Effect of mutations and their combinations on the C5-binding property.

C5-binding activity of the humanized antibody or each antibody variant containing single or multiple mutations was measured by Biacore. Mutations in the heavy chain (am1–am4) and the light chain (am5, am6) cumulatively improved the C5-binding affinity (A and B). The combination of an affinity-matured heavy chain (H-AM) with an affinity-matured light chain (L-AM) further enhanced the affinity (C). Non-histidine mutations found in the heavy chain (pm1, pm2) or the light chain (pm3), and their combinations did not worsen ka at pH 7.4 (D) and provided even better pH dependency (E). The crystal structure of the interaction site of SKY59 and the MG1 domain in human C5 is shown using PyMOL (Schrödinger, LLC) (F). Orange, light blue, and light green represent human C5, the heavy chain, and the light chain of SKY59, respectively. The lysine introduced in the CDR-L3 to improve pH dependency, H100c in the CDR-H3, T47, E48, K109, and H110 in human C5 are shown with thick sticks. The structure of SKY59 and the MG1 domain complex has been shown previously [17] and registered in the RCSB Protein Data Bank with PDB ID: 5B71.