High-throughput screening for FVIIIa-mimetic activity. An overview of the pursued mechanism of action of Mim8 (A) and screening and optimization strategy (B) is shown. Assays used to investigate functional, structural, and biophysical properties of biAb, anti-FIXa OA, and mAb variants are indicated. (C) Number of unique amino acid substitutions explored at individual positions (using consecutive numbering) in the heavy (V_H) and light (V_L) chain variable domains of the Mim8 anti-FIXa arm. Complementarity-determining region (CDR) loops are highlighted in gray. (D) Evolution of FIXa stimulation of variants of the parental Mim8 anti-FIXa arm during rounds of mutational optimization. FIXa stimulation was measured in high throughput at pH 7.4 in the presence of 0.15 to 1 nM FIXa, 100 nM FX, 500 µM PS:PC vesicles, and a single concentration (800 nM) of anti-FIXa OA. A total of 1308 variants were investigated, with the distribution of activities within each round shown as a violin plot. (E) Plasma activity of a representative subset of biAbs from cycles of anti-FIXa arm optimization. Variant anti-FIXa arms (identified by symbol and color coding as in panel D) were assembled with the parental anti-FX arm. Activity (mean, n = 2) was measured in a TF-triggered thrombin generation assay in human congenital HA plasma at 4 biAb concentrations and normalized to the response obtained with 333 nM emicizumab SIA. Green triangles and blue circles represent biAbs containing the parental (biAb46376) and final Mim8 anti-FIXa arm, respectively. The final engineered Mim8 is included for reference and shown by blue diamonds and a dashed line.