DOI: 10.5061/dryad.zpc866tgv
title: Data from: TCR/CD3-based synthetic antigen receptors (TCC) convey superior antigen sensitivity combined with high fidelity of activation

description: Low antigen sensitivity and a gradual loss of effector functions limit the
 clinical applicability of chimeric antigen receptor (CAR)-modified T-cells
 and call for alternative antigen receptor designs for effective
 T-cell-based cancer immunotherapy. Here we applied advanced microscopy to
 demonstrate that TCR/CD3-based synthetic constructs (TCC) outperform
 second-generation CAR formats with regard to conveyed antigen
 sensitivities by up to a thousand-fold. TCC-based antigen recognition
 occurred without adverse non-specific signaling, which is typically
 observed in CAR-T-cells, and did not depend - unlike sensitized
 peptide/MHC detection by conventional T-cells - on CD4- or CD8-coreceptor
 engagement. TCC-endowed signaling properties may prove critical when
 targeting antigens in low abundance and aiming for a durable anti-cancer
 response.
description: # TCR/CD3-based synthetic antigen receptors (TCC) convey superior antigen
 sensitivity combined with high fidelity of activation
 [https://doi.org/10.5061/dryad.zpc866tgv](https://doi.org/10.5061/dryad.zpc866tgv) Data underlying Fig.1-9 in the main text as well as Fig. S2-S9 of the supplementary material. ## Deposited data DNA sequences are accessible on GenBank under the following accession numbers: BankIt2823001 c58c61_1G4hi_TCR_T2A_copGFP: PP746584 BankIt2823001 T1_STARnat_T2A_copGFP: PP746585 BankIt2823001 T1_STARmir_T2A_copGFP: PP746586 BankIt2823001 T1_eTRuC_T2A_copGFP: PP746587 BankIt2823001 T1_BBz_CAR_T2A_copGFP: PP746588 BankIt2823001 1G4wt_TCR_CRISPR_construct: PP746589 BankIt2823001 c58c61_1G4hi_TCR_CRISPR_construct: PP746590 BankIt2823001 RA14_TCR_CRISPR_construct:  PP746591 BankIt2823001 T1_STARnat_T2A_copGFP_CRISPR_construct: PP746592 BankIt2823001 T1_STARmir_T2A_copGFP_CRISPR_construct: PP746593 BankIt2823001 T1_BBz_CAR_T2A_copGFP_CRISPR_construct: PP746594 Images are deposited onFigshare: * Fig. 1B access via: 10.6084/m9.figshare.25712871 * Fig. 1D access via: 10.6084/m9.figshare.25712868 * Fig. 1E access via: 10.6084/m9.figshare.25712883 * Fig. 1F access via: 10.6084/m9.figshare.25712919 * Fig. 3C access via: 10.6084/m9.figshare.25712931 * Fig. 9A access via: 10.6084/m9.figshare.25712943 * Fig. S3A access via: 10.6084/m9.figshare.25713009 Flow cytomertry data (FCS files) are deposited on Flow Repository under the accession number FR-FCM-Z7DD. ## Description of the data and file structure of the source data file We provide an Excel file containing the source data exactly as used for the generation of all main figures and supplementary figures. Each separate Excel sheet contains the data of one figure panel (e.g. Fig. 1 C). The labels and units within the table are equal to the figure axis labels and described either below or in the figure legends or results section of the article. All datasets labeled with "no antigen" are performed on supported lipid bilayers featuring no antigen but ICAM-1 for cell adhesion. Independent experiments/replicates are indicated as "Experiment 1, Experiment 2, ...". **Main Figures:** **Fig. 1:** Protein-functionalized supported lipid bilayer (SLB)-based platform for high throughput analysis of TCR- and synthetic antigen receptor-mediated activation and downstream signaling events. **SoureData.xls, tab 1 "Figure 1C":** Data underlying Fig. 1C in the main text. * Fluorescence recovery after photobleaching (FRAP): * Column A: Time (min) = x-axis of the plot shown in Fig. 1C. - Y-axis data of blue lines (filled circles) * Column B: Normalized intensity (%) of A2/CMV - Y-axis data of blue lines (empty