Probing a protein-protein interaction by in vitro evolution

Abstract

In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.

Fig. 1.

Comparison of the amino acid substitution frequency after affinity maturation of BAK1 by using ribosome display and the frequency observed after somatic hypermutation of human antibody sequences in vivo. Mutation frequencies for ribosome display (gray bars) and in vivo hypermutation (white bars) are shown for the V_H region (A) and the V_L region (B), with CDRs marked. Somatic hypermutation data are unavailable for CDR3 and framework 4 of both the V_H and V_L regions, and these sections are excluded from analysis. In vivo somatic hypermutation frequencies are reproduced from refs. and .

Fig. 2.

Inhibition of IL-13-induced proliferation in TF-1 cells by the anti-IL-13 parent IgG4 BAK1 (IC₅₀ of 78.9 nM) and the optimized IgG4 variants BAK1.1 (IC₅₀ of 388 pM), BAK1.29 (IC₅₀ of 274 pM), and BAK1.45 (IC₅₀ of 229 pM). To obtain an IC₅₀, triplicate titrations of IgG4 were preincubated with recombinant human IL-13 before both were added to TF-1 cells, and levels of cell proliferation were measured by uptake of tritiated thymidine.

Fig. 3.

Mapping of key residues affecting IL-13 binding onto a space-filling structural model of the BAK1.1 Fv from the viewpoint of the antigen. The model shows the V_H chain on the left and the V_L chain on the right, and the six CDRs composing the antigen-combining site are labeled. Hotspot positions (in which residues were found to be mutated independently in two or more variants of improved potency) derived from in vitro evolution are shown in red. CDR residues that, upon mutation to alanine, were shown to reduce their maximal binding (R_max) to an IL-13-coated Biacore surface by >70%, are shown in dark blue. The two populations show distinct distributions over the antibody V-region surface.