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The vector of anti-GPC3 chimeric antigen receptor (CAR) is constructed for the engineering of T cells to target Human GPC3. The T cells are genetically modified through transduction with a lentiviral vector expressing scFv of anti-GPC3 antibody linked to 4-1BB and CD3ζ signaling domains. And the vector product was designed for the treatment of hepatocellular carcinoma.
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CAR Construction :
Fig.1 FC analysis of clone YP7 binding on A431-GPC3 cells treated with heparinase I, II and III. . A431-GPC3 cells were treated with different heparinases for 2 hours at 37°C. Cells were then stained with HS20. Gao, W., Kim, H., Feng, M., Phung, Y., Xavier, C. P., Rubin, J. S., & Ho, M. (2014). Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican‐3 for liver cancer therapy. Hepatology, 60(2), 576-587. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.2 Antitumor activity of GPC3-specific CAR-T cells in vitro. LDH assays to show the cytotoxicity of hYP7 CAR-T cells on A431 cells and A431-GPC3 cells. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.3 Antitumor activity of GPC3-specific CAR-T cells in vitro. LDH assays to show the cytotoxicity of hYP7 CAR-T cells on native liver cancer cells. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.4 Characterization of cytokine production of hYP7 CAR-T cells. ELISA to measure the released cytokines of hYP7 CAR-T cells when targeting HCC cells for 24 hours at an E/T ratio of 1:1. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.5 In vivo antitumor activity of anti-GPC3 CAR-T cells. NCG mice with Hep3B-luciferase xenograft tumors were intravenous injected with hYP7 CAR-T cells when the average tumor size reached approximately 100 mm3. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.6 Bioluminescence imaging weekly in vivo antitumor activity of anti-GPC3 CAR-T cells. Tumor size was measured via bioluminescence imaging weekly. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.7 Detection of tumor size in vivo antitumor activity of anti-GPC3 CAR-T cells. Tumor size was measured with a digital caliper every other day. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.8 sGPC3 inhibited the cytotoxicity of GPC3-specific CAR-T cells in vitro. LDH assays to detect the cytotoxicity of GPC3-specific CAR-T cells targeting Hep3B-Mock cells and Hep3B-sGPC3 cells at the indicated E/T ratio for 16 Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.9 sGPC3 inhibited the cytotoxicity of GPC3-specific CAR-T cells in vitro. LDH assays to detect the cytotoxicity of GPC3-specific CAR-T cells targeting Hep3B cells in the presence of recombinant sGPC3-his protein at an E/T ratio of 2:1 for 16 hours. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.10 sGPC3 inhibited the activation of GPC3-specific CAR-T cells in vitro. The detection of the cytokine release of hYP7 CAR-T cells targeting Hep3B-sGPC3 cells for 16 hours at the indicated E/T ratio. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.11 sGPC3 inhibited the activation of GPC3-specific CAR-T cells in vitro. The detection of the cytokine release of hYP7 CAR-T cells targeting Hep3B cells in the presence of recombinant sGPC3-his protein at an E/T ratio of 2:1 for Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.12 Cytotoxicity of hYP7 CAR-T cells targeting Hep3B cells with 5% serum from HCC patients. LDH assays to detect the cytotoxicity of hYP7 CAR-T cells targeting Hep3B cells at E/T ratio of 1:1 for 12 hours in the presence of 5% serum from HCC patients. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.13 Cytotoxicity of hYP7 CAR-T cells treated with HCC serum samples which were pre-cleaned sGPC3. Cytotoxicity of hYP7 CAR-T cells treated with HCC serum samples which were pre-cleaned by 1 µg/mL anti-GPC3 antibody (32A9) or human IgG. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.14 sGPC3 induced a worse response to GPC3-specific CAR-T cell treatments when mice were under a greater tumor burden. NCG mice were inoculated with luciferase-expressing Hep3B-Mock and Hep3B-sGPC3 cells subcutaneously. When the average tumor size of each group reached ~400 mm3, hYP7 CAR-T cells were infused into mice by intravenous injection. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.15 Kaplan-Meier survival curve of the hYP7 CAR-T cell-treated groups. NCG mice were inoculated with luciferase-expressing Hep3B-Mock and Hep3B-sGPC3 cells subcutaneously. When the average tumor size of each group reached ~400 mm3, hYP7 CAR-T cells were infused into mice by intravenous injection. