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Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single-chain Fv (scFv) antibodies for increased affinity and thermal stability and, more recently, for the display and screening of a non-immune scFv and immune Fab libraries. A major strength of yeast display as a novel antibody discovery platform is the ability to characterize the binding properties, i.e., the affinity and epitope binding characteristics, of a clone without the need for subcloning, expression and purification of the scFv. This review focuses on novel attributes of yeast display for antibody engineering endeavors. [Copyright &y& Elsevier]

Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library. [Copyright &y& Elsevier]

In order to more productively utilize the rich source of antigen-specific reagents present in the previously described non-immune single chain fragment variable (scFv) yeast display library [Nat. Biotechnol. 21 (2003) 163], one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi Macs™ system to recover greater than 90% of the antigen-specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen-specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope-specific scFv. Combining these two improvements in library screening allowed isolation and characterization of three epitope-specific scFv, including a previously uncharacterized epitope to a 6-kDa protein, epidermal growth factor. [Copyright &y& Elsevier]