Comparison of the GA101-CD20 epitope-binding topology with other CD20 antibodies. (A) Superposition of CD20 epitope peptide bound rituximab (left) and C2H7 (right) onto the GA101 complex with respect to the peptide. In relation to both type I antibodies, GA101 was rotated 90° around the Fab middle axis and tilted ∼ 70° toward the carboxyl terminus of the epitope peptide. The GA101 Fab is shown with the light chain in blue, the heavy chain in green, and the CD20 epitope peptide in orange. The Fab fragment of rituximab is colored pink and that of C2H7 in yellow. (B) Stereo view of the position of the peptide residue N¹⁷¹ within the antigen-antibody interaction surface of GA101 (left) versus rituximab (right). In the epitope peptide complex with rituximab, the side chain of Asn176 made no contact with the antibody and therefore did not contribute to binding. In the crystal structure with GA101, the side chain of Asn176 extended toward a pocket formed by several residues (W33, F51, D57, and D59) of the heavy chain of GA101. Exchanging Asn176 to alanine substantially reduced the binding signal in Pepscan experiments and caused a small but reproducible impairment of GA101 binding on intact cells. (C) Superposition of the V_H domains of GA101, rituximab, and C2H7 with respect to the constant region. Whereas rituximab and C2H7 had a similar elbow angle of ∼ 140°, GA101 had an elbow angle of 167°. Color coding is the same as in panel A.