Figure 2.

Characterization of affinity maturated human antibodies compared to human mAb E1. (A) Monoclonal antibodies were tested for binding to hNKG2D expressed on NKL cells. Titration curves of mAbs purified from supernatants of transiently transfected HEK293-F cells are shown: mAb C3 (▲), mAb B1 (▽), mAb E1 (□), mAb E4 (○), mAb H5 (■), mAb C12 (◆), mAb hIgG1 isotype control (*). Cell-associated mean fluorescence intensity (MFI) was monitored using FACS analysis. The Graph shows one of three representative experiments. (B) Neutralization of NKG2D-dependent cytotoxicity. mAb E4 (○), mAb E1 (□), mAb B1 (▽) and a hIgG1 isotype control mAb (▲) were purified from supernatants of stable CHO-transfectants and tested in a ⁵¹Cr-release assay for their potency to inhibit NKG2D-dependent cytolytic activity of NKL cells against BaF/3-MICA target cells. (C) NKG2D-dependend cytolytic induction activity in a redirected lysis assay. All mAbs (symbols as in B) induced lysis of FcR⁺ P815 target cells by NKL cells. Error bars indicate standard deviation of triplicates. Data are representative of three independent experiments. E:T = 40:1 in both experimental setups.