circles) * Column C: Normalized intensity (%) of A2ΔCD8/CMV - Y-axis data of red lines * Column D: Normalized intensity (%) of recombinant CD19 **SoureData.xls, tab 2 "Figure 1F":** Data underlying Fig. 1F in the main text. * X-axis of the plot shown in Fig. 1F. * Column A: Time after making contact with the bilayer (min) * Columns B-E are plotted on the y-axis of the plot. Y-axis data of blue lines (empty circles): * Column B: Fura-2 ratio values RA14 TCR on SLBs without antigen * Y-axis data of blue lines (filled circles) * Column C: Fura-2 ratio values RA14 TCR on SLBs with A2/CMV * Y-axis data of red lines (empty rectangles): * Column D: Fura-2 ratio values CD19 CAR T-cells on SLBs without antigen * Y-axis data of red lines (filled rectangles): * Column E: Fura-2 ratio values CD19 CAR T-cells on SLBs with CD19 **Fig. 2:** Calcium and downstream signaling response of T-cells equipped with CMV-specific RA14 TCR and anti-CD19 second generation CAR T-cells. **SoureData.xls, tab 3 "Figure 2A":** Data underlying Fig. 2A in the main text. * X-axis of the plot shown in Fig. 2A. * Column A: Fura-2 ratio values * Columns B-R: y-axis of the plot shown as histograms in shades of blue * Column B: Ligand density (molecules µm-2) on SLBs without antigen * Column C-R: Ligand densities (molecules µm-2) on SLBs featuring a titration series of antigen **SoureData.xls, tab 4 "Figure 2B":** Data underlying Fig. 2B in the main text. * Top panel: Antigen densities are plotted on the x-axis and proportion of cells fluxing calcium (%) are shown on the y-axis of the plot shwon in Fig. 2B (top panel) CD19 CAR T-cells on SLBs featuring CD19 Experiment 1: * Column A: Ligand density (molecules µm-2) * Column B: Proportion of cells fluxing calcium (%) Experiment 2: * Column D: Ligand density (molecules µm-2) * Column E: Proportion of cells fluxing calcium (%) RA14 T-cells on SLBs featuring A2/CMV Experiment 1: * Column G: Ligand density (molecules µm-2) * Column H: Proportion of cells fluxing calcium (%) Experiment 2: * Column J: Ligand density (molecules µm-2) * Column K: Proportion of cells fluxing calcium (%) RA14 T-cells on SLBs featuring A2ΔCD8/CMV Experiment 1: * Column M: Ligand density (molecules µm-2) * Column N: Proportion of cells fluxing calcium (%) Experiment 2: * Column P: Ligand density (molecules µm-2) * Column Q: Proportion of cells fluxing calcium (%) * Middle panel: Antigen densities are plotted on the x-axis and the mean Fura-2 ratio values of activated T-cells plotted on the y-axis of the plot shwon in Fig. 2B (middle panel) CD19 CAR T-cells on SLBs featuring CD19 Experiment 1: * Column T: Ligand density (molecules µm-2) * Column U: Mean Fura-2 ratio value of activated T-cells (%) Experiment 2: * Column W: Ligand density (molecules µm-2) * Column X: Mean Fura-2 ratio value of activated T-cells (%) RA14 T-cells on SLBs featuring A2/CMV Experiment 1: * Column Z: Ligand density (molecules µm-2) * Column AA: Mean Fura-2 ratio value of activated T-cells (%) Experiment 2: * Column AC: Ligand density (molecules µm-2) * Column AD: Mean Fura-2 ratio value of activated T-cells (%) RA14 T-cells on SLBs featuring A2ΔCD8/CMV Experiment 1: * Column AF: Ligand density (molecules µm-2) * Column AG: Mean Fura-2 ratio value of activated T-cells (%) Experiment 2: * Column AI: Ligand density (molecules µm-2) * Column AJ: Mean Fura-2 ratio value of activated T-cells (%) * Bottom panel: Antigen densities are plotted on the x-axis and Interferon-γ secretion (pg/ml) on the y-axis of the plot shwon in Fig. 2B (bottom panel) - RA14 TCR on A2/CMV: * Column AM: Ligand density (molecules µm-2) * Column AN: Interferon-γ secretion (pg/ml), measurement 1 * Column AO: Interferon-γ secretion (pg/ml), measurement 2 * CD19 CAR T-cells