Integrating SpyCatcher/SpyTag covalent fusion technology into phage display workflows for rapid antibody discovery

Julie K Fierle,Matteo Brioschi,George Coukos,Johan Abram-Saliba,Steven M Dunn,Mariastella Detiani

doi:10.1038/s41598-019-49233-7

Julie K Fierle, Matteo Brioschi + Show 4 more

Open Access

https://doi.org/10.1038/s41598-019-49233-7

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Journal: Scientific Reports	Publication Date: Sep 6, 2019
Citations: 15	License type: open-access

Affiliation: Ludwig Cancer Research, University of Lausanne

Abstract

An early bottleneck in the rapid isolation of new antibody fragment binders using in vitro library approaches is the inertia encountered in acquiring and preparing soluble antigen fragments. In this report, we describe a simple, yet powerful strategy that exploits the properties of the SpyCatcher/SpyTag (SpyC/SpyT) covalent interaction to improve substantially the speed and efficiency in obtaining functional antibody clones of interest. We demonstrate that SpyC has broad utility as a protein-fusion tag partner in a eukaryotic expression/secretion context, retaining its functionality and permitting the direct, selective capture and immobilization of soluble antigen fusions using solid phase media coated with a synthetic modified SpyT peptide reagent. In addition, we show that the expressed SpyC-antigen format is highly compatible with downstream antibody phage display selection and screening procedures, requiring minimal post-expression handling with no sample modifications. To illustrate the potential of the approach, we have isolated several fully human germline scFvs that selectively recognize therapeutically relevant native cell surface tumor antigens in various in vitro cell-based assay contexts.

Highlights

Over the past 20 years display technologies have become established as robust and powerful approaches for the in vitro isolation of protein and nucleic acid-based ligands with utility for both clinical and non-clinical applications[1,2,3]
For solid-phase immobilization, we synthesized a biotinylated minimal SpyT peptide with an additional tetra-peptide ‘linker’ extension and a C-terminal lysine-biotin to allow a pre-coating of streptavidin-coated beads or plate wells (Fig. 1a)
As experimental test ‘antigens’ we first selected extracellular domain components (ECDs) of the human CD3 complex assembled in a single chain format, and a series of corresponding anti-CD3 scFv antibody fragments

Summary

Introduction

Over the past 20 years display technologies have become established as robust and powerful approaches for the in vitro isolation of protein and nucleic acid-based ligands with utility for both clinical and non-clinical applications[1,2,3]. Despite considerable advances in library construction and binder enrichment strategies, certain aspects of display-based discovery technologies remain inefficient and potentially rate limiting Among these is the need to generate recombinant, functionally immobilized antigens or antigen-fragments to sustain the needs of multiple in vitro selection and screening activities. Antigen material is provided chromatographically purified, often with oligo-histidine or IgG Fc affinity tags present In this format, it can be passively adsorbed directly to plastic well or tube surfaces, or biotinylated to permit essentially stable binding to streptavidin-coated matrices. Certain engineered enzyme fusion tags, despite their potential for direct stable covalent capture via catalytic cycle trapping, are typically large (eg. 34 kDa for Halotag7) which may explain why they have not been widely adopted for display technologies[19,20,21]

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