title: A combination of in vitro techniques for efficient discovery of functional monoclonal antibodies against human CXC chemokine receptor-2 (CXCR2).
abstract: BACKGROUND
  Development of functional monoclonal antibodies against intractable GPCR targets.
  
  
  RESULTS
  Identification of structured peptides mimicking the ligand binding site, their use in panning to enrich for a population of binders, and the subsequent challenge of this population with receptor overexpressing cells leads to functional monoclonal antibodies.
  
  
  CONCLUSIONS
  The combination of techniques provides a successful strategic approach for the development of functional monoclonal antibodies against CXCR2 in a relatively small campaign.
  
  
  CONCLUSIONS
  The presented combination of techniques might be applicable for other, notoriously difficult, GPCR targets.
  
  
  CONCLUSIONS
  The CXC chemokine receptor-2 (CXCR2) is a member of the large 'family A' of G-protein-coupled-receptors and is overexpressed in various types of cancer cells. CXCR2 is activated by binding of a number of ligands, including interleukin 8 (IL-8) and growth-related protein α (Gro-α). Monoclonal antibodies capable of blocking the ligand-receptor interaction are therefore of therapeutic interest; however, the development of biological active antibodies against highly structured GPCR proteins is challenging. Here we present a combination of techniques that improve the discovery of functional monoclonal antibodies against the native CXCR2 receptor. The IL-8 binding site of CXCR2 was identified by screening peptide libraries with the IL-8 ligand, and then reconstructed as soluble synthetic peptides. These peptides were used as antigens to probe an antibody fragment phage display library to obtain subpopulations binding to the IL-8 binding site of CXCR2. Further enrichment of the phage population was achieved by an additional selection round with CXCR2 overexpressing cells as a different antigen source. The scFvs from the CXCR2 specific phage clones were sequenced and converted into monoclonal antibodies. The obtained antibodies bound specifically to CXCR2 expressing cells and inhibited the IL-8 and Gro-α induced ß-arrestin recruitment with IC50 values of 0.3 and 0.2 nM, respectively, and were significantly more potent than the murine monoclonal antibodies (18 and 19 nM, respectively) obtained by the classical hybridoma technique, elicited with the same peptide antigen. According to epitope mapping studies, the antibody efficacy is largely defined by N-terminal epitopes comprising the IL-8 and Gro-α binding sites. The presented strategic combination of in vitro techniques, including the use of different antigen sources, is a powerful alternative for the development of functional monoclonal antibodies by the classical hybridoma technique, and might be applicable to other GPCR targets.
authors.affiliation: a Pepscan Therapeutics B.V. ; Lelystad , The Netherlands.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Boshuizen
authors.forename: Catherine
authors.initials: C
authors.lastname: Marsden
authors.forename: Johan
authors.initials: J
authors.lastname: Turkstra
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Rossant
authors.forename: Jerry
authors.initials: J
authors.lastname: Slootstra
authors.forename: Clive
authors.initials: C
authors.lastname: Copley
authors.forename: Klaus
authors.initials: K
authors.lastname: Schwamborn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D061505:Cell Surface Display Techniques / Q000379:methods*; D054360:Chemokine CXCL1 / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D007114:Immunization; D016209:Interleukin-8 / Q000276:immunology / Q000378:metabolism; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding / Q000276:immunology; D023063:Receptors, Interleukin-8B / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D054360:Chemokine CXCL1; D016209:Interleukin-8; D019151:Peptide Library; D023063:Receptors, Interleukin-8B
keywords: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); BSA, bovine serum albumin; CLIPS™, Chemical LInkage of Peptides onto Scaffolds; CXCR2; ECL, extracellular loop; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunoabsorbent assay; Fmoc, fluorenylmethyloxycarbonyl; GPCR; GPCR, G-protein coupled receptor; Gro-α, growth-related protein α; IL-8, interleukin 8; IPTG, isopropyl β-D-1-thiogalactopyranoside; MFI, mean fluorescence intensity; PBS, phosphate buffer saline; PCR, polymerase chain reaction; PEG, polyethyleneglycol; TES, 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid; TRIS, tris(hydroxymethyl)aminomethane; ligand inhibition; monoclonal antibody; phage display library; scFv, single-chain variable fragment