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

Abstract

Abstract 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.