pCDCAR1 PSCA h(ζ) (CAR-YF411)

CAR Construction : bm2B3 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.

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.

CAR Construction : bm2B3 scFv-CD28-4-1BB-CD3ζ

Fig.2 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.

CAR Construction : bm2B3 scFv-CD28-4-1BB-CD3ζ

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

Immunohistochemical analysis of FAP in PSCA-28Z CAR-treated tumor and control GFP-treated tumor.

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.

CAR Construction : bm2B3 scFv-28ζ

Fig.4 iMC Provides Rimiducid-Dependent Costimulation for CAR-T Cells

T cells transduced (n = 3) with iMC plus PSCA.z were cultured in media without exogenous IL-2 and stimulated with 10 nM Rim on a weekly basis, where indicated.

Foster, A. E., Mahendravada, A., Shinners, N. P., Chang, W. C., Crisostomo, J., Lu, A., ... & Spencer, D. M. (2017). Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40. Molecular therapy, 25(9), 2176-2188.

CAR Construction : bm2B3 scFv-28ζ

Fig.5 T cells were transduced with EGFPluc or with both EGFPluc and iMC-PSCA.z-encoding-retrovirus and subsequently cultured with HPAC-RFP tumor cells at a 1:10 effector-to-tumor (E:T) cell ratio for 48 hr with variable Rim concentrations (0–250 nM) (n = 3).

Data are represented as mean±s.d.

Foster, A. E., Mahendravada, A., Shinners, N. P., Chang, W. C., Crisostomo, J., Lu, A., ... & Spencer, D. M. (2017). Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40. Molecular therapy, 25(9), 2176-2188.

CAR Construction : bm2B3 scFv-28ζ

Fig.6 EGFPluc and iMC-PSCA.z/EGFPluc-modified T cells were cultured with HPAC-RFP tumor cells at an E:T ratio of 1:20 and imaged over 7 days (n = 2). T cells transduced with iMC-PSCA.z/EGFPluc were stimulated with or without 2 nM Rim on day 0 of culture initiation.

Validation of a Unified Vector Encoding iMC and PSCA.z CAR

Foster, A. E., Mahendravada, A., Shinners, N. P., Chang, W. C., Crisostomo, J., Lu, A., ... & Spencer, D. M. (2017). Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40. Molecular therapy, 25(9), 2176-2188.

CAR Construction : bm2B3 scFv-28ζ

Fig.7 Tumor BLI was assessed weekly (dotted lines indicate individual animals and full circles show group average).

These data are representative of three independent experiments.

Foster, A. E., Mahendravada, A., Shinners, N. P., Chang, W. C., Crisostomo, J., Lu, A., ... & Spencer, D. M. (2017). Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40. Molecular therapy, 25(9), 2176-2188.

CAR Construction : bm2B3 scFv-28ζ

Fig.8 T cells were cotransduced with iMC-PSCA.z and EGFPluc and injected i.v. into tumor-free NSG mice. Mice (n = 5 per group) were either left untreated or treated with a single i.p. injection of Rim at 5 mg/kg 24 hr post-T cell injection.

Radiance was calculated from in vivo BLI measurements on days +1, +8, and +30 post-T cell injection (LoD or limit of detection for luminescence).

Foster, A. E., Mahendravada, A., Shinners, N. P., Chang, W. C., Crisostomo, J., Lu, A., ... & Spencer, D. M. (2017). Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40. Molecular therapy, 25(9), 2176-2188.

CAR Construction : bm2B3 scFv-28ζ

Fig.9 NSG mice (n = 5 per group) were engrafted with HPAC-EGFPluc and treated with 1.0 106 iMC-PSCA.z-modified T cells or NT cells after 7 days.

Mice subsequently received weekly i.p. vehicle (Veh) or Rim injections of 5 mg/kg

Foster, A. E., Mahendravada, A., Shinners, N. P., Chang, W. C., Crisostomo, J., Lu, A., ... & Spencer, D. M. (2017). Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40. Molecular therapy, 25(9), 2176-2188.