The STEAP1130/HLA-A*02:01-specific TCR transgene was introduced into T cells using the retroviral vector pMP-71 [16 (link)]. Purified CD4+ and CD8+ T cells were activated with anti-CD3/CD28 Dynabeads™ (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) according to manufacturer’s recommendation and 100 U/mL recombinant human (rh) IL-2 (Novartis) and 2 ng/mL rh IL-15 (Bio-Techne, Minneapolis, MI, USA) for CD8+ T cells or 50 U/mL rh IL-2 and 5 ng/mL rh IL-7 (Bio-Techne, Minneapolis, MI, USA) for CD4+ T cells was added. Two days later, spin infection with virus supernatant from the packaging cell line RD114 was performed, as previously published [28 (link)]. On the next day, T cells were re-infected with the same approach. After verifying successful transduction via FACS staining, T cell subsets were purified with anti-phycoerythrin (PE) magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) coupled to STEAP1130/HLA-A*02:01-specific PE labeled multimers (in-house production), as described previously [29 (link)]. Detailed assessment of STEAP1130/HLA-A*02:01-specific TCR functionality and potential cross reactivity was published previously [30 ].
Recombinant human il 2
Manufactured by Novartis
Sourced in Switzerland, United States
Recombinant human IL-2 is a laboratory reagent used for in vitro research purposes. It is a synthetic version of the human cytokine interleukin-2, which plays a role in the activation and proliferation of T cells. The core function of this product is to serve as a tool for researchers to study the biological functions and signaling pathways of interleukin-2 in controlled experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Retronectin, by Takara Bio (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Rpmi1640, by Novartis (4 mentions)
Recombinant human il 15, by CellGenix (3 mentions)
Cd3 cd28 beads, by Thermo Fisher Scientific (3 mentions)
Recombinant murine il 7, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Easysep mouse t cell isolation kit, by STEMCELL (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Counting beads, by Thermo Fisher Scientific (2 mentions)
Nylon wool fiber, by Polysciences (2 mentions)
Accu prep, by Axis-Shield (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Il 15, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Cd3 cd28 dynabead, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Lonza (2 mentions)
Streptomycin, by Lonza (2 mentions)
44 protocols using recombinant human il 2
1
Engineered T Cells Targeting STEAP1
2
IMiDs Treatment Optimization Protocol
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Lenalidomide and pomalidomide are from Sigma-Aldrich. IMiDs were dissolved at the concentration of 80 mM in DMSO (Sigma-Aldrich) and stored at −80°C until further use. Except for dose-effect experiments, IMiDs were used at a final concentration of 10 µM. In all experiments, corresponding concentrations of DMSO were used as a negative control. Recombinant human (rh) IL-2 was purchased from Novartis, and rh-IL-15 from R&D systems.
3
Expansion of Sorted Cells with IL-2 and TAC
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Expansion protocol was adapted from previous protocols with minor modifications.10 (link) Sorted cells were expanded in Roswell Park Memorial Institute 1640 media supplemented with l -glutamine, penicillin/streptomycin, sodium pyruvate, and 10% of human AB pooled serum, in the presence of recombinant human IL-2 (1000 U/mL) (Novartis) and Dynabead Human T-activator CD3/CD28 (Life Technologies) in a 1:1 cell to bead ratio over two 7-day rounds of expansion. During the second round of expansion, 8 ng/mL TAC (Sigma) dissolved in dimethyl sulfoxide (DMSO) (Sigma), based on the published trough level in patients with stable liver or kidney allografts,6 (link),15 (link),16 (link) or empty DMSO control added to cell media (final concentration of DMSO in culture was 1.92 × 10−4% volume).
4
Generation and Validation of CAR T Cells
5
Expansion and Activation of Tumor-Infiltrating Lymphocytes
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Initially, cells isolated from tumours/ascites were cultured in 24-well plates at a concentration of 0.5 × 106/ml in T-cell media (RPMI-1640 500 ml supplemented with 10% heat-inactivated FCS, 1% l -glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin (all Life Technologies, Paisley, UK), 25 mM HEPES and 50 µM β-mercaptoethanol [both from Sigma-Aldrich, Dorset, UK)] with 3000 IU/ml recombinant human IL-2 (Novartis, UK). TILs were mitogenically stimulated by the addition of Dynabeads® Human T-Activator CD3/CD28 (Life Technologies, Paisley, UK) at 1:1 bead to T-cell ratio. Cells were incubated at 37 °C, 5% CO2, 95% humidity. On alternate days, half of the media was removed and replaced with fresh media supplemented with 3000 IU/ml of IL-2.
After 7 days in culture, the Dynabeads® were removed using a magnet (Invitrogen, Norway). TILs were counted and reseeded at 1 × 106 cells/ml in T-cell media supplemented with 1000 IU/ml of IL-2. TILs were counted and reseeded in fresh T-cell media and IL-2 on alternate days, to maintain a density of 1 × 106 cells/ml. Cultures were maintained for a maximum of 20 days, at which time functional assays were performed.
After 7 days in culture, the Dynabeads® were removed using a magnet (Invitrogen, Norway). TILs were counted and reseeded at 1 × 106 cells/ml in T-cell media supplemented with 1000 IU/ml of IL-2. TILs were counted and reseeded in fresh T-cell media and IL-2 on alternate days, to maintain a density of 1 × 106 cells/ml. Cultures were maintained for a maximum of 20 days, at which time functional assays were performed.
