Nanobodies are single V_HH domain camelid antibodies which have had wide applications in biochemistry and cell biology, including the stabilisation of G protein-coupled receptors (GPCR) in the active state.  These structures have shed light on receptor activation mechanisms, and may aid in rational drug design, particularly for peptide receptors which have more complex activation mechanisms.  Current techniques for nanobody screening rely on llama immunisation with purified receptor, in a particular conformational state. This method is costly, time consuming and depends on good quality receptor protein preparations. Recently, in vitro platforms using yeast surface display and synthetic libraries have been created, however these still rely upon purified receptor protein for screening. GPCRs are lowly expressed, conformationally dynamic proteins that are unstable upon detergent solubilisation, which limits the use of these nanobody screening methods for generating GPCR-binding nanobodies.

Thus here, we aim to develop a mammalian cell-based system to identify novel GPCR-binding nanobodies without the need to purify receptor protein. We are currently applying this system to two peptide receptors:  the tachykinin receptor 1 and the neurotensin receptor 1, both of which are therapeutically relevant targets, for which there are currently no active-state structures.

We use a lentivirus to transduce a synthetically designed library of intracellularly expressed nanobodies into HEK293F cells, stably expressing our GPCR of interest. Intracellular binding of an active state selective nanobody should increase the affinity of a cognate fluorescent peptide ligand. We can then use fluorescence activated cell sorting to select for cells containing these nanobodies and enrich this population through multiple rounds of sorting.

Selective antibodies against these peptide receptors may enable structure determination, as well as have other applications including use as fluorescent biosensors that can detect receptor activation in cells, as has been done for the opioid receptors. Furthermore, the lentiviral system can be applied to other binding molecules such as peptides, to identify novel GPCR modulatory ligands.