Dataset Information

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-? responses in calcium flux and ?-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.

SUBMITTER: Rossant CJ

PROVIDER: S-EPMC4622561 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

Rossant Christine J CJ Carroll Danielle D Huang Ling L Elvin John J Neal Frances F Walker Edward E Benschop Joris J JJ Kim Eldar E EE Barry Simon T ST Vaughan Tristan J TJ

mAbs 20140101 6

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 libra ...[more]

PMID: 25484064

Similar Datasets

Project description:BackgroundPrecise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.ResultsWe present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu), the antibody mAb Bo2C11 targeting the C(2) domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.AvailabilityUsers can access the EpiSearch from our web server http://curie.utmb.edu/episearch.html.

Dataset Information

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

Publications

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets