Complete CAR data FCM

Fig.1 FACS analysis of CAR expression in primary human T cells. OKT3-stimulated peripheral blood mononuclear cells were transduced twice with retroviral vectors encoding the indicated CAR constructs.

CAR Construction : Ha1-4.117 scFv-CD28-4-1BB-CD3ζ

Fig.1 FACS analysis of CAR expression in primary human T cells. OKT3-stimulated peripheral blood mononuclear cells were transduced twice with retroviral vectors encoding the indicated CAR constructs.

A Herceptin-based anti-Her2/neu CAR was used as a positive control. SA-PE: cells incubated with secondary staining (streptavidin-PE) alone as isotype control.

Abate-Daga, D., Lagisetty, K. H., Tran, E., Zheng, Z., Gattinoni, L., Yu, Z., ... & Morgan, R. A. (2014). A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer. Human gene therapy, 25(12), 1003-1012.

Complete CAR data FCM

Fig.2 Surface expression of anti-PSCA and anti-MSLN CARs.

CAR Construction : Ha1-4.117 scFv-CD28-4-1BB-CD3ζ

Fig.2 Surface expression of anti-PSCA and anti-MSLN CARs.

Histograms showing results of protein-L staining, analyzed by FACS, of primary human T lymphocytes engineered to express the two
versions of Ha1-4.117-based anti-PSCA CARs or the antiMSLN CAR.

Abate-Daga, D., Lagisetty, K. H., Tran, E., Zheng, Z., Gattinoni, L., Yu, Z., ... & Morgan, R. A. (2014). A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer. Human gene therapy, 25(12), 1003-1012.

Complete CAR data IHC

Fig.3 Immunohistochemistry staining of PSCA CAR-treated tumors.

CAR Construction : Ha1-4.117 scFv-CD28-4-1BB-CD3ζ

Fig.3 Immunohistochemistry staining of PSCA CAR-treated tumors.

Immunohistochemistry of tumor sections showing strong PSCA staining in control groups and greatly reduced staining in PSCA CAR-treated mice. Scale bars represent 2000 lm in the top row and 200 lm in the bottom row.

Abate-Daga, D., Lagisetty, K. H., Tran, E., Zheng, Z., Gattinoni, L., Yu, Z., ... & Morgan, R. A. (2014). A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer. Human gene therapy, 25(12), 1003-1012.

Complete CAR data FuncS

Fig.4 PSCA CARs effectively treat established tumors in NSG mice.

CAR Construction : Ha1-4.117 scFv-CD28-4-1BB-CD3ζ

Fig.4 PSCA CARs effectively treat established tumors in NSG mice.

Growth curves after treatment with CARexpressing human T cells or controls, in two independent experiments.

Abate-Daga, D., Lagisetty, K. H., Tran, E., Zheng, Z., Gattinoni, L., Yu, Z., ... & Morgan, R. A. (2014). A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer. Human gene therapy, 25(12), 1003-1012.

Complete CAR data FCM

Fig.5 Flow cytometry analysis of CD69 and CD25 abundance on preinfusion (day 12) CD8+ T cells that expressed GFP or CARs, as indicated.

CAR Construction : Ha1-4.117 scFv-CD28-4-1BB-CD3ζ

Fig.5 Flow cytometry analysis of CD69 and CD25 abundance on preinfusion (day 12) CD8+ T cells that expressed GFP or CARs, as indicated.

Histograms are representative of 4 independent experiments.

Ramello, M. C., Benzaïd, I., Kuenzi, B. M., Lienlaf-Moreno, M., Kandell, W. M., Santiago, D. N., ... & Abate-Daga, D. (2019). An immunoproteomic approach to characterize the CAR interactome and signalosome. Science signaling, 12(568), eaap9777.

Complete CAR data WB

Fig.6 Western blot analysis of CD3ζ interaction in lysates of T cells
transduced with PSCA-28t28Z, PSCA-8t28BBZ, or GFP and immunoprecipitated for the
CAR scFv.

CAR Construction : Ha1-4.117 scFv-CD28-4-1BB-CD3ζ

Fig.6 Western blot analysis of CD3ζ interaction in lysates of T cells transduced with PSCA-28t28Z, PSCA-8t28BBZ, or GFP and immunoprecipitated for the CAR scFv.

Data are represented as mean±s.d.

Ramello, M. C., Benzaïd, I., Kuenzi, B. M., Lienlaf-Moreno, M., Kandell, W. M., Santiago, D. N., ... & Abate-Daga, D. (2019). An immunoproteomic approach to characterize the CAR interactome and signalosome. Science signaling, 12(568), eaap9777.

Complete CAR data FCM

Fig.7 Flow cytometry analysis of CAR abundance on PSCA-8t28Z (green) and PSCA-8t(+24)28Z (light blue) and GFP (gray)-transduced T cells.

CAR Construction : Ha1-4.117 scFv-28ζ

Fig.7 Flow cytometry analysis of CAR abundance on PSCA-8t28Z (green) and PSCA-8t(+24)28Z (light blue) and GFP (gray)-transduced T cells.

Histograms (left) are representative of 5 independent experiments.

Ramello, M. C., Benzaïd, I., Kuenzi, B. M., Lienlaf-Moreno, M., Kandell, W. M., Santiago, D. N., ... & Abate-Daga, D. (2019). An immunoproteomic approach to characterize the CAR interactome and signalosome. Science signaling, 12(568), eaap9777.

Complete CAR data WB

Fig.8 Western blot analysis of CD3ζ interaction in lysates of T cells
transduced with PSCA-28t28Z, PSCA-8t28BBZ, or GFP and immunoprecipitated for the
CAR scFv.

CAR Construction : Ha1-4.117 scFv-28ζ

Fig.8 Western blot analysis of CD3ζ interaction in lysates of T cells transduced with PSCA-28t28Z, PSCA-8t28BBZ, or GFP and immunoprecipitated for the CAR scFv.

Data are represented as mean±s.d.

Ramello, M. C., Benzaïd, I., Kuenzi, B. M., Lienlaf-Moreno, M., Kandell, W. M., Santiago, D. N., ... & Abate-Daga, D. (2019). An immunoproteomic approach to characterize the CAR interactome and signalosome. Science signaling, 12(568), eaap9777.

Complete CAR data FuncS

Fig.9 CAR signaling upon antigen binding.

CAR Construction : Ha1-4.117 scFv-28ζ

Fig.9 CAR signaling upon antigen binding.

Proteomics data analysis of the 40/751 phospho-proteins that were differentially abundant in CAR-T cells when compared to
background controls.

Ramello, M. C., Benzaïd, I., Kuenzi, B. M., Lienlaf-Moreno, M., Kandell, W. M., Santiago, D. N., ... & Abate-Daga, D. (2019). An immunoproteomic approach to characterize the CAR interactome and signalosome. Science signaling, 12(568), eaap9777.