Fig. 4: Affinity maturation of the anti-HER2 scFv G98A.
a, Construct schematic of G98A, showing its CDR H3 sequence and a depiction of how the six scanning-window NNS sub-libraries were structured. b, Experiment statistics from the deep-screening component. c, PCA plot showing all 236,000 CDR H3 protein sequences projected into two dimensions and coloured by FImean at 100 nM of HER2. A red dot shows the position of G98A wild type relative to the library. d, CDR H3 sequences of G98A, ML3-9 and three of the top-scoring clones identified by deep screening. As we were unable to obtain a 1:1 model fit to the BLI data of clone G98A at 20 nM of Fab, we opted to use the published surface plasmon resonance (SPR) KD value. Next to the sequences are binding KDs identified via BLI, and the deep-screening-fitted equilibrium-binding \({K}_{{\rm{D}}}^{{\rm{app}}}{\rm{s}}\). e, Deep-screening equilibrium-binding and kinetic dissociation curves showing G98A, ML3-9 and three of the top-scoring clones. Error bars are s.e.m. and n ≥ 12 technical replicates of a given UMI. f, BLI kinetics of G98A, ML3-9 and three of the top-scoring clones at 20 nM of each clone in the Fab format on a HER2-loaded tip. The grey dashed line denotes the separation of the association (left) and dissociation (right) phases collected during BLI kinetics measurements.
