Extended Data Fig. 3: Rank and correlation plots from deep screening for the HER2 ML vs. Random library.
a) Rank plots of 199k anti-HER2 scFv clones from the equilibrium binding assay, showing their mean fluorescent intensities at 0 nM, 0.1 nM, 0.3 nM, 1 nM, 3.3 nM, 10 nM, 33.3 nM and 100 nM Her2. Clones were selected for a variety of reasons (see Extended Data 1) for subsequent conversion to Fab, expression, purification, and characterisation. b) PCA plot from the ‘HER2Affmat’ library, showing all 236k CDR H3 protein sequences projected into two dimensions and coloured by mean fluorescent intensity at 100 nM of HER2. A red dot shows the position of G98A wild-type relative to the library. c) PCA plot from the HER2 ML vs. Random library, showing all 199k CDR H3 protein sequences projected into two dimensions and coloured by mean fluorescent intensity at 100 nM of HER2. d) Correlation between BLI characterised binding affinities (KD) and deep screening mean FI at 0 nM, 0.1 nM, 0.3 nM, 1 nM, 3.3 nM, 10 nM, 33.3 nM, 100 nM HER2 and the 5 minute wash condition. Error bars are s.e.m. and n ≥ 12. The grey vertical line is showing the mean library intensity at each respective concentration. Correlations are shown as Spearman’s rank correlation constant (rs) and p-values were determined by a two-tailed test. e) Zoomed correlation between BLI characterised binding affinities (KD) and deep screening mean FI at 3.3 nM and 10 nM HER2. Error bars are s.e.m. and n ≥ 12. f) Correlation between BLI characterised binding affinities (KD) and deep screening determined equilibrium binding constants (\({{\rm{K}}}_{{\rm{D}}}^{{\rm{app}}}\)). Correlations are shown as Spearman’s rank correlation constant (rs) and p-values were determined by a two-tailed test. Linear regression was used to show a line of best fit.
