1 Introduction
Membrane proteins (MPs) are important therapeutic targets as they are responsible for facilitating a wide range of biological functions critical to maintaining homeostasis. MPs transduce signals into cells, catalyze biochemical reactions, adhere to surfaces, and transfer ions across the cell membrane. The ability to manipulate cellular processes, particularly when these processes are dysregulated in the context of disease, therefore rests on our capacity to specifically engage MPs. Indeed, over 60% of all clinically approved drugs target MPs.1 Due to their high affinity, specificity, and favorable pharmacokinetic properties, antibodies represent a particularly desirable and growing class of drugs against MPs.2,3 Antibodies are generally discovered either through immunization of animals or using in vitro display technologies such as phage or yeast display.4–8In vitro display methods enable the screening of large combinatorial protein libraries by linking genetic information to phenotypic responses. Antibodies are typically screened in display platforms through presentation of their minimal binding moieties, known as single-chain variable fragments (scFvs). Discovered scFvs can later be formatted into full-length antibodies as potential therapeutics.9 In vitro display technologies allow for fine tuning of the biophysical properties of antibodies such as target affinity and specificity.10–13 Although the phage display platform allows for greater library diversity compared to yeast display, the latter has several important advantages, including: (i) more accurate recapitulation of native post-translational modifications in mammalian proteins through use of a eukaryotic system; (ii) more sophisticated secretory apparatus to allow for expression of disulfide-bonded proteins; and (iii) capacity to perform dual-color sorts using fluorescently activated cell-sorting (FACS) to enhance selection of high affinity clones.10
In order to select for translationally relevant antibodies against MPs, the antigen must be displayed in a conformation that closely represents its native state in the cell membrane. Although there has been great success in generating antibodies against relatively straightforward MP targets such as single-pass membrane proteins, generation of antibodies targeting complex multipass transmembrane proteins such as G protein-coupled receptors (GPCRs) or ion channels has been more challenging. This is due to poor expression or solubility of multipass transmembrane proteins when produced recombinantly, which complicates their use as selection targets for immunization or display strategies. Addition of detergents to stabilize multipass MPs or selections against truncated peptide sequences found within MPs have been implemented; however, these approaches can enrich for antibodies against intracellular rather than extracellular epitopes or antibodies that bind non-native conformations of the protein.14–16Alternative approaches such as nanodisk technology have facilitated display of MPs, although such methods require significant optimization.17
Another approach to achieve native antigen presentation for multipass transmembrane proteins involves the use of whole cells expressing the MP of interest. Whole cell screening approaches allow for the target to be displayed in its native conformation, with appropriate post-translational modifications, and in an orientation that is directly relevant for antibody targeting. Tillotson et al established a technique known as yeast biopanning, which involves incubating a yeast-displayed library of proteins with monolayers of mammalian cells expressing the target MP for discovery of specific binders.18 In addition, the authors established a solubilized whole cell lysate-based approach to interface yeast-displayed protein libraries with detergent-treated MP-expressing mammalian cells. Cell lysate-based evolution has proven to be an effective engineering approach, although it requires significant optimization of detergent conditions.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. This approach detects yeast/mammalian cell interactions via fluorescently-labeled antibody staining of an epitope tag commonly incorporated into the yeast display platform and intracellular staining of MP-presenting mammalian cells. In contrast with previous approaches, biofloating enables interrogation of yeast/mammalian cell interactions in a fully suspension-based system without the need for genetic incorporation of fluorescent proteins. We characterize and optimize the biofloating platform by studying the interaction between scFvs on the surface of yeast and the MP programmed death-ligand 1 (PD-L1) on mammalian cells, comparing the performance of our platform to traditional biopanning techniques using a range of affinity and avidity conditions.
To extend our suspension cell-based approach to high-throughput library screening, we used magnetic-activated cell sorting (MACS) to enrich for specifically interacting scFvs against a target MP. 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 (109 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.