Fig 1: Ex vivo analysis of the T cell phenotype and AsPC1-derived tumor tissues upon treatment with CAR T cells.a Number of CAR T cells in the spleen at the end of the experiment 27 days post CAR T cell injection (CD66c S: n = 4, CD318 XS: n = 6, TSPAN8 S: n = 4, TSPAN8 L: n = 5). b Phenotype of CAR T cells in the spleen at the end of the experiment 27 days post CAR T cell injection, as demonstrated by the percentage of TCM and TEM (n equal to a). All data are shown as mean ± s.e.m. c Representative immunofluorescence images of (CAR) T cell tumor infiltration, macrophage tumor infiltration and target expression (CD318 XS tumor 9 days post CAR T cell injection, CD66c S tumor 27 days post CAR T cell injection). Staining was performed on one tumor of the respective treatment group and each image is representative for at least two regions of interest. Regions of interest during cyclic IF were chosen based on manual prestaining of DAPI and EpCAM. d Density plots of a dissociated AsPC1 xenograft showing CD66c expression 35 days post injection of 5e6 CD66c S Vl-Vh CAR T cells (top) and the unstained control (bottom). Scale bar = 100 µm. Source data are provided as a Source Data file.
Fig 2: Off-tumor target expression.a Quantification of target expression within different healthy tissues. Box-and-Whisker Plots show the distribution of the background corrected mean fluorescence intensity of identified cells in the respective tissues. The lower and upper hinges in the Box-and-Whisker Plots correspond to the first and third quartile (25th and 75th percentiles). The bar in the box depicts the median. Whiskers extend to the 5th and 95th percentile. CD66c: n (left to right) = 4603, 1695, 716, 6591, 5746, 13301, 8682, 1731, 13391, 4066, 5878, 2966, 2737, 10844, 11595, 7405, 3671, 3371. CD318: n (left to right) = 9341, 6542, 5282, 11438, 10593, 18148, 18395, 6578, 18238, 8913, 10725, 7813, 7584, 13933, 16442, 12252, 7112, 8218. TSPAN8: n (left to right) = 9341, 6554, 5563, 11438, 10593, 18148, 18395, 6578, 18238, 8913, 10725, 7813, 7584, 13933, 16442, 12252, 7112, 8218. b Flow cytometric analysis of target expression on lysed blood samples. Data represent mean ± SD of three donors. Source data are provided as a Source Data file.
Fig 3: Evaluation of CAR T cell in vivo functionality in an AsPC1 xenograft model.a Representative bioluminescence images of tumor-bearing NSG mice. Tumors were induced by subcutaneously transplanting luciferase expressing AsPC1 cells (color scale for all images, min = 1 × 109, max = 1 × 1011). Mice were randomized and treated upon established solid tumors reached 25 mm² (day 7) by intravenous infusion of 5 × 106 CAR T cells or Mock T cells. b Development of tumor burden for individual mice treated either with Mock T cells or with the respective CAR T cells (Mock: n = 6, CD66c S: n = 5, CD318 XS: n = 7, TSPAN8 S: n = 4, TSPAN8 L: n = 6). c Average bioluminescence signal ± s.e.m. of the respective treatment groups (n equal to b). d Average tumor size ± s.e.m. of the respective treatment groups (n equal to b). Source data are provided as a Source Data file.
Fig 4: Ex vivo analysis of the T cell phenotype and AsPC1-derived tumor tissues upon treatment with CAR T cells.a Number of CAR T cells in the spleen at the end of the experiment 25 days (CD66c S) and 27 days post CAR T cell injection (CD66c S: n = 2, CD318 XS: n = 2, TSPAN8 S: n = 4, TSPAN8 L: n = 4). b Phenotype of CAR T cells in the spleen at the end of the experiment 25 days (CD66c S) and 27 days post CAR T cell injection, as demonstrated by the percentage of TCM and TEM (n equal to a). All data are shown as mean ± s.e.m. c Representative immunofluorescence images of (CAR) T cell tumor infiltration, macrophage tumor infiltration and target expression (CD318 XS tumor 10 days post CAR T cell injection, CD66c S tumor 25 days post CAR T cell injection). Staining was performed on one tumor of the respective treatment group and each image is representative for at least two regions of interest. Regions of interest during cyclic IF were chosen based on manual prestaining of DAPI and EpCAM. Scale bar = 100 µm. Source data are provided as a Source Data file.
Fig 5: Evaluation of CAR T cell in vivo functionality in a BxPC3 xenograft model.a Representative bioluminescence images of tumor-bearing NSG mice. Tumors were induced by subcutaneously transplanting luciferase expressing BxPC3 (color scale for all images, min = 1 × 108, max = 1 × 1010). Mice were randomized and treated upon established solid tumors reached 25 mm² (day 15) by intravenous infusion of 1 × 107 CAR T cells or Mock T cells. b Development of tumor burden for individual mice treated either with Mock T cells or with the respective CAR T cells (Mock: n = 6, CD66c S: n = 6, CD318 XS: n = 6, TSPAN8 S: n = 6, TSPAN8 L: n = 6). c Average bioluminescence ± s.e.m. of the respective treatment groups (n equal to b). d Average tumor size ± s.e.m. of the respective treatment groups (n equal to b). Source data are provided as a Source Data file.
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