J. Hanes, L. Jermutus, S. Weber-Bornhauser, H. R. Bosshard and A. Plückthun
Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries
Proc. Natl. Acad. Sci. USA 95, 14130-14135 (1998)

Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 +/- 1) x 10(-11) M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coil periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.

C. Berger, S. Weber-Bornhauser, J. Eggenberger, J. Hanes, A. Plückthun and H. R. Bosshard,
Antigen recognition by conformational selection
FEBS Letters 450: 149-153 (1999).

Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an `induced fit', which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an `induced fit' requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross-reacts with the wild-type dimeric leucine zipper. Kinetic and equilibrium data show thatthe single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.

Wörn, A., Auf der Maur, A., Escher, D., Honegger, A., Barberis, A. and Plückthun, A.
Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors
J. Biol. Chem. 275, 2795-803 (2000)

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the GCN4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate
that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.