Figure 1.
Isolation of high-affinity monoclonal antibodies from immunized mice.
Spleen and draining lymph nodes were harvested from mice immunized with the C345C fragment of complement protein C5. After RNA isolation and cDNA synthesis, (D)J diversity was amplified from HCs and cloned into nine human germline variable regions (IGHV 1-2, 1-69, 3-7, 3-23, 3-30, 4-34, 4-59, 5-51, and 6-1). These were paired with a fully human LC library, composed of five germline IGκV regions (IGKV 1-33, 1D-39, 2D-30, 3-20, and 4-1), combined with CDR3 diversity obtained from a pool of normal human donors. Libraries were transfected into HEK 293 c18 cells as four sub-library pools for cell surface display and FACS selection of antigen specific clones. Subsequent affinity maturation by AID-induced mutagenesis and FACS resulted in a high-affinity antibody with demonstrated functional activity.
Figure 2.
Discovery and affinity maturation of C5-C345C-specific antibodies.
(A) 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. (B) Plot comparing stability late (value corresponds to amount of antibody-bound antigen) versus binding late (value corresponds to off-rate, kd) 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. (C) Full kinetic analysis of APE777 binding to C5-C345C (first panel) with a KD of 200 nM (ka = 1.4×105 M−1 s−1, kd = 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.
Figure 3.
Kinetic and functional characterization of isolated C5-C345C-specific antibodies.
(A) Plot of on-rate (ka) versus off-rate (kd) depicting anti-C5-C345C clones throughout the affinity maturation process as determined by Biacore T200 analysis. Iterative rounds of AID-induced mutagenesis and SHM resulted in mutations that improved the KD 1000-fold from the starting antibody (APE777) with the majority of improvement obtained by a reduction in kd. Key mutation events are indicated by blue arrows with HC mutations indicated in black and LC mutations indicated in blue. Note that arrows represent affinity progression of improved antigen binding as independent mutations resulting from SHM were recombined and tested in different antibody contexts; changes shown do not represent phylogenetic relationships. (B) Summary of Biacore blocking assay demonstrating inhibition of MAC assembly, in vitro. Varying concentrations of antibody or control protein were incubated with a constant concentration (100 nM) of C5b6, and this was flowed over a CM5 surface with C7 captured via an anti-C7 antibody. Fraction C5b6 bound to C7 was calculated by comparing the maximum signal (RU) obtained in the presence and absence of inhibitor. (C) Hemolysis assay demonstrating inhibition of opsonized sheep red blood cell lysis mediated by the classical complement pathway. Graph depicts concentration of antibody, control isotype-matched antibody or control C5-C345C protein relative fraction red blood cell lysis using 1% serum. Percent specific lysis was normalized to lysis observed in the absence of added inhibitor, control antibody, or control protein.
Table 1.
Summary of observed enriching mutations.