A cell–cell interaction format for selection of high-affinity antibodies to membrane proteins

Abstract

Generating and improving antibodies and peptides that bind specifically to membrane protein targets such as ion channels and G protein-coupled receptors (GPCRs) can be challenging using established selection methods. Current strategies are often limited by difficulties in the presentation of the antigen or the efficiency of the selection process. Here, we report a method for obtaining antibodies specific for whole cell membrane-associated antigens which combines a cell–cell interaction format based on yeast display technology with fluorescence-activated cell sorting of dual fluorescent complexes. Using this method, we were able to direct the affinity maturation of an antagonist antibody specific for the proton-gated ion channel ASIC1a and showed that both the affinity and potency were improved. We were also able to use this method to do kinetic selections to generate clones with better dissociation profiles. In addition, this method was employed successfully to handle the difficult problem of selecting antibodies specific to a GPCR target, the mu-opioid receptor.

FACS-Based Functional Protein Screening via Microfluidic Co-encapsulation of Yeast Secretor and Mammalian Reporter Cells

“…In addition, it is compatible with both immune and combinatorial libraries 40,41 . Simultaneous secretion and display of the library variants on the yeast surface could be achieved by utilizing an inefficient ribosomal skipping (F2A) 42 , enabling alternating affinity-based 43 and functional selection using the same mammalian target (reporter) cell www.nature.com/scientificreports www.nature.com/scientificreports/ line. Furthermore, human-like N-glycosylation could successfully be engineered in S. cerevisiae 44 , which is an important factor influencing the bioactivity, physicochemical properties, and pharmacokinetics of the majority of biologics [45][46][47] .…”

A review of antibody-based therapeutics targeting G protein-coupled receptors: an update

“…Recent developments designed to enhance the ability for antibody discovery and functional screening include increasing cell surface expression and methods for detection of GPCRexpressing cell lines [21,22]; access to new generation detergents such as calixarenes [23], GPCR stabilization for improved expression [24,25], enhanced thermostability or conformation [26], the implementation of lipoparticles [27], magnetic proteoliposomes [28] or virus-like particles [29], spherical-supported bilayer lipid membranes [30] and the generation of nanodiscs [31] have all added to the arsenal of antigen formats that can be applied not only in the generation of antibodies but also associated downstream discovery processes and compliment established tool reagents, such as whole cells or membranes over-expressing the target of interest. The effectiveness of DNA immunization expression constructs or GPCR-expressing cells can be enhanced using adjuvants [32,33] with inventive selection strategies [34] and affinity maturation cell-based approaches devised, for example, CHO cell display libraries of single-chain variable fragments The typical structure for each major GPCR family is presented where the blue horizontal lines represent the lipid bilayer of the cell membrane and numbers 1-7 each represents a GPCR helix or individual transmembrane (TM) domain. An example of each family member is provided alongside the respective endogenous ligand(s), e.g., in the case of CCR5, there are three main chemokine ligands.…”

A suspension cell‐based interaction platform for interrogation of membrane proteins

“…18 A recently reported yeast/mammalian cell interaction platform allowed for live cell incubation in suspension. 19 This platform enriched for specific clones within a yeast-displayed library using FACS, through detection of fluorescent proteins genetically incorporated into both the yeast and mammalian cells. In this study, we establish a platform denoted "biofloating," which enables quantitative analysis of the interaction between proteins on the surface of yeast and MPs on the surface of mammalian cells.…”

“…MACS was recently shown to be a useful technique for yeast/mammalian cell screening, 20 given its superior library sampling capacity compared to FACS. 19 Here, we demonstrate the use of MACS-based yeast/mammalian cell selections to enrich for a specific clone spiked into a naïve yeast-displayed scFv library (10 9 diversity). The biofloating technique complements this suspension cell-based MACS selection approach, providing a high-throughput flow cytometry-enabled screening approach that can be integrated into the protein engineering workflow.…”

“…Enrichment of atezolizumab scFv-expressing yeast using suspension versus adherent methodsIn addition to characterizing yeast/mammalian cell binding interactions on the biofloating platform, we explored whether a soluble yeast/mammalian cell interaction system could be exploited for enrichment of a specific clone spiked into a naïve scFv library. Enrichment using a soluble yeast/mammalian cell interaction platform has been conducted previously via FACS to enrich specific yeast clones from a library of diversity 2.5 × 10 7 19. To increase the throughput of enrichment and allow for selection of higher diversity libraries, we F I G U R E 3 Effects of scFv affinity on equilibrium yeast/mammalian cell interactions for biofloating versus biopanning platforms.…”