on CD19: * Column AQ: Ligand density (molecules µm-2) * Column AR: Interferon-γ secretion (pg/ml), measurement 1 * Column AS: Interferon-γ secretion (pg/ml), measurement 2 **Fig. 3:** Design and surface expression of synthetic antigen receptor constructs. **SoureData.xls, tab 5 "Figure 3B":** Data underlying Fig. 3B in the main text * CRISPR Cas9-modified T-cells: Color code: RA14 TCR (column C, filled blue circles), 1G4 TCR (column D, filled yellow circles), 1G4hi TCR (column D, filled brown circles), STARnat (column F, filled green circles), STARmir (column G, filled green circles), T1 BBz CAR (column I, filled green circles) Experiment 1: * Row 4: Mean number of receptors per cell Experiment 2: * Row 5: Mean number of receptors per cell Experiment 3: * Row 6: Mean number of receptors per cell Experiment 4: * Row 7: Mean number of receptors per cell Experiment 5: * Row 8: Mean number of receptors per cell * Lentivirally transduced T-cells: Color code: STARnat (column F, empty green circles), STARmir (column G, empty green circles), eTRuC (column H, empty green circles), T1 BBz CAR (column I, emtpy green circles), CD19 BBz CAR (column J, empty red circles), CD19 eTRuC CAR (column K, empty red circles) Experiment 1: * Row 11: Mean number of receptors per cell Experiment 2: * Row 12: Mean number of receptors per cell Experiment 3: * Row 13: Mean number of receptors per cell **SoureData.xls, tab 6 "Figure 3D":** Data underlying Fig. 3D in the main text CRISPR/Cas9- modified T-cells: RA14 TCR: column A: Antigen receptor surface density (molecules /µm2) 1G4 TCR: column B: Antigen receptor surface density (molecules /µm2) 1G4hi TCR: column C: Antigen receptor surface density (molecules /µm2) STAR mir: column D: Antigen receptor surface density (molecules /µm2) STAR nat: column E: Antigen receptor surface density (molecules /µm2) T1 BBz CAR: column F: Antigen receptor surface density (molecules /µm2) Lentivirally- transduced T-cells: STAR nat: column H: Antigen receptor surface density (molecules /µm2) STAR mir: column I: Antigen receptor surface density (molecules /µm2) eTRuC: column J: Antigen receptor surface density (molecules /µm2) T1 BBz CAR: column K: Antigen receptor surface density (molecules /µm2) **Fig. 4:** Calcium response of T-cells equipped with NY-ESO-1-specific TCR, TCC or CARs targeting peptide-loaded HLA.A2. **SoureData.xls, tab 7 "Figure 4":** Histogram data underlying Fig. 4 in the main text * Data of 1G4 TCR: * Experiment 1: Row 7-17 * Experiment 2: Row 21-31 * Data of 1G4hi TCR: * Experiment 1: Row 37-45 * Experiment 2: Row 49-59 * Experiment 2: Row 63-73 * Data of T1 STARnat: * Experiment 1: Row 78-89 * Experiment 2: Row 93-103 * Data of T1 STARmir: * Experiment 1: Row 109-118 * Experiment 2: Row 123-133 * Experiment 3: Row 137-147 * Data of T1 eTRuC: * Experiment 1: Row 153-161 * Experiment 2: Row 165-174 * Data of T1 BBz CAR: * Experiment 1: Row 181-191 * Experiment 2: Row 195-204 * Experiment 3: Row 209-219 * Experiment 4: Row 223-233 * Experiment 5: Row 237-247 - X-axis of plots in Fig. 4: * Column A: A2/NY-ESO-1 density on SLBs - Y-axis of plots in Fig. 4, left panels: * Columns B, G and L: Proportion of activated T-cells (%) - Y-axis of plots in Fig. 4, right panels: * Columns C, H and M: Mean Fura-2 ratio value of activated T-cells **Fig. 5:** Calcium response of T-cells equipped with NY-ESO-1-specific TCR, TCC or CARs targeting peptide-loaded CD8-binding-deficient HLA.A2ΔCD8. **SoureData.xls, tab 8 "Figure 5":** Histogram data underlying Fig. 5 in the main text * Data of 1G4 TCR: * Experiment 1: Row 7-17 * Experiment 2: Row 21-31 * Data of 1G4hi TCR: * Experiment 1: Row 37-45 * Experiment 2: Row 49-59 * Experiment 2: Row 63-73 * Data of T1 STARnat: * Experiment 