Untouched CD8+ T cells were isolated (Miltenyi Biotec) from PBMCs from HLA-B*35-positive donors and activated with anti-CD3/CD28 beads (Thermo Fisher Scientific) at a ratio of 1:1 in TexMACS (Miltenyi Biotec) with 3% human serum (c.c.pro; CTL medium) supplemented with 12.5 ng/mL IL-7 and IL-15 (PeproTech). After 1 day, T cells were transduced with lentiviral particles by spinoculation using an multiplicity of infection (MOI) of 3 and addition of 5 µg/mL Polybrene Infection/Transfection Reagent (Merck). Untransduced T cells were treated equally and served as controls. The anti-CD3/CD28 beads were removed on the following day. On day 7 or 9, EGFRt+ T cells were enriched as described30 (link) or via fluorescence-activated cell sorting (FACS). After a total expansion time of 9 to 17 days, functionality of the transduced T cells was assessed by co-culturing them with target cells. For this, 5×104 target cells and effector cells according to the specified effector-to-target (E:T) ratios were seeded in 200 µL CTL medium. Since they do not express IL-12, iEGFP-expressing TÜ165 CAR-Ts (TÜ165 EGFP-TRUCKs) were expected to show the same functionality as TÜ165 CAR-Ts and were both used to compare functionality with iIL-12-secreting TÜ165 CAR-Ts (TÜ165 TRUCKs).
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Functional Evaluation of Engineered CAR-T Cells
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Corresponding Organization : Medizinische Hochschule Hannover
Other organizations : Universitätsklinikum Würzburg, Bayer (Germany), Fraunhofer Institute for Cell Therapy and Immunology, University Hospital Regensburg, Boston Children's Hospital
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Variable analysis
independent variables
- Transduction of CD8+ T cells with lentiviral particles (MOI = 3)
- Enrichment of EGFRt+ T cells
- Co-culturing transduced T cells with target cells at different effector-to-target (E:T) ratios
- Functionality of the transduced T cells
- PBMCs from HLA-B*35-positive donors
- Activation of CD8+ T cells with anti-CD3/CD28 beads (1:1 ratio)
- Culture conditions (TexMACS medium with 3% human serum, 12.5 ng/mL IL-7 and IL-15)
- Removal of anti-CD3/CD28 beads on the following day
- Total expansion time of 9 to 17 days
- Untransduced T cells treated equally as controls
- IEGFP-expressing TÜ165 CAR-Ts (TÜ165 EGFP-TRUCKs)
- Untransduced T cells
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