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Yeast surface display platform for rapid discovery of conformationally selective nanobodies.

ABSTRACT: Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.

SUBMITTER: McMahon C

PROVIDER: S-EPMC5839991 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Yeast surface display platform for rapid discovery of conformationally selective nanobodies.

McMahon Conor C Baier Alexander S AS Pascolutti Roberta R Wegrecki Marcin M Zheng Sanduo S Ong Janice X JX Erlandson Sarah C SC Hilger Daniel D Rasmussen Søren G F SGF Ring Aaron M AM Manglik Aashish A Kruse Andrew C AC

Nature structural & molecular biology 20180212 3

Camelid single-domain antibody fragments ('nanobodies') provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin V<sub>HH</sub> domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date hav ...[more]

PMID: 29434346

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Project description:BackgroundNeutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19.ResultsIn this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable 'switch' through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB.ConclusionThe antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.

Dataset Information

Yeast surface display platform for rapid discovery of conformationally selective nanobodies.

Publications

Yeast surface display platform for rapid discovery of conformationally selective nanobodies.

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