title: DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops.
abstract: The identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated 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 mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.
authors.affiliation: a Argenx BVBA , Zwijnaarde , Belgium.
authors.forename: Bas
authors.initials: B
authors.lastname: van der Woning
authors.affiliation: a Argenx BVBA , Zwijnaarde , Belgium.
authors.forename: Gitte
authors.initials: G
authors.lastname: De Boeck
authors.affiliation: a Argenx BVBA , Zwijnaarde , Belgium.
authors.forename: Christophe
authors.initials: C
authors.lastname: Blanchetot
authors.affiliation: a Argenx BVBA , Zwijnaarde , Belgium.|b AIMMS, Division Medicinal Chemistry, VU University Amsterdam , The Netherlands.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Bobkov
authors.affiliation: c Dept. of Cell Biology , Science Faculty, Utrecht University , Utrecht , The Netherlands.
authors.forename: Alex
authors.initials: A
authors.lastname: Klarenbeek
authors.affiliation: a Argenx BVBA , Zwijnaarde , Belgium.
authors.forename: Michael
authors.initials: M
authors.lastname: Saunders
authors.affiliation: d LCBN, Université Libre de Bruxelles , Brussels , Belgium.
authors.forename: Magali
authors.initials: M
authors.lastname: Waelbroeck
authors.affiliation: e Confotherapeutics , Brussels , Belgium.
authors.forename: Toon
authors.initials: T
authors.lastname: Laeremans
authors.affiliation: e Confotherapeutics , Brussels , Belgium.|f VIB Structural Biology Research Center , Brussels , Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: a Argenx BVBA , Zwijnaarde , Belgium.
authors.forename: Anna
authors.initials: A
authors.lastname: Hultberg
authors.affiliation: a Argenx BVBA , Zwijnaarde , Belgium.
authors.forename: Hans
authors.initials: H
authors.lastname: De Haard
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D023181:Antimicrobial Cationic Peptides; D016466:CHO Cells; D002161:Camelids, New World / Q000276:immunology; D054804:Cathelicidins / Q000276:immunology; D061505:Cell Surface Display Techniques; D002478:Cells, Cultured; D003412:Cricetulus; D005347:Fibroblasts; D057809:HEK293 Cells; D006801:Humans; D007114:Immunization; D016056:Immunodominant Epitopes / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000097:blood / Q000276:immunology; D008565:Membrane Proteins; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D018027:Receptors, Glucagon / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000097:blood / Q000276:immunology*; D058425:Vaccines, Virus-Like Particle / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D023181:Antimicrobial Cationic Peptides; D054804:Cathelicidins; D016056:Immunodominant Epitopes; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D018027:Receptors, Glucagon; D057127:Single-Chain Antibodies; D058425:Vaccines, Virus-Like Particle
keywords: DNA immunizations; glucagon receptor; llama; phage display; single-chain Fv; virus-like particles