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pSBCAR1 GD2 (3F8) h(28BBζ) (CAR-SB-02LX220)

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All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

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.

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Details

  • Target
  • GD2
  • Targeting Cell Type
  • T cell
  • Targeting Diseases
  • Neuroblastoma
  • Generation
  • Third
  • Vector Name
  • pSBCAR1
  • Vector Length
  • ~6kb
  • Vector Type
  • Sleeping Beauty (SB) transposon
  • Receptor Construction
  • scFv-CD28-41BB-CD3ζ
  • Discription of Signaling Cassetes
  • CD28
    CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide costimulatory signals required for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins which are expressed on antigen-presenting cells (APC). CD28 modulates the primary TCR/CD3ζ signal in a different fashion than the late costimulatory elements OX40 and 4-1BB. CD28 enhances the expression of downstream regulators that impact on T-cell proliferation, death, differentiation, and effector functions. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced sustained T cell activation, growth, survival. And CD28 results in a brightly expressed, stable receptor as the transmembrane domain. Including CD28 costimulatory domains in CARs led to enhanced anti-malignancy efficacy.
    41BB
    CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein originally described as present on activated T lymphocytes, which belongs to the tumor necrosis factor (TNF) receptor superfamily. It is expressed mainly on activated CD4+ and CD8+ T cells, and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. On the basis of preclinical observation, this molecule can promote the persistence of antigen-specific and antigennonspecific chimeric antigen receptor T-cells to significantly increases antitumor activity.
    CD3ζ
    CD3ζ, also known as T-cell receptor zeta, which together with T-cell receptor and CD3γ, δ, ε chain, forms the TCR-CD3 complex. ζ was expressed independently from the complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. CD3-zeta, which contains 3 ITAMs, is the most commonly used endodomain component of CARs. It transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric 4-1BB and OX40 can be used with CD3-zeta to transmit a proliferative/survival signal, or all three can be used together.

Target

  • Clone
  • 3F8
  • Host
  • Mouse
  • Target Species
  • Human
  • Gene Name
  • beta-1,4-N-acetyl-galactosaminyltransferase 1
  • Synonyms
  • GD2;B4GALNT1; beta-1,4-N-acetyl-galactosaminyltransferase 1; GALGT; SPG26; GALNACT; GalNAc-T;

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  • Published Data
Complete CAR data FuncS

Fig.1 Cytolytic activity of CAR-GD2 T cells against Y79RB-Wasabi cell line.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.2 Restimulation of GD2-specific CAR T cells promotes hypofunctional killing activity.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.3 Restimulation of GD2-specific CAR T cells promotes hypofunctional killing activity.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.4 GD2-CAR T cell killing assay.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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,
and plated into 96 or 48 wells for 2 hours before adding the different CAR T cells (Jurkat or primary T cells, at E:T ratio 3:1).

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.

Complete CAR data FuncS

Fig.5 Anti-GD2 CART cells are depleted upon stimulation with GD2(C) target cells in vitro.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.6 Anti-GD2 CART cells are depleted upon stimulation with GD2(C) target cells in vitro.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.7 Lowering CAR affinity cannot prevent CART cell depletion upon antigen exposure.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.8 Lowering CAR affinity cannot prevent CART cell depletion upon antigen exposure.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.9 GD2-specific CART cell depletion severely compromises their function in short-term killing assays.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.10 Surviving CART cells retain a significant antitumor cytotoxicity in long-term killing assays.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.11 Surviving CART cells retain a significant antitumor cytotoxicity in long-term killing assays.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

Complete CAR data FuncS

Fig.12 Anti-GD2 CAR-T cells cure melanoma tumors in vivo.

CAR Construction : 3F8 scfv-CD28-41BB-CD3ζ-icasp9 Latest CAR Construction

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.

More Published Data More Published Data

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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