Binding of rituximab and GA101 to mutant CD20 variants on intact cells. (A) FACS-binding curves of rituximab and GA101. Point-mutated CD20 variants were transiently transfected in Freestyle 293-F cells. By gating on live cells with high transfection efficiency, FACS-binding curves were determined for the indicated antibodies. It was readily apparent that binding of the type I antibodies rituximab and LT20 was completely abolished by each of the 8 different substitutions of the N171 residue, whereas exchanges at the more peripheral positions E168, N176, and S177 were well tolerated. In contrast to this, binding of the type II antibodies GA101 and H299/B1 was less affected by many substitutions of N171, but was weakened by the N176A exchange. (B) Table of normalized EC₅₀ values for rituximab, LT20, GA101, and H299/B1. The average EC₅₀ values from 3 independent experiments were calculated for different CD20 variants using 2 type I (rituximab and LT20) and 2 type II antibodies (GA101 and H299/B1). To average data from separate experiments, all values were normalized by calculating the ratios of EC₅₀ mutant:EC₅₀ wt. The standard deviations for the ratios calculated from the 3 separate experiments are indicated.