1: Row 79-89 * Experiment 2: Row 93-103 * Data of T1 STARmir: * Experiment 1: Row 109-119 * Experiment 2: Row 123-133 * Data of T1 eTRuC: * Experiment 1: Row 139-147 * Experiment 2: Row 151-161 * Data of T1 BBz CAR: * Experiment 1: Row 167-177 * Experiment 2: Row 181-291 - X-axis of plots in Fig. 5: * Column A: A2/NY-ESO-1 density on SLBs - Y-axis of plots in Fig. 5, left panels: * Columns B, G and L: Proportion of activated T-cells (%) - Y-axis of plots in Fig. 5, right panels: * Columns C, H and M: Mean Fura-2 ratio value of activated T-cells **Fig. 6**: Comparative heat map of T-cell-antigen sensitivities conveyed by antigen receptors. **SoureData.xls, tab 9 "Figure 6":** Heat map data underlying Fig. 6 in the main text Column A: Description of the different TCRs/ CARs/ TCCs tested Row 2, 16 and 29: Description of the different ligands tested Data for confidence intervals (bottom values) are provided in row 17-25. Data for confidence intervals (top values) are provided in row 30-38. Data are provided as log fold increase in activation threshold normalized to RA14: A2/CMV. **Fig. 7:** Calcium response of T-cells equipped with a CD19-reactive second-generation CAR or an εTRuC. **SoureData.xls, tab 10 "Figure 7A":** Data underlying Fig. 7A in the main text CD19 eTRuc data, right panel: row 4-804 * X-axis of the plot shown in Fig. 7A. * Column A: Fura-2 ratio values * Columns B-L: y-axis of the plot shown as histograms in shades of blue * Column B-K: Ligand densities (molecules µm-2) on SLBs featuring a titration series of antigen * Column L: Ligand density (molecules µm-2) on SLBs without antigen CD19 BBz CAR data, left panel: row 808-1608 * X-axis of the plot shown in Fig. 7A. * Column A: Fura-2 ratio values * Columns B-I: y-axis of the plot shown as histograms in shades of blue * Column B-H: Ligand densities (molecules µm-2) on SLBs featuring a titration series of antigen * Column I: Ligand density (molecules µm-2) on SLBs without antigen **SoureData.xls, tab 11 "Figure 7B":** Data underlying Fig. 7B in the main text CD19 eTRuC data: violet datapoints Experiment 1: row 5-12 Experiment 2: row 16-26 Experiment 3: row 30-40 CD19 BBz CAR data: red datapoints Experiment 1: row 45-52 Experiment 2: row 56-66 Experiment 3: row 70-80 Experiment 4: row 84-94 * X-axis of plots in Fig. 7B: * Column A: A2/NY-ESO-1 density on SLBs * Y-axis of plots in Fig. 7B, top panel: * Columns B: Proportion of activated T-cells (%) * Y-axis of plots in Fig. 7B, bottom panel: * Columns C: Mean Fura-2 ratio value of activated T-cells **Fig. 8**: *Ex vivo* cytotoxic capacity of engineered T-cells with low antigen receptor expression levels. **SoureData.xls, tab 12 "Figure 8":** Data underlying Fig. 8 in the main text Rows 2, 11 and 20: Description of the different TCRs/ CARs/ TCCs tested Column A: NY-ESO-1/9V peptide concentration (nM) Columns B-K: Target cell killing (%) 6h timepoint measurements: row 4-9 8h timepoint measurements: row 13-18 24h timepoint measurements: row 22-27 **Fig. 9:** Immunofluorescence-based analysis of synapse-associated CD3ζ pITAM#2 and pZAP-70. **SoureData.xls, tab 13 "Figure 9B":** Data underlying Fig. 9B in the main text * Bar graph data of Fig 9B, left panel: * Row 2: Description of the different TCRs/ CARs/ TCCs tested * Row 3: Integrated fluorescence (x 10^4) * Columns B, D, F, H, J, L: Mean integrated fluorescence intensity * Columns C, E, G, I, K, M: standard error of the mean of measured integrated fluorescence intensity * Line graph data of Fig 9B, right panel: row 8-52 * Column A: Antigen density (molecules µm-2) * Column B: Mean integrated fluorescence * Column C: standard error of the mean of measured integrated fluorescence intensity * RA14 TCR data: row 8-12, blue line * eTRuC