Discovery and affinity maturation of C5-C345C-specific antibodies.

<p>(<i>A</i>) FACS scattergrams depicting isolation of a C5-C345C-specific population from the sub-library pool containing IGHV3-30-3 and IGHV4-34 (panels I–III), and subsequent affinity maturation of APE777 in strategy B (panels IV–VI). Approximately 5×10⁷ cells were sorted per round. IgG expression is shown on the y-axis and C5-C345C binding on the x-axis. (I) The first sort round (5 µM C5-C345C-Myc) showed little detectable binding; (II) round 4 (100 nM C5-C345C-Myc); and (III) round 9 (3 nM C5-C345C-Myc) which included a room-temperature incubation to increase stringency. Affinity maturation FACS scattergrams of isolated C5-C345C-specific antibody, APE777, are shown for strategy B starting with (IV) round 1 (20 nM C5-C345C-Myc), (V) round 5 (1.5 nM C5-C345C-Dyl-650), and (VI) round 8 (100 pM C5-C345C-Dyl-650). Each sort round consisted of AID-induced SHM and selection of highest antigen binding and antibody expressing cells by FACS. Note emergence of new cell populations that exhibited increased antigen binding relative to the original population over time, and which were able to recognize sequentially decreasing antigen concentrations. (<i>B</i>) Plot comparing stability late (value corresponds to amount of antibody-bound antigen) versus binding late (value corresponds to off-rate, k_d) capture-adjusted report points for Biacore 4000 screen of single cell clone supernatants. Higher values correspond to more antigen bound per unit antibody (binding late) and slower off-rates (stability late). Highlighted points indicate highest affinity C5-C345C binding clones further characterized by Biacore T200 and sequencing. (<i>C</i>) Full kinetic analysis of APE777 binding to C5-C345C (first panel) with a K_D of 200 nM (k_a = 1.4×10⁵ M⁻¹ s⁻¹, k_d = 2.4×10⁻² s⁻¹). Binding to C5 and C5b6 complement proteins was detectible (second and third panels), while APE777 shows no significant binding to the C7 negative control.