GNS cells were cultured in StemPro NSC medium (Thermo Fisher) as described previously.27 (link) The neuroblastoma line LAN-1 was maintained in Dubesco’s Modified Eagle Medium (DMEM)-F12 media with 10% Fetal Calf Serum, 1% Glutamax, 1% Pen/Step. Peripheral T cells were separated from blood samples and used to generate GD2-specific third-generation CAR-T cells using GD2-iCAR retroviral vector SFG.iCasp9.2A.14g2a.CD28.OX40.zeta supernatant (Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA) and our clinical manufacturing protocol as described previously.29 (link) The second-generation GD2-CAR-IL-15 retrovector38 58 59 (link) was obtained under a material transfer agreement with Baylor College of Medicine with the kind assistance of Professor Leonid Metelitsa. Post-thawing, CAR-T-cell products were maintained in TEXMACS media (Miltenyi) with 10 ng/mL IL-7 and 5 ng/mL IL-15 for a maximum of 10 days.
Protocol detail
Generating GD2-specific CAR-T Cells
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Blood
Cell
Clinical protocol
Eagle
Fetal calf serum
Gene therapy
Icar
Il 15
Line 1
Medicine
Neuroblastoma
Ox40
Retroviral
T cell
Vector
Corresponding Organization :
Other organizations : Royal Adelaide Hospital, Centre for Cancer Biology, South Australia Pathology, University of Adelaide, Translational Research in Oncology, Cancer Research UK Clinical Trials Unit, Cancer Clinic, Flinders Medical Centre, Flinders University, Occupational Cancer Research Centre, Sydney Children's Hospital
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Variable analysis
independent variables
- Culture media for GNS cells (StemPro NSC medium)
- Culture media for neuroblastoma line LAN-1 (DMEM-F12 with 10% Fetal Calf Serum, 1% Glutamax, 1% Pen/Step)
- Generation of GD2-specific third-generation CAR-T cells using GD2-iCAR retroviral vector and clinical manufacturing protocol
- Use of second-generation GD2-CAR-IL-15 retrovector
- Maintenance of CAR-T-cell products in TEXMACS media with 10 ng/mL IL-7 and 5 ng/mL IL-15
- Not explicitly mentioned
- Not explicitly mentioned
- Positive control: Not mentioned
- Negative control: Not mentioned
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