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Fig. 4 | Journal for ImmunoTherapy of Cancer

Fig. 4

From: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

Fig. 4

The human PD1-Fc-OX40L ARC has functional activity in vitro and ex vivo in both tumor co-culture and SEB super-antigen assays. a Schematic of the tumor/T cell co-culture assay. CD3+ human T cells stimulated for 48 h with suboptimal levels of CD3/CD28/IL-2, were plated on mitomycin-c treated PD-L1low (PC3) and PD-L1high (HCC827) tumor cells ± the PD1-Fc-OX40L ARC, for an additional 3–5 days (days 5–7 of the entire time-course). b On day 6 of the assay, culture supernatant was collected and analyzed by human IL-2 ELISA. c On days 5 (top) and 7 (bottom) of the assay, floating T cells were collected and subjected to extra- and intra-cellular flow cytometry in order to assess proliferation (Ki67) and markers of T cell activation (IFNγ & TNFα). d Workflow of the SEB super-antigen assay. Total primary human PBMCs were harvested and treated with Staphylococcal enterotoxin B ± the PD1-Fc-OX40L ARC and benchmark antibody controls. Culture supernatants were collected 3 days later and assessed for secreted levels of IL-2 by ELISA

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