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 IC 50 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.affinity maturation ͉ human antibodies ͉ IL-13 ͉ protein evolution ͉ ribosome display

Mining human antibody repertoires

“…[167][168][169] Ribosome display has been employed to both accomplish and guide the affinity maturation of human mAbs. 170 Other less common display technologies, such as retroviral display, 171 have also been used for mining synthetic repertoires. In addition to the conventional scFv and Fab formats, synthetic repertoires have facilitated the display and selection of single human variable domains 81,172 and, recently, human constant domains with diversified sequences in non-CDR loops of the Ig fold.…”

Parallel Processing of Complex Biomolecular Information: Combining Experimental and Computational Approaches

“…Another approach is the indiscriminate mutation of nucleotides using the low-fidelity Taq DNA polymerase (Hanes et al, 2000), error-prone PCR (Hawkins et al, 1992;Daugherty et al, 2000;Jermutus et al, 2001;van den Beucken et al, 2003), the error-prone Qbeta RNA replicase or E. coli mutator strains (Irving et al, 1996;Low et al, 1996;Coia et al, 2001) before and in-between rounds of selection. Shuffling and random point mutagenesis are particularly useful when used in conjunction with targeted approaches because they enable the simultaneous evolution of non-targeted regions (Thom et al, 2006); in addition, they are powerful when performed together because individual point mutations can recombine and cooperate, again leading to synergistic potency improvements. This has created some of the highest affinity antibodies produced so far, with dissociation constants in the low picomolar range (Zahnd et al, 2004) and in a study using yeast display, even in the femtomolar range .…”

“…To this end, a variant library generated by error-prone PCR, for example, might be subjected to affinity selections followed by the sequencing of improved scFvs. In a manner similar to somatic hypermutation, this method leads to the accumulation of mutations responsible for potency gains mainly in CDRs, despite having been introduced randomly throughout the whole scFv coding sequence (Thom et al, 2006).…”

“…In addition, the indiscriminate mutation of these residues creates many variants that no longer bind the antigen, reducing the functional library size. Scientists have therefore restricted the number of mutations by targeting only blocks of around six consecutive residues per library (Thom et al, 2006) or by mutating four variants in all the CDRs (Laffly et al, 2008) or by mutating only the CDRs 1 and 2 (Hoet et al, 2005). Mutagenesis has also been focussed on natural hotspots of SHM (Ho et al, 2005).…”

‘In-Format’ screening of a novel bispecific antibody format reveals significant potency improvements relative to unformatted molecules

“…These cells were used to measure the ability of antibodies to neutralise NGF, similar to methods described previously. 31,32 Briefly, various concentrations of anti-IL4/anti-NGF mAbdAbs and appropriate controls were pre-incubated for 15 minutes at room temperature with NGF (R&D Systems) in RPMI 1640 media (Invitrogen). The antibody/ligand mixture was then added to TF-1 cells (10,000 cells/well) in RPMI 1640 media in a 96-well tissue culture treated plate (Costar Ò , Cambridge, MA, USA) and incubated for 4 d at 37…”