Integral membrane proteins are important drug targets and monoclonal antibodies (MAbs) directed against them are highly sought for both research and therapeutic purposes. However, the complex structure of membrane protein targets makes the discovery and characterization of these MAbs especially challenging. We have generated potent antibody antagonists targeting the ligand-gated ion channel P2X3, a validated target for neuropathic pain, using virus-like particles (VLPs) embedded with structurally-intact P2X3. Thorough characterization of the P2X3 MAbs reveals that they bind diverse conformational epitopes on P2X3 with high specificity. Using VLPs coupled to an optical biosensor, we measured the sub-nanomolar affinity and kinetics of the binding of the MAbs to P2X3. The P2X3 MAbs inhibited ATP-activated calcium influx in transfected cells and, more importantly, blocked 90% of neuronal currents in primary cells <i>ex vivo</i>, enabling further development of lead MAb candidates. We additionally show the use of VLPs for the generation of antibodies against complex epitopes on diverse structural classes of membrane proteins including G protein-coupled receptors, transporters, and ion channels. VLPs containing non-viral membrane proteins have proven to be useful reagents both for the generation and isolation of MAbs against membrane proteins and for the detailed characterization of interaction of MAbs with their membrane-bound targets.

Discovery and characterization of antibodies against membrane proteins using virus-like particles

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.