Assistant Professor Johns Hopkins University, United States
Introduction:: Membrane proteins are critical for a vast array of cellular functions, and they carefully regulate the cell’s ability to react to and exchange with its external environment. As a result, over 60% of all clinically approved drugs target membrane proteins, over half of which target G-protein coupled receptors (GPCRs)1. One class of drugs that has risen to prominence in recent years is that of antibody therapeutics. However, there currently exists a gap in the antibody drug landscape with respect to GPCR targeting, as only 2 FDA-approved antibodies target GPCRs.1 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 discovery methods for antibodies against complex membrane proteins, such as GPCRs and ion channels, will enhance the ability of researchers to study these proteins in a variety of biomedical contexts and ultimately broaden the range of accessible targets for therapeutics. We have developed a method we call “biofloating,” in which mammalian cells expressing a target membrane protein are co-incubated with yeast expressing an antibody library.2 This method overcomes the challenges of GPCR expression and presents the target protein in its native form, exposing physiologically relevant epitopes. Recently, we published results demonstrating discovery of target-specific binders for 4 GPCRs.3 However, these GPCRs are targets for which diagnostic antibodies already exist, and additional studies are being conducted to demonstrate application of the platform to targets for which there are not commercially available antibodies.
Materials and Methods:: Antibody libraries were expressed on EBY-100 yeast using the pCTCon2 expression vector. Target membrane proteins were stably expressed on mammalian (HEK293T or NIH/3T3) cells using lentiviral transduction. Enrichment for specific antibody binders to target proteins is achieved through iterative rounds of selection via magnetically-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS).
MACS was conducted by biotinylating the mammalian cell surface with EZ-Link Sulfo-NHS-SS-Biotin and incubating with streptavidin-coated magnetic beads. The cells were sorted using an LS magnetic column (Miltenyi Biotec). FACS was conducted by staining mammalian cells with CellTrace Violet dye and staining yeast with anti-cmyc Alexa647 antibody. The cell mixture was sorted on an SH800S SONY cell sorter for the double positive population. Selection rounds were characterized using a Beckman Coulter CytoFLEX.
Once yeast clones of interest were identified, plasmids were extracted with a ZymoPrep miniprep kit and antibody fragments were reformatted into an IgG1 backbone. Nanobody-Fc fusions were expressed in HEK293F cells. Resulting Fc fusion constructs were then titrated against target-expressing and target-null cells and labeled with an APC-conjugated anti-human IgG1 Fc antibody.
Results, Conclusions, and Discussions:: Directed evolution via in vitro display has enabled discovery of target-specific antibodies from a naive library. Nanobody-Fc fusions specific to 4 different GPCRs were discovered: CXCR2, GLP1R, GCGR, and CXCR43. For these 4 GPCRs: 2 unique binders were found against CXCR2; 4 unique binders were found against GLP1R; 6 unique binders were found against GCGR; 3 unique binders were found against CXCR4. These results demonstrate that the biofloating platform is capable of generating specific antibodies against a wide range of GPCRs. However, since these experiments were designed as proof of concept, commercial antibodies against all 4 of the GPCR targets are available and were used to characterize cell surface expression levels. Thus, we are currently working on discovering antibodies against novel targets for which commercial antibodies are not available. One of these targets is a ligand-gated ion channel, P2RX5. Another target is an adhesion molecule, NRCAM.
Acknowledgements (Optional): :
References (Optional): : 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. Krohl, P. J. et al. A suspension cell-based interaction platform for interrogation of membrane proteins. AIChE J.66, e16995 (2020).
3. Krohl, P. J. et al. Discovery of antibodies targeting multipass transmembrane proteins using a suspension cell-based evolutionary approach. Cell Rep. Methods3, 100429 (2023).
