Human GCGR Stable Cell Line - HEK293

Identifying functional monoclonal antibodies against G protein-coupled receptors (GPCRs) is challenging due to the membrane-embedded topology of these molecules. Here, researchers report the successful combination of camel DNA immunization, scFv phage display, and virus-like particle (VLP) screening with the recombinant extracellular domain of the GPCR glucagon receptor (GCGR) to generate glucagon receptor-specific antagonistic antibodies. Hybrid camels were immunized with plasmid DNA containing the human GCGR gene in an attempt to activate their immune systems and generate a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying monoclonal antibodies that bind to the GCGR ECL region is challenging because in the energetically most favorable "closed conformation" of GCGR, the larger ECD covers the smaller ECL. Comparison of Fab and scFv phage display demonstrated that the multivalent nature of scFv display was crucial for identifying GCGR-specific clones via VLP screening, as strong interactions were observed between the two.

To demonstrate that mAbs 6A5 and 6C6 bind to GCGR without the ECD, researchers constructed a truncated GCGR lacking the ECD (GCGRΔECD, aa146-447). Constructs encoding GCGR and GCGRΔECD were transfected into HEK293E cells. mAb binding assays in GCGR overexpressing HEK293E cells and GCGRΔECD overexpressing HEK293E cells confirmed previous results: 6C6 and 6A5 bound to both GCGR and GCGRΔECD, while 6B3 bound only to GCGR (Figure 1). After preincubation with excess recombinant ECD-GCGR, 6B3 lost binding to GCGR overexpressing HEK293E cells, while mAbs 6C6 and 6A5 still showed GCGR binding (Figure 1).

Figure 1. FACS binding of mAbs to GCGR- (dark blue) or GCGRΔECD- (green) expressing HEK293 cells with and without ECD-GCGR competition (light blue). (van der Woning B, et al., 2016)