Supplementary Figure 1: Engineering of C1q-specific IgG antibodies.
(a) Schematic illustration of aglycosylated IgG display system for Fc engineering. Soluble C1q-PE competes with non-fluorescent FcγRs-GST for binding to the displayed IgG variants on the surface of bacterial spheroplasts before FACS sorting. (b) Schematic illustration of the construction of the three sub-libraries of mutated Fc domains: S-library: randomization of the focused 15 amino acids; E-library: random mutagenesis on Fc domain by error prone PCR with 1% error rate: SE-library: random mutagenesis of the S-library gene pool by error prone PCR with 1% error rate. The sizes of sub-libraries were 2 × 108 (S-library), 3 × 108 (SE-library), and 1 × 109 (E-library). (c) C1q does not bind to IgG-displaying E.coli spheroplasts in high salt buffer (50 mM phosphate, 330 mM NaCl, pH 7.4). Since E.coli cannot synthesize glycosylated antibodies, E.coli cells were engineered to display an antigen (PA domain 4) on the inner membrane and then the spheroplasted cells were incubated with the very high affinity anti-PA domain 4 antibody, M18. Binding to C1q to the surface-bound C1q was detected by FACS using 10 nM C1q-PE. (d) Flow cytometry analysis of C1q binding onto IgG-displaying E.coli spheroplasts in high salt buffer. (e) Flow cytometry analysis of E.coli library spheroplasts labeled with 10 nM C1q-PE before (Red) and after sorting (Cyan). (f-g) Fluorescent histogram of isolated IgG variants binding to 10 nM of C1q-PE in high salt phosphate buffer (f) or to 10 nM of tetrameric FcγRIIIaV158-PE in PBS (g). (h) MFI and fold increase in MFI following incubation with C1q or tetrameric FcγRIIIaV158 relative to cells unmutated aglycosylated IgG.