data: row 16-20, violet line * CD19 BBz CAR data: row 24-28, red line * T1 BBz CAR data: row 32-36, yellow line * STARnat data: row 40-44, light green line * STARmir data: row 48-52, dark green line **SoureData.xls, tab 14 "Figure 9C":** Data underlying Fig. 9C in the main text * Bar graph data of Fig 9C, left panel: * Row 3: Description of the different TCRs/ CARs/ TCCs tested * Row 5: Integrated fluorescence (x 10^4) * Columns B, D, F, H, J, L: Mean integrated fluorescence intensity * Columns C, E, G, I, K, M: standard error of the mean of measured integrated fluorescence intensity * Line graph data of Fig 9C, right panel: row 11-55 * Column A: Antigen density (molecules µm-2) * Column B: Mean integrated fluorescence * Column C: standard error of the mean of measured integrated fluorescence intensity * RA14 TCR data: row 11-15, blue line * eTRuC data: row 19-23, violet line * CD19 BBz CAR data: row 27-31, red line * T1 BBz CAR data: row 35-39, yellow line * STARmir data: row 43-47, dark green line * STARnat data: row 51-55, light green line **SoureData.xls, tab 15 "Figure 9D":** Data underlying Fig. 9D in the main text * X-axis of plot in Fig. 9D: * Column A: Antigen density (molecules µm-2) * Y-axis of plot in Fig. 9D: * Column B: pZAP70/pCD3z ITAM#2 integrated fluorescence (normalized to CD19 BBz CAR) RA14 TCR: row 4-7, blue datapoints STARnat: row 10-13, light green datapoints STAR mir: row 16-19, dark green datapoints eTRuC: row 22-25, violet datapoints T1 BBz CAR: row 28-30, yellow datapoints CD19 BBz CAR: row 34-37, red datapoints **Supplementary Figures:** **Fig. S2.** Flow cytometric analysis of antigen receptor surface expression. **SoureData.xls, tab 16 "Fig S2C":** Data underlying Fig. S2C in the supplementary material * Column A: Concentration of staining probe (µg/ml) * Column B: Mean fluorescence intensity (MFI) * Anti-FMC63 -CD19-AF647 data: row 4-8, light grey datapoints * Anti-T1-A2/9V-AF647 data: row 12-16, dark grey datapoints * Anti-TCR(IP26)-AF647 data: row 20-24, black datapoints **SoureData.xls, tab 17 "Fig S2D":** Data underlying Fig. S2D in the main text * Column A: Number of equivalent soluble AF647 molecules (µg/ml) * Column B: Mean fluorescence intensity (MFI) of bead population **Fig. S3.** SPR-based analysis and production of A2/NY-ESO-1 variants for SLB functionalization. **SoureData.xls, tab 18 "Fig S3B":** Data underlying Fig. S3B in the supplementary material * Column A: Time (min) * Column B: Normalized intensity (%), A2/9V * Column C: Normalized intensity (%), A2ΔCD8/9V * Column D: Normalized intensity (%), A2/6T * Column E: Normalized intensity (%), A2ΔCD8/6T * Column F: Normalized intensity (%), A2/4D * Column G: Normalized intensity (%), A2ΔCD8/4D **Fig. S4.** Surface expression of lentivirally introduced T1 STARnat and T1 STARmir improves substantially after CRISPR/Cas9-mediated genetic ablation of endogenous TCRαβ, but does not majorly affect the sensitivity of T1 STARnatT-cells towards the A2/4D low affinity ligand. **SoureData.xls, tab 19 "Fig S4B":** Data underlying Fig. S4B in the supplementary material * Row 6: Mean fluorescence intensity (MFI) * Row 11: Percentage of GFP expressing and A2/9V stained T-cells (%) * Column A: STAR nat = light green bar * Column B: STAR nat + TCR knockout = light green squared bar * Column C: STARmir = dark green bar * Column D: STAR mir + TCR knockout = dark green squared bar **SoureData.xls, tab 20 "Fig S4C":** Data underlying Fig. S4C in the supplementary material * Column A: Ligand density (molecules µm-2) * Column B: Proportion of activated T-cells (%) * A2/4D dataset: * T1 STARnat: Row 5-13 * T1 STARnat + TCR knockout: Row 16-23 * A2ΔCD8/4D dataset: * T1 STARnat: Row 28-34 * T1 STARnat + TCR knockout: Row 38-44 **Fig. S5.