Fig. 6: Epitope Binning-seq for analyzing various qAbs.

a Schematic diagram of Epitope Binning-seq. A cell library is generated by mixing K562/HER2 cells expressing 14 different scFvs, followed by incubation with an rAb and a collection of the rAb-non-binding [rAb(−)] cell population using a cell sorter. The genome is extracted from these cells and read counts of each scFv are analyzed by NGS. The enrichment ratio of each scFv is calculated from the occupancy of each sequence before and after sorting. b Occupancy of each scFv before and after sorting using Pert-AF647 at 0.1 nM. Cell library was incubated with 0.1 nM Pert-AF647 and the rAb(−) cell population was collected for NGS analysis. c Relative enrichment ratio of each scFv calculated from the occupancy in (b), using Niv as a reference. d Occupancy of each scFv before and after sorting using Pert-AF647 at 10 nM. The same cell library in (b) was incubated with 10 nM Pert-AF647 and the rAb(−) cell population was collected for NGS analysis. e Relative enrichment ratio of each scFv calculated from the occupancy in (b) (library cells) and (d), using Niv as a reference. f Parallel qAb classification via Epitope Binning-seq. The 14 qAbs were classified into three bins, pertuzumab-epitope, trastuzumab-epitope, and ungrouped.