Anti-CD171 scFv h(CD28) CART, pCDCAR1 (CAR-T-1-M307-2)

CAR Construction :

Fig.1 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 clone CE7.

Mean fluorescent intensity (MFI) and percentages of cells with positive staining (%, red histograms) over secondary reagent alone (black histograms) are indicated.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction :

Fig.2 Flow cytometric analysis of surface expressed Fc-containing CE7-CAR, CD19t, CD4, or CD8 (grey histogram) compared to staining with either isotype controls or SA-PE alone (open histograms).

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.3 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.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.4 IFN-γ and TNF-α release quantification of CE7 CAR-Ts co-cultured with the indicated tumor lines.

CE7-CAR+ 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.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.5 Flow cytometric analysis of CE7-CAR TCM cells prior to use in vivo.

Percentages of positive cells in each quadrant are indicated.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.6 CE7-CAR+ TCM Cells Inhibit Intraperitoneal Ovarian Tumor Growth in Vivo.

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 CE7-CAR transduced T cells i.p. (i.e, days 5 and 12).

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.7 CE7-CAR+ TCM Cells Inhibit Intraperitoneal Ovarian Tumor Growth in Vivo.

Quantitative bioluminescence imaging of for each group over the time. Mean ± S.E. of total flux levels of luciferase activity were measured.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.8 Flow cytometric detection of T cells in peripheral blood 8 days after the second dose of CE7-CART cells were administered.

Representative histograms and percentages of human CD45+ cells (mean + S.D.) are depicted for each group of mice.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.9 Kaplan-Meier analysis of survival for CE7-CAR treatment group.

p ≤ 0.001 when the CE7-CAR+ T cell treated group was compared to any other group using the Log-rang (Mantel-Cox) test.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.10 Treatment with CE7-CAR+ T Cells Results in Less Ascites Rormation and Reduced L1-CAM Expression on Residual Tumors.

Representative images of ascites formation in control mice (PBS, mock or CD19R+ T cell treated groups) versus CE7R+ T cell treated mice.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.11 Treatment with CE7-CAR+ T Cells Results in Less Ascites Rormation and Reduced L1-CAM Expression on Residual Tumors.

Tumor nodules were resected from each mouse upon euthanasia when mice became moribund and subjected to immunohistochemical staining via CE7 monoclonal antibody.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.12 CE7-CAR+ T Cells Specifically Recognized and Killed L1-CAM-Expressing Primary Ovarian Cancer Cells Derived from Malignant Ascites.

CE7-CAR+ 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.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.

CAR Construction : CE7 scfv-CD3ζ

Fig.13 CE7-CAR+ T Cells Specifically Recognized and Killed L1-CAM-Expressing Primary Ovarian Cancer Cells Derived from Malignant Ascites.

CE7-CAR+ T cells against the indicated cancer cell lines targets was determined by 4-hr 51Cr-release assay.

Hong, H., Brown, C. E., Ostberg, J. R., Priceman, S. J., Chang, W. C., Weng, L., ... & Forman, S. J. (2016). L1 cell adhesion molecule-specific chimeric antigen receptor-redirected human T cells exhibit specific and efficient antitumor activity against human ovarian cancer in mice. PLoS One, 11(1), e0146885.