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Recombinant Moloney murine leukemia virus (MMLV) retroviral vectors serve as efficient viral vector tools for introducing genes permanently into a wide variety of dividing cells, and provide an alternative to modify primary T cells. Creative biolabs has developed retroviral CAR vector pMMLV GD2 (14.G2a) h(BBζ), which is constructed for the engineering of T cells to target human GD2. The T cells are genetically modified through transduction with a retroviral vector expressing scFv of anti-GD2 antibody linked to CD137 (4-1BB) and CD3-zeta signaling domains. And the vector product was designed for the treatment of Neuroblastoma.
CAR Construction : FIG.1 Relative affinity and specificity of 14.G2a scFv variants toward GD2. Representative histograms showing binding of WT and HGlu101Lys variants of scFv (5 g/ml) to tested neuroblastoma cells (dark gray) overlaid with Horwacik, I., Golik, P., Grudnik, P., Kolinski, M., Zdzalik, M., Rokita, H., & Dubin, G. (2015). Structural basis of GD2 ganglioside and mimetic peptide recognition by 14G2a antibody. Molecular & Cellular Proteomics, 14(10), 2577-2590. |
CAR Construction : FIG.2 Relative affinity and specificity of 14.G2a scFv variants toward GD2. MFI signals for single cell populations stained with particular scFv and FITC-conjugated anti-His tag antibody, or the latter antibody alone (control). Horwacik, I., Golik, P., Grudnik, P., Kolinski, M., Zdzalik, M., Rokita, H., & Dubin, G. (2015). Structural basis of GD2 ganglioside and mimetic peptide recognition by 14G2a antibody. Molecular & Cellular Proteomics, 14(10), 2577-2590. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.3 Cell viability calculation of Glioblastoma cell lines (dsRED T98G GD2high, dsRED A172 GD2low) co-cultures at 2:1 E:T ratio with GD2 CAR T and GFP control T cells. Co-cultures at 2:1 E:T ratio with GD2 CAR T. After 48 h tumor cell viability is calculated by fluorescence viability assay. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.4 Cell viability calculation of patient-derived primary glioblastoma cells (dsRED C3, dsRED C6, dsRED C10, and dsRED C12) co-cultures at 2:1 E:T ratio with GD2 CAR T and GFP control T cells derived from five PBMC donors. Co-cultures at 2:1 E:T ratio with GD2 CAR T. After 48 h tumor cell viability is calculated by fluorescence viability assay. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.5 GD2 CAR T cells exert specific killing in a time- and dose-dependent manner against several GD2high patient-derived primary glioblastoma cells. Glioblastoma cells (dsRED C3, dsRED C6, dsRED C10, and dsRED C12) co-cultures with GD2 CAR T and GFP control T cells at three different E:T ratios. After 24, 48, and 72 h tumor viability is calculated by fluorescence viability assay. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.6 GD2 CAR T cells generate clusters to eradicate patient-derived primary glioblastoma cells. Representative fluorescence micrographs highlight a robust cluster activation and killing activity of GD2 CAR T population against primary glioblastoma cells. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.7 Autologous GD2 CAR T cells kill glioblastoma in vitro. Glioblastoma cells (dsRED C6) co-cultures with either autologous or allogeneic GD2 CAR T and GFP control T cells at 5:1 E:T ratios. After 48 and 72 h tumor viability is calculated by fluorescence assay. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.8 Autologous GD2 CAR T cells kill glioblastoma in vitro. Representative fluorescence micrographs show clusters of activation and killing activity of GD2 CAR T cells. GFP T cell population show an absence of reactivity of autologous cytotoxic lymphocytes. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.9 Granzyme release analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 7days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.10 MIP-1α release analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 8days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.11 IFNγ release analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 9days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.12 TNFα release analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 10days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.13 sFAS-Ligand analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 11days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.14 TRAIL analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 12days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.15 GM-CSF release analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 13days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.16 IL-6 release analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 14days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.17 CD137 analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 15days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.18 TGF β1 analysis during GD2 CAR T cells in co-culture with glioblastoma cells. Co-culture supernatants of glioblastoma cell lines and primary glioblastoma cells with either GD2 CAR T (light gray) or GFP control T cells (light black) at 2:1 E:T ratio are assessed for cytokine release at 48 h and 16days. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.19 Survival curve of Intracerebral GD2 CAR T cell treatment in the orthotopic glioblastoma model with E:T=2:1. Kaplan-Meier survival curve and log-rank tests are used to measure differences among groups. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.20 Mean total flux values of Intracerebral GD2 CAR T cell treatment in the orthotopic glioblastoma model with E:T=2:1. Mean total flux values (photons/s) of either GD2 CAR T or GFP T control-treated mice and control animals monitored by IVIS system. Data are shown as a mean ± SD; p values are calculated by unpaired two-tailed t-test. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.21 Tumor progression monitored with intracerebral GD2 CAR T cell treatment in the orthotopic glioblastoma model with E:T=2:1. Tumor progression is monitored weekly by a luminescence signal with IVIS imaging measuring. DPI day post-injection. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.22 Survival curve of Intracerebral GD2 CAR T cell treatment in the orthotopic glioblastoma model with E:T=5:1. Kaplan-Meier survival curve and log-rank tests are used to measure differences among groups. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.23 Mean total flux values of Intracerebral GD2 CAR T cell treatment in the orthotopic glioblastoma model with E:T=5:1. Mean total flux values (photons/s) of either GD2 CAR T or GFP T control-treated mice and control animals monitored by IVIS system. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
CAR Construction : 14.G2a/14g2a-CD3ζ Fig.24 Tumor progression monitored with intracerebral GD2 CAR T cell treatment in the orthotopic glioblastoma model with E:T=5:1. Tumor progression is monitored weekly by a luminescence signal with IVIS imaging measuring. DPI day post-injection. Prapa, M., Chiavelli, C., Golinelli, G., Grisendi, G., Bestagno, M., Di Tinco, R., ... & Dominici, M. (2021). GD2 CAR T cells against human glioblastoma. NPJ precision oncology, 5(1), 1-14. |
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