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Engineered Cell Lines for Immunotherapies

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Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells were transfected to stably express CTLA-4 and/or OX40. The following cell lines were generated and used: CHO-CTLA-4 (0.1 × 106 receptors/cell), HEK-CTLA-4 (0.8 × 106 receptors/cell), CHO-CTLA-4-OX40 (0.7 × 106 CTLA-4 receptors/cell; 4.8 × 106 OX40 receptors/cell) and CHO-OX40 (1.1 × 106 receptors/cell). Moreover, CHO-FcγRI (0.1 × 106 receptors/cell) were used for crosslinking of ATOR-1015. All cell lines were cultured in RPMI-1640 with Glutamax (Gibco) supplemented with 10% fetal bovine serum (FBS, GE Healthcare), 10 mM HEPES (Gibco), zeocin (250 μg/ml) and/or geneticin (600 μg/ml, Gibco) depending on selection pressure.
MB49 bladder cancer cells (EMD Millipore) were cultured in modified DMEM (high glucose, Glutamax and sodium pyruvate) supplemented with 10% FBS. MC38 colon carcinoma cells (Kerafast) were cultured in RPMI-1640 with Glutamax supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate and 0.05 mM 2-mercaptoethanol.
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2

Cell Line Culture and Authentication

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Cell lines were kept in a humidified incubator at 37 °C with 5% CO2 unless otherwise denoted. SKW-3 (derived from a male with T cell leukaemia), Jurkat T cells (derived from a male with acute T cell leukaemia), Raji B lymphocytes (derived from a male with Burkitt’s lymphoma), THP-1 monocyte cell line (derived from a male with acute monocytic leukaemia), K562 cells and primary human T cells were cultured in RPMI supplemented with 2 mM GlutaMAX (Invitrogen), 10% FBS (Sigma),10 mM HEPES pH 8.0 (Thermo Fisher), 1 mM sodium pyruvate (Gibco) and 50 U ml−1 penicillin and streptomycin (Thermo Fisher). SKW-3 cells were purchased from DSMZ. Jurkat T cells, Raji, THP-1 and K562 cells were purchased from ATCC. Validation of T cell lines was performed by staining with known markers pre- and post-transfection or transduction. HEK293T (LentiX) cells (female-derived kidney cell line) were grown in DMEM complete media (Thermo Fisher) supplemented with 10% FBS, 2 mM L-glutamine, and 50 U ml−1 of penicillin and streptomycin. MC38 cells were purchased from Kerafast, and cultured in DMEM complete media containing 10% FBS, 2 mM L-glutamine, 0.1 mM NEAA, 1 mM sodium pyruvate, 10 mM HEPES, 50 U ml−1 penicillin/streptomycin and 50 μg ml−1 gentamycin sulfate. Cell lines tested negative for mycoplasma (MycoAlert Mycoplasma Detection kit, Lonza).
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