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.