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. 2023 Feb 24;32(3):e4593. doi: 10.1002/pro.4593

FIGURE 2.

FIGURE 2

Fc‐silenced EGFR × NKp46 NKCEs trigger NK cell‐mediated lysis of EGFR‐overexpressing A431 cells. (a) Fluorescence based killing assays were conducted using A431 cells and freshly isolated PBMC‐derived NK cells derived from healthy donors at an effector‐to‐target cell (E:T) ratio of 5:1. Bispecific NKp46‐specific VHH SEEDbodies harboring a humanized version of the Fab arm of Cetuximab as well as an effector‐silenced Fc region were added at a concentration of 50 nM. As positive control, the monoclonal antibody Cetuximab, activating NK cells exclusively via FcγRIIIa was included. Mean values ± SEM of four independent experiments with biological duplicates are indicated. Data were normalized to the maximum concentration of Cetuximab to allow for comparison. (b) Fluorescence based killing assays of 11 selected NKCEs in a dose‐dependent manner were conducted with A431 cells and freshly isolated PBMC‐derived NK cells from healthy donors at E:T = 5:1. Cetuximab and a one‐armed effector competent SEEDbody lacking the NKp46 VHH domain (oa_hu225 SEEDbody eff+) as well as the corresponding effector‐silenced counterpart (oa_hu225 SEEDbody eff−) were included as controls. Mean values ± SEM of seven independent experiments with biological duplicates are indicated. Data were normalized to the maximum concentration of Cetuximab to allow for comparison.