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. 2020 Apr 28;8(1):e000413. doi: 10.1136/jitc-2019-000413

Figure 3.

Figure 3

SRF231 induces caspase-independent tumor cell death that is dependent on antibody scaffolding. (A) Phagocytosis induction (left panel) evaluated in the presence of SRF231 or B6H12 using Jurkat cell targets and human macrophages. Anti-CD47 mAb-induced cell death (right panel) measured as % of dead cells (as identified by uptake of LIVE/DEAD dye) within the non-phagocytosed (CFSE+CD14-) target cell population. (B) Macrophages were pretreated with 10 µg/mL CD32a blocking antibody prior to addition of the indicated concentrations of hIgG4 or SRF231. SRF231-induced cell death was evaluated as described in figure 3A. (C) Jurkat cell death induction (measured by Annexin V/PI) on 24 hours treatment with 10 µg/mL soluble (top panel) or protein G-bound (bottom panel) anti-CD47 mAb or isotype controls. (D) Jurkat cells were pretreated for 30 min at 37°C with 100 µM pan-caspase inhibitor (Z-VAD-FMK) prior to culture with protein G-bound SRF231. CFSE, carboxyfluorescein succinimidyl ester; DMSO, dimethyl sulfoxide; hIgG4, human IgG4; mAb, monoclonal antibody; mIgG1, mouse IgG1; PI, propidium iodide.