ogy, a scFv fragment of an antibody with picomolar affinity for a GCN4-variant peptide was isolated from a library prepared from immunized mice8. Alternative in vitro selection methods such as “RNA–peptide fusion”9,10, in which an in vitro-synthesized polypep-

1287 Ribosome display1,2 is a technology for the in vitro selection and evolution of very large protein libraries. The main feature distinguishing this technique from other selection techniques, such as phage display3–5, is that the entire procedure is performed in vitro, without using cells at any step. Ribosome display was developed and applied first for peptide libraries6, and was then systematically improved to be suitable for screening and selection of folded proteins7. The principle of ribosome display is depicted in Figure 1. In ribosome display genotype and phenotype are linked through ribosomal complexes, consisting of messenger RNA (mRNA), ribosome, and encoded protein, that are used for selection. Using this technology, a scFv fragment of an antibody with picomolar affinity for a GCN4-variant peptide was isolated from a library prepared from immunized mice8. Alternative in vitro selection methods such as “RNA–peptide fusion”9,10, in which an in vitro-synthesized polypeptide is covalently attached to its encoded message, have been demonstrated to work for peptide libraries. If a high-fidelity proofreading DNA polymerase is used during the PCR amplification steps (Fig. 1), the repertoire of the library employed is virtually maintained11. However, a particularly interesting feature of the ribosome display technology is that it can also be used for the directed evolution and affinity maturation that occurs during the selection process, if a low-fidelity DNA polymerase is used that introduces mutations during amplification (Fig. 1). A directed evolution can also be achieved when in vivo selection technologies are used, for example by transforming into particular mutator strains12 when using phage display. However, this entails the risk of simultaneously introducing unwanted and possibly detrimental mutations in the plasmid or the host genome. For more efficient and controlled mutagenesis, it is usually necessary to switch between the diversification steps in vitro and the selection steps in vivo, which includes additional laborious cloning and transformation after each cycle13–15. Thus, an important advantage of ribosome display is that selection and evolution can easily be performed entirely in vitro and thus these impediments can be avoided. In this study we applied the ribosome display technology for the selection and evolution of scFvs from the synthetic HuCAL, which contains 2 × 109 independent members and has been designed to cover most of the antibody structure space16. The HuCAL is several orders of magnitude more diverse than a typical library from immunized mice and is not enriched for specific binders before selection. We show here that by ribosome display it is possible to select a range of different scFvs from a naive library, which have affinities up to 82 pM. All the selected antibodies accumulated mutations through amplification with low-fidelity DNA polymerase, and thereby improved their affinities for antigen up to 40-fold when compared to the progenitor sequences originally present in the HuCAL and thus evolved during ribosome display selection.