6
Tumor Cell Oxygen and Glucose Deprivation Protocol
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Tumor cells were seeded in 6-well plates and cultured overnight until fully attached (t0). Next, tumor cells were cultured under normal (21% oxygen, 25 mM glucose), glucose deprivation (GD, 21% oxygen, 0.5 mM glucose), oxygen deprivation (OD, 1% oxygen, 25 mM glucose), or oxygen and glucose deprivation (OGD, 1% oxygen, 0.5 mM glucose) conditions with, our without IFNy (20 U/mL) for 24 h. Tumor cell medium (TCM) contained IMDM media (Invitrogen) supplemented with 100μg/mL streptomycin, 100 U/mL penicillin, 2 mM L-glutamine (Invitrogen) and 10% FCS (Gibco). Tumor cells were maintained in humidified air at 37 °C and with 5% CO2. Pharmacological inhibition of PI3K was done by adding LY294002 (Seleckchem) or wortmannin (Seleckchem) at timepoint t0 for 24 h.
T cell medium contained IMDM media (Invitrogen) and 10% FCS (Greiner), 0.2% β2ME, 100μg/mL streptomycin, 100 U/mL penicillin, 2 mM L-glutamine (Invitrogen), and 10 IU/mL recombinant human IL-2 (Novartis). B16F10 specific CD8 T cells (CTL clone LP9 [26 (link)]) were stimulated once a week with irradiated B16F10-B7 tumor cells and splenocytes from C57BL/6 mice. TC1 specific CD8 T cells (CTL clone 9.5c3 [27 (link)]) were stimulated once a week with irradiated TC1-B7 tumor cells and splenocytes from C57BL/6 mice.
T cell medium contained IMDM media (Invitrogen) and 10% FCS (Greiner), 0.2% β2ME, 100μg/mL streptomycin, 100 U/mL penicillin, 2 mM L-glutamine (Invitrogen), and 10 IU/mL recombinant human IL-2 (Novartis). B16F10 specific CD8 T cells (CTL clone LP9 [26 (link)]) were stimulated once a week with irradiated B16F10-B7 tumor cells and splenocytes from C57BL/6 mice. TC1 specific CD8 T cells (CTL clone 9.5c3 [27 (link)]) were stimulated once a week with irradiated TC1-B7 tumor cells and splenocytes from C57BL/6 mice.
7
Activation and Transduction of T Cells for CAR Therapy
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T cell activation and transduction was performed as described previously.35 (link) Briefly, freshly thawed CD14– or whole PBMCs were washed once and cultured in X-VIVO-15 (Lonza) with 10% FBS (complete X-VIVO) containing 100 U/mL recombinant human IL-2 (Novartis Oncology) and 0.5 ng/mL recombinant human IL-15 (CellGenix). For CAR lentiviral transduction, T cells were cultured with CD3/CD28 Dynabeads (Life Technologies), protamine sulfate (APP Pharmaceuticals), cytokine mixture (as stated above), and desired lentivirus at a 0.1–1 multiplicity of infection the day following stimulation. Cells were then cultured in and replenished with fresh complete X-VIVO containing cytokines every 2–3 days. After 7 days, beads were magnetically removed, and cells were further expanded in complete X-VIVO containing cytokines to achieve desired cell yield. CAR T cells were positively selected for truncated CD19 using the EasySep CD19 Positive Enrichment Kit I or II (StemCell Technologies) (for PSCA-CAR T cells) or positively selected for truncated EGFR using a custom EasySep EGFR Positive Enrichment Kit (for CD19-CAR T cells) according to the manufacturer’s protocol. Following further expansion, cells were frozen in CryoStor CS5 prior to in vitro and in vivo studies. Purity and phenotype of CAR T cells were verified by flow cytometry.
8
Isolation and Culture of Immune Cells
9
Isolation and Expansion of PBMCs
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Fresh peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consent according to the University of Wuerzburg institutional review board (Gz. 20220927 01). Tubes preloaded with Histopaque-1077 (SIGMA, 10711) were layered with whole blood and centrifuged at 400*g for 20 mins at room temperature with no acceleration or brakes. The opaque interface containing PBMCs was aspirated after centrifugation and was washed twice at 461*g for 5 mins. PBMCs were cultivated with RPMI containing heat inactivated 10% FCS, 100 IU/mL recombinant human IL-2 (Novartis Pharma) and 10 nM BrHBPP in 106 cells/mL density in a 96 well plate round bottom plate. After 10 days, cells were pooled and washed twice, and cultured in a 6 well plate in 106 cells/mL for 3 days without rhIL-2. Such rested cells were subjected to further experiments.
10
Tumor-Infiltrating Lymphocyte Isolation and Characterization
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Fresh tumor tissues were washed with RPMI 1640 (Lonza, Basel, Switzerland), and fatty, connective parts were removed. Tissues were minced into little pieces with a sterile scalpel and placed in culture in RPMI 1640 with the addition of 5% human serum and 60 U/mL recombinant human IL-2 (Novartis, Varese, Italy). After 4–7 days, cells emigrated from tissue were collected and placed in starvation with minimal IL-2 supply before phenotypic characterization, functional assays, and T cell cloning by limiting dilution (0.6 cells/well) in the presence of irradiated allogeneic feeder cells plus 1% phytohemagglutinin-M (Roche, Mannheim, Germany). For selected experiments, TIL clones, maintained in culture in RPMI 1640 plus IL-2 (80 U/mL), were stimulated for 5 days with IL-6 (10 µg/µL) and IL-1β (10 µg/µL) (R&D Systems, Minneapolis, MN, USA), following previous indications [30 (link),31 (link),32 (link)], and then examined by flow cytometry. PBMC were isolated from whole blood samples of the same patients from which the tumor biopsy was taken by Ficoll-Paque PLUS (Lonza, Basel, Switzerland) centrifugation.
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