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 non-profit research.

Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds

“…As a consequence, OrthoRep readily integrates with the existing rich ecosystem of cell-based and in vivo yeast genetic selections. For example, OrthoRep is already being used in our lab and others to evolve novel antibodies via yeast surface display (Boder and Wittrup, 1997; McMahon et al, 2018), protein-protein interactions (PPIs) or PPI inhibitors via yeast two-hybrid systems (Fields and Song, 1989), and heterologous metabolic enzymes like Pf DHFR that can replace essential yeast functions. The last of these applications may be especially useful in efforts to create humanized yeast models (Kachroo et al, 2015; Truong and Boeke, 2018) or to improve enzymes from difficult-to-transform hosts like plants.…”

A Nanobody‐Conjugated DNA Nanoplatform for Targeted Platinum‐Drug Delivery

“…[15] Thus,t he targeted delivery of platinum drugs will both improve therapeutic effects and reduce systemic toxicity. [17] Thesmall size and spatial structure endow Nb with the capability of epitope recognition with high affinity and specificity,even when target antigens are densely packed and cryptic. [17] Thesmall size and spatial structure endow Nb with the capability of epitope recognition with high affinity and specificity,even when target antigens are densely packed and cryptic.…”

“…[16] AD NA nanocarrier with active targeting groups may be an ideal platform for targeted platinum-drug delivery.T he camelid-derived singledomain antibody,r eferred to as an anobody (Nb), is the smallest (about 15 kDa) among all types of antibody analogues. [17,18] After antigen recognition, Nbs can subsequently block signal transduction of at arget receptor to inhibit proliferation for combined therapy. [17,18] After antigen recognition, Nbs can subsequently block signal transduction of at arget receptor to inhibit proliferation for combined therapy.…”

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…Nanobodies fold into their native and functional structure once secreted at the surface of bacterial or yeast cells and nanobody display have been used for both screening purposes and for preparing wholecell biosensors [88][89][90][91]. The advantages represented by displaying functional macromolecules on cell surface are multiple and have been…”