Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1–CH3 fusion protein

doi: 10.1007/s00262-018-2221-1

Figure Lengend Snippet: GPC3-28Z and GPC3-28Z-sPD1 CAR constructs and T-cell transfection. a Schematic representation of soluble PD-1–CH3 fusion protein (sPD1) and GPC3-28Z CAR; GPC3-28Z was fused with sPD1 using the ‘self-cleaving’ F2A peptide; b Expression of GPC3-28Z and GPC3-28Z-sPD1 on T-cell surface were detected with the indicated antibodies; c CAR expression data pooled from three independent experiments with individual donors (n = 3, mean ± SEM; not significant, P > 0.05); d Concentration of sPD1 in the medium of transduced T cells was measured by Human PD/CD1 ELISA Quantitation Set (Sino Biological); data are pooled from three independent experiments with individual donors (n = 12, mean ± SEM); e sPD1 in the cell lysate and supernatant was determined by Western blot

Article Snippet: Open in a separate window Fig. 1 GPC3-28Z and GPC3-28Z-sPD1 CAR constructs and T-cell transfection. a Schematic representation of soluble PD-1–CH3 fusion protein (sPD1) and GPC3-28Z CAR; GPC3-28Z was fused with sPD1 using the ‘self-cleaving’ F2A peptide; b Expression of GPC3-28Z and GPC3-28Z-sPD1 on T-cell surface were detected with the indicated antibodies; c CAR expression data pooled from three independent experiments with individual donors (n = 3, mean ± SEM; not significant, P > 0.05); d Concentration of sPD1 in the medium of transduced T cells was measured by Human PD/CD1 ELISA Quantitation Set (Sino Biological); data are pooled from three independent experiments with individual donors (n = 12, mean ± SEM); e sPD1 in the cell lysate and supernatant was determined by Western blot. . High-titer lentivirus stocks were produced by the transient transfection of transfer and packaging vectors into 293T cells using polyethyleneimine (PEI) [ 19 ].

Techniques: Construct, Transfection, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1–CH3 fusion protein

doi: 10.1007/s00262-018-2221-1

Figure Lengend Snippet: In vitro behavior of CAR-T cells after repetitive stimulation with SK-HEP-1-GPC3 cells. a CAR expression on the T-cell surface after three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells. b Each data set is pooled from four independent experiments with individual donors (n = 4, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001); c The ratio of CD8+ versus CD4+ T cells after three rounds of antigen-specific stimulation (n = 3, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01); d Proliferation of both CAR-T cells after three rounds of antigen-specific stimulation. The two groups of CAR-T cells were prestained with CFSE before the third stimulation, and then the stained CAR-T cells were cocultured with SK-HEP-1-GPC3 at a 1:1 effector-to-target ratio for 96 h. The intensity of CFSE in each group was measured by flow cytometry. e After three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells, the expression of exhaustion biomarkers on the T-cell surface, including PD-1, TIM-3 and LAG-3, was determined using flow cytometry with the indicated antibodies; f Each data set is pooled from three independent experiments with individual donors (n = 3, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001); g Cytokine release of the engineered T cells after three rounds of antigen-specific stimulation with SK-HEP-1-GPC3 cells. A total of 1 × 106 engineered T cells were cocultured with 1 × 106 tumor cells for 24 h. The levels of IL-2, IFN-γ and TNF-α in the supernatants were evaluated by ELISA (n = 6, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Open in a separate window Fig. 1 GPC3-28Z and GPC3-28Z-sPD1 CAR constructs and T-cell transfection. a Schematic representation of soluble PD-1–CH3 fusion protein (sPD1) and GPC3-28Z CAR; GPC3-28Z was fused with sPD1 using the ‘self-cleaving’ F2A peptide; b Expression of GPC3-28Z and GPC3-28Z-sPD1 on T-cell surface were detected with the indicated antibodies; c CAR expression data pooled from three independent experiments with individual donors (n = 3, mean ± SEM; not significant, P > 0.05); d Concentration of sPD1 in the medium of transduced T cells was measured by Human PD/CD1 ELISA Quantitation Set (Sino Biological); data are pooled from three independent experiments with individual donors (n = 12, mean ± SEM); e sPD1 in the cell lysate and supernatant was determined by Western blot. . High-titer lentivirus stocks were produced by the transient transfection of transfer and packaging vectors into 293T cells using polyethyleneimine (PEI) [ 19 ].

