Genotyped functional screening of soluble Fab clones enables in-depth analysis of mutation effects

: Oksanen Sami, Saarinen Roope, Korkiakoski Anttoni, Lamminmäki Urpo, Huovinen Tuomas

Publisher: Nature Publishing Group

: 2023

: Scientific Reports

: Scientific reports

: Sci Rep

: 13107

: 13

: 2045-2322

DOI: https://doi.org/10.1038/s41598-023-40241-2

: https://doi.org/10.1038/s41598-023-40241-2

: https://research.utu.fi/converis/portal/detail/Publication/180921338

Monoclonal antibodies (mAbs) and their fragments are widely used in therapeutics, diagnostics and basic research. Although display methods such as phage display offer high-throughput, affinities of individual antibodies need to be accurately measured in soluble format. We have developed a screening platform capable of providing genotyped functional data from a total of 9216 soluble, individual antigen binding fragment (Fab) clones by employing next-generation sequencing (NGS) with hierarchical indexing. Full-length, paired variable domain sequences (VL-VH) are linked to functional screening data, enabling in-depth analysis of mutation effects. The platform was applied to four phage display-selected scFv/Fab screening projects and one site-saturation VH affinity maturation project. Genotyped functional screening simultaneously enabled the identification of affinity improving mutations in the VH domain of Fab 49A3 recognizing Dengue virus non-structural protein 1 (NS1) serotype 2 and informed on VH residue positions which cannot be changed from wild-type without decreasing the affinity. Genotype-based identification revealed to us the extent of intraclonal signal variance inherent to single point screening data, a phenomenon often overlooked in the field. Moreover, genotyped screening eliminated the redundant selection of identical genotypes for further study and provided a new analysis tool to evaluate the success of phage display selections and remaining clonal diversity in the screened repertoires.

s41598-023-40241-2.pdf