** Assessment of antigen sensitivity conveyed by NY-ESO-1-specific antigen receptor constructs. **SoureData.xls, tab 21 "Fig S5":** Data underlying Fig. S5 in the supplementary material * Columns A and O: Median Fura-2 ratio (Peak + 10 frames) * Columns B-M: Antigen densities of A2 titration series on SLBs (molecules µm-2) * Columns P-AA: Antigen densities of A2ΔCD8 titration series on SLBs (molecules µm-2) * 1G4 TCR data, 9V: row 5-805 * 1G4hi TCR data, 9V: row 810-1610 * T1 STARnat data, 9V: row 1615-2415 * T1 STARnat data, 6T: row 2419-3219 * T1 STARnat data, 4D: row 3223-4023 * T1 STARmir data, 9V: row 4028-4828 * T1 STARmir data, 6T: row 4832-5632 * T1 STARmir data, 4D: row 5636-6436 * T1 eTRuC data, 9V: row 6441-7241 * T1 eTRuC data, 6T: row 7245-8045 * T1 eTRuC data, 4D: row 8049-8849 * T1 BBz CAR data, 9V: row 8854-9654 * T1 BBz CAR data, 6T: row 9659-10459 * T1 BBz CAR data, 4D: row 10463-11263 **Fig. S6.** Assessment of calcium signaling of T-cells equipped with NY-ESO-1-specific TCCs and CARs. **SoureData.xls, tab 21 "Fig S6":** Data underlying Fig. S6 in the supplementary material * A2 data: * Column A: A2/NY-ESO-1 density (molecules µm-2) * Column B: Proportion of activated T-cells (%) * Column C: Mean Fura-2 ratio value of activated T-cells * A2ΔCD8 data: * Column H: A2/NY-ESO-1 density (molecules µm-2) * Column I: Proportion of activated T-cells (%) * Column J: Mean Fura-2 ratio value of activated T-cells * T1 STARnat data: row 5-38 * T1 STARmir data: row 44-76 * T1 BBz CAR data experiment 1: row 83-115 * T1 BBz CAR data experiment 2: row 120-143 **Fig. S7**. Assessment of antigen sensitivity conveyed by lentivirally transduced NY-ESO-1-specific antigen receptor constructs. **SoureData.xls, tab 22 "Fig S7":** Data underlying Fig. S7 in the supplementary material * Column A and L: Fura 2 ratio * Columns B-J: Ligand density of A2 titration series * Columns M-U: Ligand density of A2ΔCD8 titration series * T1 STARnat data, 9V: row 5-805 * T1 STARnat data, 6T: row 809-1609 * T1 STARnat data, 4D: row 1614-2414 * T1 STARmir data, 9V: row 2419-3219 * T1 STARmir data, 6T: row 3223-4023 * T1 STARmir data, 4D: row 4027-4827 * T1 BBz CAR data, 9V: row 4832-5632 * T1 BBz CAR data, 6T: row 5637-6437 * T1 BBz CAR data, 4D: row 6441-7242 **Fig. S8.** IFN-γ secretion by engineered T-cells responding to SLBs decorated with ICAM-1 and antigen at indicated densities. **SoureData.xls, tab 23 "Fig S8":** Data underlying Fig. S8 in the supplementary material * Left ELISA panel, lentivirally transduced T-cells: * Column A: (molecules µm-2) * Column B + C: duplicates of INF-γ production (pg ml-1) * Right ELISA panel, CRISPR Cas9- modified T-cells: * Column E: A2/9V (molecules µm-2) * Column F: Standard deviation of A2/9V densities (molecules µm-2) * Column G + H: duplicates of INF-γ production (pg ml-1) * STARnat data, light green datapoints: row 4-9 * STARmir data, dark green datapoints : row 13-18 * T1 eTRuC data, violet datapoints: row 22-27 * T1 BBz CAR data, yellow datapoints: row 31-36 * 1G4 TCR data, blue datapoints : row 40-44 **Fig. S9.** *Ex vivo* cytotoxic capacity of engineered T-cells with high antigen receptor expression levels. **SoureData.xls, tab 24 "Fig S9":** Data underlying Fig. S9 in the supplementary material * Column A: NY-ESO-1 peptide concentration (nM) * 1G4 TCR: * Column B+C: duplicates, target cell killing (%) * T1 BBz TCR: * Column D+E: duplicates, target cell killing (%) * T1 STARnat TCR: * Column F+G: duplicates, target cell killing (%) * T1 STARmir TCR: * Column H+I: duplicates, target cell killing (%) * T1 eTRuC TCR: * Column J+K: duplicates, target cell killing (%) * 6h timepoints: row 4-9 * 8h timepoints: row 14-19 * 24h timepoints: row 24-29