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

FIGURE 4.

FIGURE 4

Format and Fc engineering approaches augment killing capacities of VHH‐based NKp46 × EGFR NKCEs. (a) Fluorescence based killing assays were conducted with EGFR‐overexpressing A431 cells and freshly isolated PBMC‐derived NK cells from healthy donors at an E:T ratio of 5:1 with increasing concentrations of strictly monovalent NKp46 and EGFR targeting NKp46.2 (green) and NKp46.26 (orange) VHH SEEDbodies with effector‐silenced (indicated as eff−, continuous lines and filled symbols) or effector competent (indicated as eff+, dotted lines and open symbols) Fc portions. (b) Schematic depiction of engineered antibody architectures for NK redirection based on a NKp46 specific VHH molecule in combination with humanized Cetuximab Fab in an effector silenced Fc backbone. Strictly monovalent N‐terminal fusion of NKp46 VHH and EGFR Fab shown as SEEDbody format A, while N‐terminal bivalent tandem arrangement of NKp46‐specific VHHs and monovalent EGFR Fab is indicated as SEEDbody format B. N‐terminal bivalent EGFR Fab fusion with C‐terminal bivalent arrangement of NKp46 VHH fused onto an IgG1 backbone is indicated as design C and monovalent C‐terminal NKp46 VHH fusion with bivalent N‐terminal EGFR Fab orientation is indicated as design format D. Schemes were generated using PyMol software version 2.3.0. (c) Fluorescence based killing assays with NKp46.2 based NKCE formats were conducted with EGFR‐overexpressing A431 cells and freshly isolated PBMC‐derived NK cells from healthy donors at an E:T ratio of 5:1 with increasing concentrations of NKp46.2 design A (green continuous line and filled symbols), design B (light blue, dotted line and open symbols), design C (blue, dotted line and open symbols) as well as design D (brown, dotted line and open symbols). (d) Fluorescence based killing assays with NKp46.26 based NKCE formats were conducted with EGFR‐overexpressing A431 cells and freshly isolated PBMC‐derived NK cells from healthy donors at an E:T ratio of 5:1 with increasing concentrations of NKp46.26 design A (orange continuous line and filled symbols), design B (pink, dotted line and open symbols), design C (dark red, dotted line and open symbols) as well as design D (purple, dotted line and open symbols). For all experiments, mean values ± SEM of eight independent experiments with biological duplicates are indicated. Data were normalized to the maximum concentration of Cetuximab to allow for comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 versus respective strictly monovalent bispecific SEEDbody eff− (design A).