Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries

Abstract

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 coli 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.

Figure 1

Analysis of pools after the third round of ribosome display. Pools of selected RNA were translated in vitro in the presence of [³⁵S]methionine, and translation mixtures containing 0 and 1 μM GCN4(7P14P) peptide antigen as inhibitor were analyzed by RIA as described in Methods. Each bar represents the average of three samples. The ribosomal complexes were enriched on immobilized peptide antigen in the presence (experiment A) or in the absence (experiment B) of 2% sterilized milk during selection (for details, see text).

Figure 2

Alignment of amino acid sequences of related scFvs binding to GCN4(7P14P) peptide, isolated by ribosome display. Only V_L and V_H are shown. Residues identical to the consensus sequence of the 22 related scFvs (cons) are represented by dashes. The upper three scFvs (g2, g5, and g14) were isolated in experiment B, and the other 19 scFvs (c1 to c23) in experiment A (see Fig. 1). Numbering of amino acid residues in V_L and V_H and the labeling of CDRs is according to Kabat et al. (23).

Figure 3

RIA analysis of selected scFvs binding to GCN4(7P14P) peptide isolated by ribosome display and c11L34Ser mutant. RNA of single scFvs was translated in vitro in the presence of [³⁵S]methionine, and translation mixtures containing 0, 1, 10, and 100 nM GCN4(7P14P) peptide were analyzed by RIA as described in Methods. Each bar represents the average of three samples. For experiments A and B, see Fig. 1.

Figure 4

Determination of antigen dissociation constants (K_d) of c11 and c11L34Ser scFvs. Purified proteins c11 (2 nM) and c11L34Ser (1 nM) were mixed with different concentration of GCN4(7P14P) peptide and incubated for 1 hour before analysis. Samples were injected over the sensor chip coated with BSA-GCN4(7P14P) conjugate. From the linear sensograms, the slopes (resonance units vs. time in sec) were plotted against the corresponding total soluble antigen concentration. The slopes correlate to uncomplexed scFv in the injected solutions. From a fit with Eq. 1, K_d was calculated. Each point is the average of four independent experiments.