Platform for rapid nanobody discoveryin vitro

Abstract

Camelid single-domain antibody fragments ("nanobodies") provide the remarkable specificity of antibodies within a single immunoglobulin V HH domain. This unique feature enables applications ranging from their use as biochemical tools to therapeutic agents. Virtually all nanobodies reported to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we developed a fully in vitro platform for nanobody discovery based on yeast surface display of a synthetic nanobody scaffold. This platform provides a facile and cost-effective method for rapidly isolating nanobodies targeting a diverse range of antigens. We provide a blueprint for identifying nanobodies starting from both purified and non-purified antigens, and in addition, we demonstrate application of the platform to discover rare conformationally-selective nanobodies to a lipid flippase and a G protein-coupled receptor. To facilitate broad deployment of this platform, we have made the library and all associated protocols publicly available. IntroductionAntibodies have had a transformative impact on science and medicine due to their exceptional specificity and biochemical versatility, enabling applications in almost every aspect of biomedical inquiry. Conventional antibodies are composed of two heavy chains and two light chains. Each chain contributes to antigen binding specificity through a variable domain, termed V H and V L for the heavy and light chain, respectively. A key exception to this general architecture is found in camelids (llamas, alpacas, and their relatives), which possess a parallel antibody repertoire composed solely of heavy chains 1,2 . Such antibodies bind to their target antigens through a single variable domain, termed V HH , which contains the entire antigen-binding surface. Unlike the antigen binding fragments of conventional antibodies (Fabs), isolated V HH domains (also called "nanobodies") can be readily expressed in bacteria as the product of a single gene, and in many cases these fragments can even fold and retain antigen specificity in the reducing environment of the cytosol. Owing to their versatility, nanobodies have found applications in protein biochemistry and structural biology, cell biology, and as potential diagnostic and therapeutic agents [2][3][4][5][6] .

A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

“…Ideally, one could replace all animal immunization by selecting binders from synthetic libraries ( Gray et al, 2016 ; Moutel et al, 2016 ; McMahon et al, 2017 ; Zimmermann et al, 2017 ). Yet, with a purely synthetic approach, it is still not straightforward to obtain high-affinity binders.…”

Development of a Cell Surface Display System inChlamydomonas reinhardtii

“…Our investigation led to the identification of anchoring systems targeting the cell membrane with the GPI anchor signal of the MAW8 protein and targeting the cell wall with the hydroxyproline-rich GP1. The cell wall represents a compelling location for surface display, and this strategy has already been reported for other organisms [66,90]. The mesh-like structure enclosed in five layers [48,91] represents a large surface matrix to host catalytic reactions, such as the ones pursued in whole cell biocatalyst applications.…”

“…Cell-surface technology remains a powerful tool for human health, being used in the development of vaccines[8], nanobodies[66], and has been studied as a delivery system[67].We envisioned C. reinhardtii as an ideal candidate for the future development of key applications in cell-surface biotechnology. A system based on C. reinhardtii would be inexpensive due to its low-cost growth requirements [77] and would represent an attractive alternative in bioprocesses in which the cost is a limiting factor.…”

A toolbox of anti-mouse and rabbit IgG secondary nanobodies

“…Ideally, one should replace all animal immunization by selecting binders from synthetic libraries (Gray et al, 2016;Moutel et al, 2016;McMahon et al, 2017;Zimmermann et al, 2017). Yet, with a purely synthetic approach it is still not straightforward to obtain high-affinity binders.…”