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.16 Flow cytometry analysis of the binding activity of the sGPC3- his protein on GPC3-specific CAR-T cells. CAR-T cells were incubated with 5 μg/ml sGPC3-his for detection. ELISA was performed to measure the secretion of IL-2 in the culture supernatants of CAR-T cells Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.17 Flow cytometry analysis of the binding activity of the sGPC3- his protein on GPC3-specific CAR-T cells. CAR-T cells were incubated with 5 μg/ml sGPC3-his for detection. ELISA was performed to measure the secretion of IL-2 in the culture supernatants of CAR-T cells Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.18 Flow cytometry analysis of the binding activity of the sGPC3- his protein on GPC3-specific CAR-T cells. CAR-T cells were incubated with 5 μg/ml sGPC3-his for detection. ELISA was performed to measure the secretion of IL-2 in the culture supernatants of CAR-T cells Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.19 Flow cytometry analysis of the binding of hYP7 CAR-T cells Cells were incubated on ice at an E/T ratio of 5:1 for 1 hour in the presence of sGPC3-his protein. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.20 Flow cytometry analysis of the binding of hYP7 CAR-T cells Cells were incubated on ice at an E/T ratio of 5:1 for 1 hour in the presence of culture supernatant of Hep3B-sGPC3 cells. Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ
Fig.21 Flow cytometry analysis of the binding of hYP7 CAR-T cells Figure shows percentage of the binding of hYP7 CAR-T cells Sun, L., Gao, F., Gao, Z., Ao, L., Li, N., Ma, S., ... & Gao, W. (2021). Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma. Journal for immunotherapy of cancer, 9(4). |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.22 Cytolytic activity of CAR (YP7) T cells from healthy donors after 24 hours of co-culture with Hep3B cells. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.23 Cytolytic activity of CAR (YP7) T cells from HCC patients after 24 hours of co-culture with Hep3B cells. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.24 GPC3-targeted CAR T-cell–mediated killing of HepG2 cells as determined by using IncuCyte zoom. HepG2 cells were incubated with CAR T cells at the effector to target ratio of 2:1 up to 140 hours. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.25 CAR (hYP7) T cells eradicate tumors in the peritoneal Hep3B xenograft mouse model. Hep3B tumorbearing NSG mice were intraperitoneal (i.p.) injected with 5, 10, and 20 million CAR (hYP7) T cells. CAR (hYP7) T cells regressed established Hep3B xenografts at high dose (20M) and inhibited tumor growth at low doses (5M and 10M). Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.26 Tumor bioluminescence imaging of the Hep3B xenograft mouse model. Tumor bioluminescence in peritoneal Hep3B xenograft mouse model. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.27 Survival curve of mice after CAR (hYP7) T cells infusion. Kaplan-Meier survival curve of mice after infusion. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.28 a-Fetoprotein (AFP) levels detection in serum collected from groups after 2 weeks of injection. a-Fetoprotein (AFP) levels in serum collected from groups after 2 weeks of injection. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.29 CAR (hYP7) T cells eliminate tumor cells in the HepG2 xenograft mouse models. (F) Experimental schematic of the orthotopic HepG2 xenograft model. HepG2 tumor-bearing female NSG mice were infused with 10 million CD19 CAR T cells and CAR (hYP7) T cells. (G) CAR (hYP7) T cells regressed growth of orthotopic HepG2 tumors. (H) Tumor bioluminescence in mice treated in FigureG. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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CAR Construction : huYP7 scfv-41BB-CD3ζ; huYP7 scfv-41BB-CD28-CD3ζ
Fig.30 Tumor eradication in the orthotopic Hep3B xenograft mouse model by CAR (hYP7) T cells from a healthy donor. (C)The orthotopic Hep3B tumor-bearing NSG mice were IV injected with 10 million CAR (hYP7) T cells. (D) CAR (hYP7) T-cell administration regressed Hep3B tumor growth in mice. Li, D., Li, N., Zhang, Y. F., Fu, H., Feng, M., Schneider, D., ... & Ho, M. (2020). Persistent polyfunctional chimeric antigen receptor T cells that target glypican 3 eliminate orthotopic hepatocellular carcinomas in mice. Gastroenterology, 158(8), 2250-2265. |
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