Techniques: In Vitro, Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1–CH3 fusion protein

doi: 10.1007/s00262-018-2221-1

Figure Lengend Snippet: In vitro cytokine release and cytotoxicity of CAR-T against target cells and the behavior after coculture with SK-HEP-1-GPC3 or Huh7 cells. a Cytokine release of the engineered T cells. A total of 1 × 106 engineered T cells were cocultured with 1 × 106 tumor cells for 24 h. The levels of IL-2, IFN-γ and TNF-α in the supernatants were evaluated by ELISA; b Cytotoxicity of the engineered T cells against tumor cells. Untransduced, GPC3-28Z and GPC3-28Z-sPD1 T cells were cocultured with the various HCC cells at the indicated effector:target ratios for 72 h. Each data set is pooled from three independent experiments with individual donors (n = 12–15, mean ± SEM); c GPC3-28Z and GPC3-28Z-sPD1 T cells were cocultured with GPC3- and PD-L1-positive cells (Huh7 or SK-HEP-1-GPC3) for 48 h. Then, T cells were isolated and subjected to Western blot analysis to measure the expression levels of phospho-Akt and Bcl-xL. The densitometry quantification of the protein levels of phospho-Akt and Bcl-xL is shown. a: GPC3-28Z + Huh7, b: GPC3-28Z-sPD1 + Huh7, c: GPC3-28Z + SK-HEP-1-GPC3, d: GPC3-28Z-sPD1 + SK-HEP-1-GPC3. Data were quantitated, and expression relative to Akt and GAPDH was plotted. Each data set is pooled from three independent experiments with individual donors (n = 3, mean ± SEM, *P < 0.05, **P < 0.01); d Proliferation capacity of the engineered T cells. A total of 1 × 106 untransduced, GPC3-28Z and GPC3-28Z-sPD1 T cells were stimulated with freshly irradiated tumor cells every week. The numbers of viable T cells were counted twice a week, and the data were pooled from three independent experiments with individual donors (n = 9, mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Open in a separate window Fig. 1 GPC3-28Z and GPC3-28Z-sPD1 CAR constructs and T-cell transfection. a Schematic representation of soluble PD-1–CH3 fusion protein (sPD1) and GPC3-28Z CAR; GPC3-28Z was fused with sPD1 using the ‘self-cleaving’ F2A peptide; b Expression of GPC3-28Z and GPC3-28Z-sPD1 on T-cell surface were detected with the indicated antibodies; c CAR expression data pooled from three independent experiments with individual donors (n = 3, mean ± SEM; not significant, P > 0.05); d Concentration of sPD1 in the medium of transduced T cells was measured by Human PD/CD1 ELISA Quantitation Set (Sino Biological); data are pooled from three independent experiments with individual donors (n = 12, mean ± SEM); e sPD1 in the cell lysate and supernatant was determined by Western blot. . High-titer lentivirus stocks were produced by the transient transfection of transfer and packaging vectors into 293T cells using polyethyleneimine (PEI) [ 19 ].

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Isolation, Western Blot, Expressing, Irradiation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1–CH3 fusion protein

