Recently, in vitro screening methods such as phage display or yeast display are commonly used for engineering antibodies, while they have Pros/Cons. Standing on this point, the development of other screening methods will contribute to engineer and make it better therapeutic antibodies. Ribosome display (RD) is expected to have several advantages over existing in vitro screening methods, so we developed our own RD (PUREfrex^® RD) using very effective fully reconstituted cell-free expression system (PUREfrex). We showed that a
scaffold, a scFab format and a Fab format without conversion to scFv could be used on PUREfrex^® RD. That was the first report of a direct Fab fragment selection on RD (PEGS 2012).

Here we report another application of PUREfrex^® RD that can be utilized for further antibody engineering such as affinity maturation on the Fab format. We applied PUREfrex^® RD to the lead therapeutic antibody for in vitro affinity maturation. We investigated the process for forming Fab displaying RD complex, which greatly influenced on the screening efficiency.

For instance, we found that several conditions, such as the amount of ribosome to add into the system, or synthesizing H and L chain at different timing, had huge impact on the screening efficiency. We prepared two kinds of randomly mutated L chain libraries for whole region or only for CDRs of the L chain by PCR-based DNA shuffling. After three rounds of off-rate selection over several days, the Fabs were expressed in E. coli and their binding affinities were evaluated by ELISA assay.

Finally, EC50 was determined for comparison in specific activity of each Fabs to the original Fab. We have successfully improved the EC50 approximately 60-fold better than the original Fab. As such, PUREfrex^® RD is the unique screening method, and it could be customized for various use. We would like to discuss the potential applications of PUREfrex^® RD here.