FIGURE 5.

Protease-resistant variants differ in the ability to mediate Fc-dependent cell killing. A, the K326A/E333A mutations restore CDC activity to the 2h backbone. CDC activity was measured using rituximab variants against WIL2-S target cells. Shown is a representative experiment of two independent experiments, each performed in duplicate. B–D, either the S239D or I332E mutation is sufficient to restore ADCC activity to the 2h protease-resistant backbone. ADCC activity was determined using anti-CD20 variants, WIL2-S targets cells, and PBMC donor cells that were FcγRIIIa 158^V/F heterozygous (B), high affinity FcγRIIIa 158^V/V homozygous (C), or low affinity FcγRIIIa 158^F/F homozygous (D) as effector cells. Shown are representative plots of two independent assays from two individual donors. Anti-CD20 (A–D) were as follows: IgG1 (black circles), IgG2 (green squares), and protease-resistant variants 2h (brown triangles), 2h-DE (red inverted triangles), 2h-AA (orange diamonds), 2h-DAA (blue open circles), and 2h-AEA (purple open squares) compared with an IgG1 isotype control (light gray crosses) (n = 2 for all assays). Error bars represent S.D.