Fig 1.
The CE7 Epitope of L1-CAM is a Clinically Relevant Ovarian Cancer Associated Antigen.
A, Cell-surface expression of L1-CAM in various ovarian tumor lines including CAOV-3, OVCAR-3, SK-OV-3, MADH2744, and A2780 were examined by flow cytometry via CE7 mAb. Mean fluorescent intensity (MFI) and percentages of cells with positive staining (%, red histograms) over secondary reagent alone (black histograms) are indicated. B, Human ovarian cancer tissue microarray of primary and metastatic tumors was immunohistochemically stained with CE7 mAb. Representative images from each histological subtype are depicted. Photomicrographs are shown at 400x magnification (ocular 10x; objective 40x). C, Fresh ascites cells derived from ovarian cancer patients were immunofluorescently stained (Left) using anti-keratin 18 mouse or CE7 mAb followed by Alexa Fluor 488–conjugated goat-anti-mouse IgG. Nuclei were counter-stained with DAPI (blue). Ascites cells were also immunohistochemically stained (Right) with the same primary antibodies. In each case, staining with goat-anti-mouse secondary antibody alone (2° Ab) served as negative controls.
Fig 2.
CE7R+ TCM Cells Specifically Target L1-CAM Positive Tumor Cells in Vitro.
A, Schematic representation of the second generation CE7-specific CAR (top) and a representative lentiviral CE7R cassette (bottom) which contains sequences coding an immunoglobulin single chain variable fragment (scFv) derived from the L1-CAM-specific murine CE7 monoclonal antibody linked through a human immunoglobulin (huγ4 Fc) hinge region to the human CD28 transmembrane and cytoplasmic signaling domains (huCD28 tm/cyt), and the human CD3 ζcytoplasmic (huCD3ζ cyt) domain followed by the ribosomal skip T2A sequence, and selection markers CD19t and double mutant DHFR (DHFRFS). Construct expression is driven by an EF-1α promoter. B, Western blots revealing both the endogenous CD3ζ (internal loading control) and the CE7R bands detected with anti-human CD3 ζ cytoplasmic tail-specific antibody. Lane 1: a known CAR-positive T cell line (positive control), Lane2: CE7R-transduced TCM cells, Lane3: mock-transduced TCM cells. C, Flow cytometric analysis of surface expressed Fc-containing CE7R, CD19t, CD4, or CD8 (grey histogram) compared to staining with either isotype controls or SA-PE alone (open histograms). D, Cytolytic activity of CE7R+ TCM cells against the indicated ovarian cancer cell line targets was determined by 4-hr 51Cr-release assay. LCL-OKT3 was used as positive control target. Mean % chromium release ± S.D. of triplicate wells are depicted. E, CE7R+ TCM cells were co-cultured with the indicated tumor lines at a 10:1 ratio for 21 hrs and supernatants were analyzed for IFN-γ and TNF-α levels by cytometric bead array. Means + S.E.M. of triplicate wells are depicted.
Fig 3.
CE7R+ TCM Cells Inhibit Intraperitoneal Ovarian Tumor Growth and Ascites Formation in Vivo.
A, Flow cytometric analysis of mock-, CE7R- and CD19R-transduced TCM cells prior to use in vivo. Percentages of positive cells in each quadrant are indicated. B, NSG mice received i.p. injection of ffLuc+ SK-OV-3 tumor cells on day 0, and were randomized into 4 groups (n = 6 mice per group) for treatment with two doses of either PBS or mock-, CE7R- or CD19R-transduced T cells i.p. (i.e, days 5 and 12). Results are representative of three independent experiments. C, Quantitative bioluminescence imaging of for each group over the time. Mean ± S.E. of total flux levels of luciferase activity were measured. Dashed lines represent days of T cell treatment. *, p < 0.05 when comparing flux values at the indicated timepoints using the unpaired Student’s t-test. D, Flow cytometric detection of T cells in peripheral blood 8 days after the second dose of T cells were administered. Representative histograms and percentages of human CD45+ cells (mean + S.D.) are depicted for each group of mice. *, p < 0.003 when the CE7R+ T cell group was compared to either the Mock or CD19R+ T cell treated groups. E, Kaplan-Meier analysis of survival for each group. *, p ≤ 0.001 when the CE7R+ T cell treated group was compared to any other group using the Log-rang (Mantel-Cox) test.
Table 1.
Ascites Formation in Each Group of Mice.
Fig 4.
Treatment with CE7R+ T Cells Results in Less Ascites Rormation and Reduced L1-CAM Expression on Residual Tumors.
A, Representative images of ascites formation in control mice (PBS, mock or CD19R+ T cell treated groups) versus CE7R+ T cell treated mice. B, Tumor nodules were resected from each mouse upon euthanasia when mice became moribund and subjected to immunohistochemical staining via CE7 monoclonal antibody. Images of representative tumors from PBS, mock-transduced T cell (Mock), CD19R+ T cell (CD19R) or CE7R+ T cell (mouse#1, #2, #3) treated mice are depicted. Photomicrographs are shown at magnification of x200 (ocular 10x; objective 20x).
Fig 5.
CE7R+ T Cells Specifically Recognized and Killed L1-CAM-Expressing Primary Ovarian Cancer Cells Derived from Malignant Ascites.
A, Representative primary culture of ovarian cancer cells derived from patient ascites depicting typical epithelial cobblestone morphology. B, Representative cytogenetic analysis of primary culture of ovarian cancer cells derived from patient ascites. Stemline: 37, X, -X, der(2)t(2;6)(q35;p11), -4, del(5)(q13q33), -6, del(8)(p21p23), 9, del(10) (q24q26), der(11)t(11;?15) (p15;q15), der(12)t(12;?13) (q24.3;q22), -13, -16, -17, -18, 19, der(22)t(12;22)(q11;p13). C, Representative images of fluorescence in situ hybridization for cultured primary ovarian cancer cells in comparison to original ascites cells using probes for 13q14.3 (red) and 13q34 (green), 5p15 (red) and 5q31 (green), or P16 (red) and ABL (green). D, Flow cytometric examination of cell-surface L1-CAM expression on the primary cultured, ascites-derived cancer cells compared to the BE2 neuroblastoma, OVCAR-3 ovarian cancer, and D283 medulloblastoma cell lines. Mean fluorescent intensity (MFI) and percentages of cells with positive staining (%) over secondary reagent alone are indicated. E, CE7R+ T cells were co-cultured overnight with the indicated tumor lines at a 10:1 ratio and supernatants were analyzed for IFN-γ and TNF-α levels by cytometric bead array. Means + S.E.M. of triplicate wells are depicted. F, CE7R+ T cells against the indicated cancer cell lines targets was determined by 4-hr 51Cr-release assay. Mean % chromium release ± SD of triplicate wells are depicted.