(217b) High Affinity Fn3 Domains Using Loop Length Diversity and Population Maturation

The tenth type III domain of human fibronectin (Fn3) is a small (10 kDa), stable protein with a beta-sandwich fold and three solvent-exposed loops (termed BC, DE, and FG for the beta-strands that they connect). It has been demonstrated as an effective protein scaffold to engineer high affinity binders to multiple targets using mRNA display, phage display, and yeast two-hybrid methods. Yet, while variability in the length of complementarity-determining regions is recognized as a critical factor in antibody diversity, past Fn3 libraries have had fixed loop lengths. The current work addresses if loop length diversity can improve the affinity and recognition capacity of the Fn3 scaffold. A second, broader question is if recurrent mutagenesis of selected populations, already a necessary component of in vitro display methods through PCR, enables rapid isolation of high affinity binders using yeast surface display libraries.

Oligonucleotides of multiple lengths containing NNB degenerate codons were used to diversify the length and amino acid composition of the BC, DE, and FG loops. The yeast surface display library had 2x10^7 Fn3 clones with four possible lengths of each loop. Clones that bound lysozyme were selected by multiple rounds of fluorescence-activated cell sorting. The enriched population was diversified every two to three rounds by error-prone PCR, loop shuffling, and homologous recombination using a simple one-day protocol. Isolated clones from several stages of the selection were sequenced and characterized in terms of affinity and association and dissociation kinetics.

Multiple clones with picomolar equilibrium dissociation constants were identified. The majority of high affinity clones had BC and DE loops one amino acid shorter than wild-type suggesting that loop length diversity is a valuable element of Fn3 library design. Isolation of such high affinity clones from a relatively small initial library was enabled by affinity maturation via continued selection and diversification as demonstrated by parallel selections without mutagenesis and shuffling. The recurrent population diversification should be advantageous to protein engineering in general.