Abstract

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires both in vivo and in vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800 bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from an in vivo selection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of the in vivo selection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during the in vivo panning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767 bp, 53 of them covering the whole insert of 977 bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939 bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.

Highlights

  • Immunoglobulin (IG) or antibody fragments displayed as single chain Fragment variable on filamentous phages are classically selected from scFv-phage combinatorial libraries to obtain human antibodies specific for a given target [1,2,3]

  • The IMGT/HighV-QUEST analysis of the scFv reads included identification of the closest V, D, and J IMGT genes and alleles, characterization of the junction, evaluation of the mutations, and complete description of both V domains with IMGT labels. These results demonstrate that both domains of the scFv reads sequenced by Pacific Biosciences (PacBio) could be fully characterized with the functionality for scFv

  • In vivo or ex vivo phage-display selections have emerged as interesting ways to identify accurate antibodies in the context of the pathologic microenvironment [11]

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Summary

Introduction

Immunoglobulin (IG) or antibody fragments displayed as single chain Fragment variable (scFv) on filamentous phages (scFvphages) are classically selected from scFv-phage combinatorial libraries to obtain human antibodies specific for a given target [1,2,3]. Classical in vitro phage display approaches involve multiple rounds of selection (or panning) for the enrichment of scFv-phages that demonstrate the desired specificity against a target followed, at the last selection round, by functional screening and characterization of selected candidates using appropriate assays. At this very last step, analysis of the selected scFv via Sanger sequencing is commonly used to identify sequences of interest, taking advantage of the genotype–phenotype linkage inherent to the display system. A few approaches have been proposed, retrieving information regarding V domain association has still not been solved [14,15,16]

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