title: Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.
abstract: Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.
authors.affiliation: a Antibody Discovery and Protein Engineering , MedImmune , Cambridge , UK.
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Rossant
authors.forename: Danielle
authors.initials: D
authors.lastname: Carroll
authors.forename: Ling
authors.initials: L
authors.lastname: Huang
authors.forename: John
authors.initials: J
authors.lastname: Elvin
authors.forename: Frances
authors.initials: F
authors.lastname: Neal
authors.forename: Edward
authors.initials: E
authors.lastname: Walker
authors.forename: Joris J
authors.initials: JJ
authors.lastname: Benschop
authors.forename: Eldar E
authors.initials: EE
authors.lastname: Kim
authors.forename: Simon T
authors.initials: ST
authors.lastname: Barry
authors.forename: Tristan J
authors.initials: TJ
authors.lastname: Vaughan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology*; D019390:Arrestins / Q000276:immunology / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects / Q000276:immunology; D002118:Calcium / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D061505:Cell Surface Display Techniques / Q000379:methods; D054360:Chemokine CXCL1 / Q000276:immunology / Q000494:pharmacology; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000276:immunology* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas; D007114:Immunization; D016209:Interleukin-8 / Q000276:immunology / Q000494:pharmacology; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding / Q000276:immunology; D023063:Receptors, Interleukin-8B / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D000071557:beta-Arrestins
chemical_list: D000911:Antibodies, Monoclonal; D019390:Arrestins; D054360:Chemokine CXCL1; D000939:Epitopes; D016209:Interleukin-8; D019151:Peptide Library; D023063:Receptors, Interleukin-8B; D000071557:beta-Arrestins; D002118:Calcium
keywords: BSA, bovine serum albumin; CDR, complementarity determining region; CXCR2; CXCR2, C-X-C Chemokine Receptor 2; ECL, extracellular loops; ENA-78, epithelial derived -neutrophil activating protein; FBS, fetal bovine serum; FMAT, Fluorescence Microvolume Assay Technology; GCP-2, granulocyte activating protein; GPCR; GPCR, G-protein coupled receptor; Gro-α, growth related oncogene- α; Gro-β, growth related oncogene- β; Gro-γ, growth related oncogene- γ; IL-8, Interleukin-8; Ig, Immunoglobulin; NAP-2, neutrophil activating protein-2, CLIPS, Chemical Linkage of Peptides onto Scaffolds; PBS, phosphate buffered saline; epitope mapping; human antibody; immunization; phage display; proteoliposomes; scFv, single chain Fv fragments