doi: 10.1007/s00262-018-2221-1

Figure Lengend Snippet: Antitumor activity of the modified T cells in established murine xenogeneic HCC models. To examine the unspecific cytotoxicity of CAR-T, a SK-HEP-1 xenograft model was established. No in vivo antitumor activity was observed after CAR-T injections (a); Mice bearing established SK-HEP-1-GPC3 (b) or Huh7 (c) xenografts were pretreated with cyclophosphamide (200 mg/ml). The next day, mice were infused with GPC3-28Z or GPC3-28Z-sPD1 T cells (8 × 106 CAR-T cells for SK-HEP-1-GPC3 xenograft models and 7 × 106 for Huh7 xenograft models). Control groups received untransduced T cells. The tumor volumes were measured with calipers every 3 days. On day 27, the mice were sacrificed, and tumor weight was measured (d–f). Quantitation of circulating human CD4+ and CD8+ T cells from mice bearing SK-HEP-1-GPC3 (g) or Huh7 xenografts (h) treated with the indicated genetically modified T cells. The mean cell level (cells/µl ± SEM) in tumor-bearing mice treated by untransduced or modified T cells are shown. The level of IFN-γ in mouse serum was evaluated by ELISA in SK-HEP-1-GPC3 (i) or Huh7 (j) xenograft models. Each data set is pooled from 3 independent experiments with individual donors (n = 9–15, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Open in a separate window Fig. 1 GPC3-28Z and GPC3-28Z-sPD1 CAR constructs and T-cell transfection. a Schematic representation of soluble PD-1–CH3 fusion protein (sPD1) and GPC3-28Z CAR; GPC3-28Z was fused with sPD1 using the ‘self-cleaving’ F2A peptide; b Expression of GPC3-28Z and GPC3-28Z-sPD1 on T-cell surface were detected with the indicated antibodies; c CAR expression data pooled from three independent experiments with individual donors (n = 3, mean ± SEM; not significant, P > 0.05); d Concentration of sPD1 in the medium of transduced T cells was measured by Human PD/CD1 ELISA Quantitation Set (Sino Biological); data are pooled from three independent experiments with individual donors (n = 12, mean ± SEM); e sPD1 in the cell lysate and supernatant was determined by Western blot. . High-titer lentivirus stocks were produced by the transient transfection of transfer and packaging vectors into 293T cells using polyethyleneimine (PEI) [ 19 ].

Techniques: Activity Assay, Modification, In Vivo, Quantitation Assay, Genetically Modified, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1–CH3 fusion protein

doi: 10.1007/s00262-018-2221-1

Figure Lengend Snippet: Immunohistochemical (IHC) analysis of CD3, Ki67 and granzyme B in tumor sections. a CD3, Ki67 and granzyme B detection on tumor sections from the SK-HEP-1-GPC3 xenograft model. CD3+ T cells were obvious in SK-HEP-1-GPC3 tumors treated with GPC3-28Z-sPD1 and GPC3-28Z T cells; Ki67 expression in the GPC3-28Z-sPD1 treatment group was significantly reduced, and granzyme B showed a higher expression in the GPC3-28Z-sPD1 treatment group than in the other two groups. b CD3, Ki67 and Granzyme B expression was detected in tumor sections after Huh7 xenografts. CD3+ T cells were found to be located in Huh7 tumor sites in CAR-T treated groups, and more cells were detected in GPC3-28Z-sPD1 treated mice; Ki67 expression in GPC3-28Z-sPD1 group showed an obvious decrease compared with GPC3-28Z and untransduced T cells; granzyme B expression was higher in the GPC3-28Z-sPD1 group than in the other two groups. Representative sections are shown at a magnification of ×400. Each data set is pooled from three independent experiments (n = 3, mean ± SEM; ns not significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: Open in a separate window Fig. 1 GPC3-28Z and GPC3-28Z-sPD1 CAR constructs and T-cell transfection. a Schematic representation of soluble PD-1–CH3 fusion protein (sPD1) and GPC3-28Z CAR; GPC3-28Z was fused with sPD1 using the ‘self-cleaving’ F2A peptide; b Expression of GPC3-28Z and GPC3-28Z-sPD1 on T-cell surface were detected with the indicated antibodies; c CAR expression data pooled from three independent experiments with individual donors (n = 3, mean ± SEM; not significant, P > 0.05); d Concentration of sPD1 in the medium of transduced T cells was measured by Human PD/CD1 ELISA Quantitation Set (Sino Biological); data are pooled from three independent experiments with individual donors (n = 12, mean ± SEM); e sPD1 in the cell lysate and supernatant was determined by Western blot. . High-titer lentivirus stocks were produced by the transient transfection of transfer and packaging vectors into 293T cells using polyethyleneimine (PEI) [ 19 ].

Techniques: Immunohistochemical staining, Expressing