-density tumor volumes after treatment with untransduced T cells, respectively (for details of control experiment, see fig. S10A). UnT, untransduced. (C) Fraction of CD3+ T cells infiltrated in high or low K562 tumors 7 days after T cell injection. Representative FACS distributions (n = 3) showing the CAR expression (mCherry tagged) in CD3+ engineered T cells are given. (D) Schematics of a dual-tumor mouse model to test the circuit T cell distribution. The doses and injection times for tumors and T cells are indicated in the gray box. A representative image of luciferase activity in dual-tumor mice treated with low-to-high–circuit T cells 9 days after T cell injection is shown. Luciferase signal was only detected in the high-HER2 tumor, indicating localized expansion (n = 2). effLuc, firefly luciferase. (E) Tumor volumes of cancer lines PC3 (low) and SKOV3 (high) after treatment with T cells expressing a two-step circuit (low-affinity synNotch to high-affinity CAR) (n = 5). The doses and injection times for tumors and T cells are indicated in the gray box. (F) Tumor volumes of cancer lines MDA-231 (low) and HCC1569 (high) after treatment with T cells expressing a two-step circuit (low-affinity synNotch to high-affinity CAR) (n = 6). The doses and injection times for tumors and T cells are indicated in the gray box. For more details and individual mouse tumor volume plots, see fig. S9. Statistical longitudinal analyses were performed over entire segments of the tumor growth curves by using TumGrowth (32). See materials and methods for more details.