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Sleeping Beauty (SB) transposon, a type of nonviral integrative vectors, provides an alternative to modify primary T cells. Creative biolabs has developed SB transposon CAR vector pSBCAR1 GD2 (3F8) h(28BBζ), which is constructed for the engineering of T cells to target human GD2. The T cells are genetically modified through transduction with a nonviral vector expressing scFv of anti-GD2 antibody linked to a CD28 transmembrane domain/ endodomain and CD137 (4-1BB), CD3-zeta signaling domains. And the vector product was designed for the treatment of Neuroblastoma.
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.1 Cytolytic activity of CAR-GD2 T cells against Y79RB-Wasabi cell line. Percent specific cell death of tumor cell after co-culturing with control T cells or CD19-specific CAR T cells or GD2 CAR T cells. Sujjitjoon, J., Sayour, E., Tsao, S. T., Uiprasertkul, M., Sanpakit, K., Buaboonnam, J., ... & Chang, L. J. (2021). GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma–assessing tumor and T cell interaction. Translational oncology, 14(2), 100971. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.2 Restimulation of GD2-specific CAR T cells promotes hypofunctional killing activity. Expression of GD2 on Y79RB tumor cells after re-stimulation. Green tumor cells after co-culturing with control T or CD19 CAR T or GD2 CAR T cells were collected for immunostaining with anti-GD2 antibody and analysis by flow cytometry. Sujjitjoon, J., Sayour, E., Tsao, S. T., Uiprasertkul, M., Sanpakit, K., Buaboonnam, J., ... & Chang, L. J. (2021). GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma–assessing tumor and T cell interaction. Translational oncology, 14(2), 100971. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.3 Restimulation of GD2-specific CAR T cells promotes hypofunctional killing activity. Expression of PD-L1 on tumor cell surface, and PD-1 on CAR T cells. Y79RB cells were co-cultured with control T or CD19 CAR or GD2 CAR T cells at an E:T ratio of 1:1. Sujjitjoon, J., Sayour, E., Tsao, S. T., Uiprasertkul, M., Sanpakit, K., Buaboonnam, J., ... & Chang, L. J. (2021). GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma–assessing tumor and T cell interaction. Translational oncology, 14(2), 100971. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.4 GD2-CAR T cell killing assay. Target cell killing analysis of the GD2 CAR T cells, Hu3F8. The OS cells transduced with a lentiviral wasabi green fluorescence reporter gene were used as target cells, Sujjitjoon, J., Sayour, E., Tsao, S. T., Uiprasertkul, M., Sanpakit, K., Buaboonnam, J., ... & Chang, L. J. (2021). GD2-specific chimeric antigen receptor-modified T cells targeting retinoblastoma–assessing tumor and T cell interaction. Translational oncology, 14(2), 100971. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.5 Anti-GD2 CART cells are depleted upon stimulation with GD2(C) target cells in vitro. T cells that were transduced with the hu3F8CAR (anti-GD2) and expanded for 8 d thereafter were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. After 1, 2, and 4 d, cells were counted and CAR expression was assessed by anti-idiotype antibody (A1G4). Dead cells were excluded by Annexin-V staining. Absolute cell number was calculated based on flow cytometry. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.6 Anti-GD2 CART cells are depleted upon stimulation with GD2(C) target cells in vitro. T cells that were transduced with the hu3F8CAR (anti-GD2) and expanded for 8 d thereafter were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. After 1, 2, and 4 d, cells were counted and CAR expression was assessed by anti-idiotype antibody (A1G4). Dead cells were excluded by Annexin-V staining. Absolute cell number was calculated based on cell count. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.7 Lowering CAR affinity cannot prevent CART cell depletion upon antigen exposure. T cells that were transduced with the 2nd-generation hu3F8 (loweraffinity) or hu3F8(D32H-E1K) (higher-affinity) CARs, and expanded thereafter for 8 d were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. One day later, cells were assessed by flow cytometry for CAR expression. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.8 Lowering CAR affinity cannot prevent CART cell depletion upon antigen exposure. T cells that were transduced with the 1st-generation or 2nd-generation hu3F8 CARs, and expanded thereafter for 8 d were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. One day later, cells were assessed by flow cytometry for CAR expression. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.9 GD2-specific CART cell depletion severely compromises their function in short-term killing assays. T cells that were transduced with the hu3F8CAR and expanded thereafter for 8 d were stimulated with irradiated IMR32luc (GD2+) or THP1 (GD2-) cells. Four days later, hu3F8CART cells were tested against IMR32luc targets in a 4-h 51Cr-release assay. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.10 Surviving CART cells retain a significant antitumor cytotoxicity in long-term killing assays. Hu3F8CART cells were exposed to GD2(+) IMR32luc cells in triplicates for 3 d at an E:T=1. On day 3, cells were harvested, counted, and mixed with fresh IMR32luc targets (E:T=1). After 4 more days, cells were counted and assessed by flow cytometry. Number of live target cells was calculated based on cell count. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.11 Surviving CART cells retain a significant antitumor cytotoxicity in long-term killing assays. Hu3F8CART cells were exposed to GD2(+) IMR32luc cells in triplicates for 3 d at an E:T=1. On day 3, cells were harvested, counted, and mixed with fresh IMR32luc targets (E:T=1). After 4 more days, cells were counted and assessed by flow cytometry. Number of live target cells was calculated based on flow cytometry. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Fig.12 Anti-GD2 CAR-T cells cure melanoma tumors in vivo. Immunodeficient DKO mice were subcutaneously inoculated with GD2(+) M14luc human melanoma xenografts (4E6 cells). CART Therapy was started after 7 d. Tumor volume was measured weekly. Hoseini, S. S., Dobrenkov, K., Pankov, D., Xu, X. L., & Cheung, N. K. V. (2017). Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2. Oncoimmunology, 6(6), e1320625. |
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