Pepscan analysis of the epitopes of rituximab and GA101. (A) Epitope footprints of type I (rituximab, 2H7, and LT20) and type II (GA101, H299/B1, and B-Ly1) antibodies: 39 different overlapping 8-mer sequences covering position 142-187 in CD20 were synthesized. The ratio between the average binding value in all peptides containing a particular 8-mer sequence and the average binding value of all 4291 peptides was calculated. When the ratio was > 1, all peptides containing that particular 8-mer sequence had a binding value above average. Consecutive 8-mer sequences with a ratio > 1 indicate the presence of the epitope. (B) Fine mapping of the binding contribution of each residue within epitopes of the same type I and II antibodies as in panel A. A peptide library was synthesized in which, with the exception of C167 and C183, the residues in every position between Y165 and S185 were replaced by all possible alternative natural amino acids except for cysteine. Therefore, 18 single substituted peptides per position were synthesized and their binding value relative to the native CD20 loop peptide (position 142-187 in CLIPS) was determined. Substitutions negatively affecting binding result in a ratio < 1, and the respective bars in the graph are colored red. If replacements clearly improved binding, the ratios were > 1 and the corresponding bars are colored green.