4 Discussion
In vitro display platforms have been employed extensively over the past 3 decades for protein discovery and characterization.7 Within the past 10 years, whole cell selection technologies have empowered selection approaches against MP targets by simulating the natural context of the protein relevant for ligand recognition.18 In this study, we build upon these whole cell selection technologies by presenting and characterizing a novel platform that we call biofloating to interrogate protein interactions within the context of a yeast/mammalian cell system. In contrast with previous yeast/mammalian cell interaction systems, our approach enables incubation of yeast with mammalian cells in suspension and utilizes quantitative flow cytometry analysis, without requirement for the incorporation of genetic fusions into either the yeast or mammalian cells. Hence, this versatile interaction system allows for compatibility with existing yeast display infrastructure. Moreover, we found that the biofloating platform demonstrated superior sensitivity compared to biopanning, both in terms of kinetics and equilibrium interactions investigated in this study. In particular, dramatic differences in binding behavior were evident in the context of low target binding affinity or reduced target expression (avidity). Of note, the biofloating approach detected high, medium, and low affinity interactions between yeast-displayed scFvs and target antigen-expressing mammalian cells, whereas biopanning detected only high and medium affinity interactions, and with significantly weaker potency (Figure 3). In addition, the biofloating method detected scFv-displaying yeast interactions with dense and medium density antigen-expressing mammalian cells (Figure 5), whereas biopanning was only able to detect interactions with antigen-dense cells, and with significantly weaker sensitivity.
Interestingly, the biofloating platform achieved maximum yeast/mammalian cell binding virtually instantaneously in the case of high and medium affinity interactions, whereas the biopanning platform exhibited a more gradual rise in binding (Figure 2). The biofloating approach also led to immediate saturation of dense and medium density target-expressing cell lines, whereas the biopanning strategy effected a time-dependent rise in binding for only the dense cell line (Figure 4). The significant differences in on-rate kinetics between the two platforms likely results from distinct spatial arrangements and mixing capacities between the 2-dimensional biopanning and 3-dimensional biofloating systems. In the biopanning platform, yeast gradually settle on top of the mammalian cell monolayer, thus manifesting a diffusive limitation in achieving cell-cell binding. In contrast, in the biofloating platform, yeast cells are well mixed in suspension with mammalian cells, thereby eliminating any diffusive barriers.
A major advantage of the biofloating platform is integration with flow cytometry, which enables precise quantification of binding dynamics. This gives us access to important molecular parameters that define yeast/mammalian cell interactions, such as the number of yeast bound per mammalian cell and the percentage of mammalian cells that interact with yeast. In addition, the biofloating platform interrogates protein interactions fully in suspension, which can be exploited for in vitro evolutionary schemes. To this end, we demonstrated enrichment of yeast displaying a specific scFv from a library of scFv-displaying yeast using a fully suspension cell-based MACS selection approach. This work builds upon a recently reported MACS-based selection platform20 to achieve specific enrichment of a clone spiked into a naïve scFv library and to elucidate the dynamics of enrichment over of multiple rounds of evolution. We showed that this suspension cell-based approach led to robust enrichment of yeast specific for the mammalian cell-expressed target antigen, and this enrichment was more rapid than for an analogous adherent cell-based selection approach (Figure 6). Importantly, substantial enrichment occurred in the first round using the suspension cell-based approach. This observation is significant since MACS is most useful in early rounds to debulk large libraries, compared to alternative approaches such as FACS, which take prohibitively lengthy amounts of time to screen highly diverse libraries. Indeed, the throughput of our approach enabled rapid screening of a 109 member scFv library incorporating a 10-fold excess of yeast cells. We ultimately envision integration our methodologies into a hybrid MACS/FACS selection workflow. Initial rounds of MACS could be implemented to accommodate large library sizes, followed by subsequent rounds using FACS for superior control over the affinities of the enriched proteins. The 96-well plate biofloating format we have developed could then be implemented for high-throughput screening of individual clones from the evolved library to monitor selection progress and identify lead clones. This post-selection screening would be logistically challenging and significantly less sensitive if implemented using the biopanning format, underscoring the advantage for our novel biofloating platform. Furthermore, although FACS was not used in this study, our characterization of yeast/mammalian cell interactions via flow cytometry offers useful insight that could inform the design of FACS-based selections in future implementations. An important limitation to keep in mind is that specific scFv-expressing yeast were spiked into the naïve yeast scFv library at a ratio of 1:1000. Representation of a binding clone within the library would likely be much lower; thus, additional rounds of selection could be required to achieve adequate enrichment. In addition, these selections were carried out using a high affinity anti-PD-L1 scFv, which may not be present in a naïve library. Indeed, Lown et al found that enrichment of yeast displaying a target-specific fibronectin using MACS-based selections against target-expressing mammalian cells was more challenging for fibronectin clones with lower affinity.20 Thus, the selection process may require optimization to enable enrichment of lower affinity clones.
Antibody discovery against MPs has immense potential to advance scientific research and therapeutic development. The ability to study protein interactions and perform selections against MPs in their native conformations in the context of the cell membrane empowers the characterization and manipulation of complex MPs such as GPCRs and ion channels, for which the native antigen display has been historically difficult. Our novel biofloating platform enables quantitative investigation of MP interactions in a fully suspension cell-based system that is compatible with existing yeast display infrastructure. This work also paves the way for development and optimization of new selection technologies that will advance MP-focused therapeutic design.