Figure 3.

Fc variants with pH-dependent binding to FcγRIIIa isolated from CHO display library.A, the Fc display library was transfected into CHO-T cells and Fc display level monitored by antihuman Fc-AF647 and FcγRIIIa binding monitored by monomeric FcγRIIIa-SA-PE. B, the library was sorted over two rounds for binding to FcγRIIIa-SA-PE at pH 6.5 (first round at 50 nM, second round at 20 nM), followed by two rounds of enrichment for stronger pH-dependence. To select for pH-dependent binding, the library was stained first with 50 nM FcgRIIIa-SA-AF647 at pH 7.4, washed, and then stained with 20 nM FcγRIIIa-SA-PE at pH 6.5. The gate shown is representative of the sorting gate used in round four. C, individual clones selected during rounds three and four were isolated and sequenced before transfection into fresh CHO-T cells, staining as above, and assessment of Fc variant display and FcγRIIIa binding at pH 6.5 and pH 7.4. The pH-selectivity of each variant was calculated as the percent of cells binding FcγRIIIa at pH 6.5 divided by percent of cells binding FcγRIIIa at pH 7.4. The data shown are pooled from three experimental replicates; these data collected with FcγRIIIa allele V158.