The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold

The design of combinatorial libraries for molecular recognition requires extensive diversity to provide high affinity binding to myriad epitopes while maintaining a high degree of functionality to enable inclusion of binders in the limited screenable library size. In the current work, we directly compare minimal and maximal amino acid diversity libraries in the context of the 10th type III domain of human fibronectin. Libraries with either serine/tyrosine or full 20 amino acid diversity were created, pooled and screened for binding to rabbit and goat immunoglobulin G (IgG), and affinity matured by directed evolution. Multiple picomolar binders to rabbit IgG and nanomolar binders to goat IgG were engineered with peak affinities of 51 ± 4 pM and 1.2 ± 0.4 nM, respectively. Sequence analysis reveals that 93 of the selected BC and FG loops, including those from the highest affinity clones, originate from the full diversity library. Thus, with a modest initial library size (∼1 × 10⁸) and an efficient affinity maturation scheme, more extensive diversity is superior to a binary serine/tyrosine code for the generation of picomolar to low nanomolar binders in the fibronectin domain. The highest affinity binders demonstrated utility in affinity purification of IgG from serum and as detection reagents in flow cytometry.