Fig. 1.

Directed Evolution of PB clones from a non-immune human scFv library. (A) A series of magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) selections were used, both including both positive (+) selections of Ag-binders and negative (−) selections against reagent binders such as secondary (2ary) Ab, streptavidin bead (Bare SA) and Mouse IgG Fc binders. Generation names x.y represent the number of x) diversification steps and y) selections after diversification. Pre-Block indicates a blocking step against the mouse IgG Fc tag using 500 nM normal msIgG prior to staining with B7H6-Ig. Concentrations of B7H6-Ig used for selections are indicated under selection arrows. Boxes indicate generations for which titration curves of antigen were tested and shown in (C). PB clones were isolated from g2.5. (B) Representative FACS plots for a subset of improving generations are shown. Binding to B7H6-Ig (y-axis) and expression tag antibodies (x-axis) are shown. (C) Titration curves depict the MFI of library generations stained with B7H6-Ig antigen at a range of concentrations to compare binding avidities.