(531g) Biofloating: An Antibody Discovery Platform for Targeting Multi-Pass Transmembrane Proteins Utilizing Suspension Cell-Based Directed Evolution

Membrane proteins are critical for a vast array of cellular functions, such as cell signaling, catalysis, and transport across the cell membrane. Through these functions, membrane proteins carefully regulate the cell’s ability to react to its external environment. When these functions are dysregulated, cells often adopt new behaviors, which can result in development or progression of disease. Over 60% of all clinically-approved drugs target membrane proteins, demonstrating that targeting of membrane proteins can lead to improvements in patient outcomes.¹ One class of drugs that has risen to prominence in recent years is that of antibody therapeutics.² Antibodies offer many advantages as therapeutics, such as high specificity, long serum half-life, low immunogenicity, and high modularity. However, there currently exists a gap in the antibody drug landscape with respect to targeting more complex, multipass transmembrane proteins. One class of such multipass transmembrane proteins that is of particular importance is that of G-protein coupled receptors (GPCRs). GPCRs are the target of 34% of all FDA-approved drugs, but only 2 FDA-approved antibodies target GPCRs.^1,2 This is in part because GPCRs have relatively small extracellular epitopes, and solution presentation of GPCRs in their native conformations as selection targets is challenging. Thus, development of effective discovery methods for antibodies against complex membrane proteins will enhance the ability of researchers to study these proteins in a variety of biomedical contexts, which will ultimately broaden the range of accessible targets for therapeutics.

Methods

To address the challenges associated with discovering antibodies targeting membrane proteins, we have developed an antibody screening method we call ‘biofloating’ (Figure 1A).³ In this platform, mammalian cells expressing a membrane protein are used for target presentation. This overcomes the challenges of solubly expressing multipass transmembrane proteins and also presents the target protein in its native form, exposing physiologically relevant epitopes with appropriate post-translational modifications. A yeast-displayed antibody library of interest is then interfaced with these mammalian cell-presented target proteins.⁴ A directed evolution workflow is then utilized to enrich for yeast expressing target-specific antibodies, comprising iterative rounds of magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). A key consideration for the biofloating platform is discrimination between target-specific binders and non-specific binders. This is achieved through interspersing positive and negative selection steps within each round against phenotypically matched cell lines that either do or do not express the target transmembrane protein. Sequential rounds of evolution lead to convergence of the antibody library towards clones that recognize the mammalian cell-presented target protein (Figure 1B).

Results

Recently, we published results demonstrating the discovery of target-specific binders against 4 GPCRs: CXCR2, GLP1R, GCGR, and CXCR4.⁵ For these 4 GPCRs: 2 unique antibodies were found against CXCR2; 4 unique antibodies were found against GLP1R; 6 unique antibodies were found against GCGR; and 3 unique antibodies were found against CXCR4. All binders showed affinities within the nanomolar range, with some binding as tightly as 0.49 nM. As these GPCRs have divergent structures and functions, the success of these selections establishes that biofloating can be used to discover antibodies specific to a wide range of complex membrane protein targets. Studies are currently underway to demonstrate application of the platform to additional targets, including ion channels, cell adhesion molecules, and other GPCRs.

Implications

We have demonstrated that biofloating can be used to discover antibodies that specifically bind to complex, multi-pass transmembrane proteins of therapeutic relevance. Thus, biofloating provides a new method for discovering antibodies against drug targets that have been previously inaccessible, enhancing capabilities for targeted disease treatment. Moreover, discovery of antibodies against these new targets opens opportunities to interrogate the biology of these proteins for advancement of scientific knowledge and to inform future drug development efforts. Overall, biofloating is a unique and innovative method for discovering antibodies against complex membrane proteins that has shown promising results and may be used for antibody discovery by both academic labs and the pharmaceutical industry for a variety of research and medical applications.

References

1. Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schiöth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat Rev Drug Discov 16, 829–842 (2017).
2. Hutchings, C. J. A review of antibody-based therapeutics targeting G protein-coupled receptors: an update. Expert Opin Biol Ther 20, 925–935 (2020).
3. Krohl, P. J. et al. A suspension cell-based interaction platform for interrogation of membrane proteins. AIChE Journal 66, e16995 (2020).
4. Chao, G. et al. Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1, 755–768 (2006).
5. Krohl, P. J. et al. Discovery of antibodies targeting multipass transmembrane proteins using a suspension cell-based evolutionary approach. Cell Reports Methods 3, 100429 (2023).