Fig. 1
Mutagenesis campaign identifies novel affinity-matured HER2-targeting IgE. (A) Mutagenesis strategy used to construct the Fab phage display library. A synthetic DNA library was designed such that each amino acid position within the CDRs was individually substituted with all other 19 amino acids (Site-Saturation Mutagenesis) or deleted. To enhance library diversity and enable the analysis of the effects of multiple simultaneous mutations on binding affinity, saturated CDR variants were assembled using conserved framework region homology. This strategy allows each clone in the library to carry between 1 and 3 mutations (random point mutations in the CDRs are indicated with *) in each of the heavy and light chain domains. The resulting DNA library was electroporated into E. coli TG1 cells and phage display selection was performed by panning against decreasing concentrations of human HER2. (B) Top: Binding affinity and mast cell degranulation potency measurements of the parental and affinity matured Fab and full length IgE clones, respectively. Binding affinities were determined using BLI. Kd values were measured against both human and rat HER2. Reported values represent the average of three independent measurements for each antigen. Hoxb8 EC50 values are from Supplementary Figure 1A. Bottom: Representative BLI sensorgrams for the parental Fab and affinity matured Fab clones 4, 5 (EPS 232) and 9 bound to human HER2. (C) Hoxb8 mast cell degranulation EC50 versus the monovalent human HER2 affinity. R2, coefficient of determination. Values are from Supplementary Fig. 1A. (D) Amino acid sequence of the heavy and light CDRs of EPS 226 and clones 4, 5 (EPS 232) and 9, with their respective modifications highlighted in red. (E) SKBR3, JIMT-1, and MDA-MB-231 cells were treated with either full length NIP IgE, EPS 226, or clones 4, 5, or 9; where bound IgE was detected using an anti-IgE FITC conjugated antibody. Data points show mean average, error bars are standard error of the mean (SEM), n = 6, three independent experiments, MFI—median fluorescence intensity, mean EC50 value shown ± SEM.
