DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops

Abstract

Haard (2016) DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops, mAbs, 8:6, 1126-1135, DOI: 10.1080/19420862.2016 ABSTRACTThe 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.

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3

“…The transmembrane topology and absence of purified protein material necessitate usage of membranes, virus-like particles, or cells presenting a target receptor for immunization and/or phage display selection (Baribaud et al, 2001;Tamura and Chiba, 2009;Silence et al, 2014;Van Hout et al, 2018). As an alternative to live cell immunization, which often results in a broad off-target immune response, DNA immunization represents another approach for transmembrane receptors (van der Woning et al, 2016;Bobkov et al, 2018a). An additional limiting factor is generally the low surface expression levels of GPCRs.…”

A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

“…Ballistic transfection of skin cells with plasmid-coated gold particles is a very efficient technique to transfect skin cells including epithelial cells (EC), endothelial cells, and professional antigen-presenting cells (APC; e.g., Langerhans cells) ( 19 , 31 , 32 ). Other techniques for cDNA transfection have been used successfully ( 33 – 35 ). Even the simple injection of plasmid DNA in saline can lead to transfection, albeit usually with lower efficiencies ( 36 ).…”

“…Even the simple injection of plasmid DNA in saline can lead to transfection, albeit usually with lower efficiencies ( 36 ). High pressure or electric pulses can be used to enhance transfection efficiencies ( 34 , 35 , 37 , 38 ).…”

A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

“…Llama immunization and library construction : Llama immunization, peripheral blood lymphocytes (PBLs) isolation, RNA extraction and the construction of scFv libraries were performed as described previously. 39 All llamas used in this study were farmed outdoors in the Ardèche region of France according to French animal welfare legislation. Animal handling included immunization by intramuscular injection and peripheral blood collection.…”

“…Subsequently, scFv libraries were made for two of the four immunized llamas as previously described. 39 In brief, VH, Vκ and Vλ libraries were independently constructed into the pSc vector, which was derived from the pCB3 phagemid vector. Re-cloning the light chain libraries in the VH library was performed, to obtain the combinatorial κ and λ library for each llama.…”