pCDCAR1 GPC3 h(28ζ) (CAR-ZP126)

CAR Construction : 9F2 scfv-CD3ζ

Fig.1 Surface expression of sFv-ε and CARs on primary human CART cells.

Flow cytometry analysis of surface expression of sFv-ε and CARs on primary human CART cells.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.2 Surface expression of CD25 and CD69 of UTD or 9F2-CART cells.

Flow cytometry analysis of surface expression of CD25 and CD69 of UTD or 9F2-CART cells.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.3 Surface expression of CD25 and CD69 of UTD or 9F2-CART cells.

Figure shows MFI of Flow cytometry analysis of surface expression of CD25 and CD69 of UTD or 9F2-CART cells.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.4 Frequencies of CD45RA+ CCR7+ naive (TN) and CD45RA+ CCR7- terminally differentiated effector memory T cell (TEMRA) subsets in CD8+ T cells.

Flow cytometry analysis of CD45RA+ CCR7+ naive (TN) and CD45RA+ CCR7- terminally differentiated effector memory T cell (TEMRA) subsets in CD8+ T cells.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.5 Frequencies of CD45RA+ CCR7+ naive (TN) and CD45RA+ CCR7- terminally differentiated effector memory T cell (TEMRA) subsets in CD8+ T cells.

Figure shows percentage of CD45RA+ CCR7+ naive (TN) and CD45RA+ CCR7- terminally differentiated effector memory T cell (TEMRA) subsets in CD8+ T cells.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.6 Surface expression of PD-1 and LAG-3 in UTD or 9F2 CART cells

Flow cytometry analysis of surface expression of PD-1 and LAG-3 in UTD or 9F2 CART cells

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.7 Surface expression of PD-1 and LAG-3 in UTD or 9F2 CART cells

Figure shows percentage of surface expression of PD-1 and LAG-3 in UTD or 9F2 CART cells

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.8 Characterization of antitumor activity of human 28z CAR, BBz CAR, and sFv-ε T cells

T cells were co-incubated with SK-Hep-1 or PLC/PRF/5 tumor cells at the indicated effector to target (E:T) ratios for 18 h (n = 3-4). Cytotoxic activities were measured using a standard nonradioactive cytotoxicity assay.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.9 Characterization of cytokine production of human 28z CAR, BBz CAR, and sFv-ε T cells

T cells were cocultured with PLC/PRF/5 cells with or without monocytes at the ratio of 1:1:1 or 1:1:0 for 48 h. IL6, IFNg, TNFa, and IL2 in co-culture supernatants were quantified by cytometric bead array (CBA) assays, and GM-CSF was examined by ELISA (n = 3).

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.10 CD3+CD8+ and CD3+ CD4+ T cells in peripheral blood were measured with Trucount tubes 7 days after treatment (n = 3).

PLC/PRF/5 tumor cells subcutaneously injected into NPG mice on day 0. On day 13, mice bearing tumors were treated with a single intravenous infusion of CART cells.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.11 Tumor Growth analysis after treatment.

Growth of PLC/PRF/5 xenografts were monitored over 29 days (n = 5).

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.12 Tumor weights analysis at the end of the experiment.

Tumor weights at the end of the experiment (n = 5).

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.13 Characterization of cytokine production of human 28z CAR, BBz CAR, and sFv-ε T cells

Tumor-bearing mice were intravenously infused with 1.5e7 9F2 CAR-T or UTD T cells. Human IFNg, TNFa, IL2, and murine IL6 in peripheral blood were measured 3 and 6 days after treatment (n = 6 from 3 different mice of each group). Cytokines were also measured in tumor tissues harvested 6 days after treatment (n = 3).

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.14 Characterization of cytokine production of human 28z CAR, BBz CAR, and sFv-ε T cells stimulated with GPC3 peptide.

T cells were stimulated with GPC3 peptide of gradient concentrations for 8 h, and the cytokines in supernatants were quantified.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.15 Characterization of cytokine production of human 28z CAR, BBz CAR, and sFv-ε T cells co-incubated with PLC/PRF/5 cells.

T cells were co-incubated with PLC/PRF/5 cells at a 1:1 ratio with or without calcineurin inhibitor FK506 (0.25 nM) for 8 h, and the cytokines in supernatants were measured (n = 3).

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.

CAR Construction : 9F2 scfv-CD3ζ

Fig.16 Flow cytometry analysis of intracellular calcium level of human 28z CAR, BBz CAR, and sFv-ε T cells co-incubated with PLC/PRF/5 cells.

The intracellular calcium level of sFv-ε or CAR T cells stimulated with PLC/PRF/5 cells were analyzed by flow cytometry.

Sun, Y., Dong, Y., Sun, R., Liu, Y., Wang, Y., Luo, H., ... & Li, Z. (2022). Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors. Molecular Therapy-Oncolytics, 25, 160-173.