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Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.
- Source :
-
MAbs [MAbs] 2014; Vol. 6 (6), pp. 1425-38. - Publication Year :
- 2014
-
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.
- Subjects :
- Amino Acid Sequence
Animals
Antibodies, Monoclonal genetics
Antibodies, Monoclonal pharmacology
Arrestins immunology
Arrestins metabolism
Biological Transport drug effects
Biological Transport immunology
Calcium immunology
Calcium metabolism
Cell Line
Cell Surface Display Techniques methods
Chemokine CXCL1 immunology
Chemokine CXCL1 pharmacology
Epitope Mapping methods
Epitopes metabolism
HEK293 Cells
Humans
Hybridomas
Immunization
Interleukin-8 immunology
Interleukin-8 pharmacology
Mice, Transgenic
Molecular Sequence Data
Peptide Library
Protein Binding immunology
Receptors, Interleukin-8B genetics
Receptors, Interleukin-8B metabolism
Signal Transduction immunology
beta-Arrestins
Antibodies, Monoclonal immunology
Antibody Specificity immunology
Epitopes immunology
Receptors, Interleukin-8B immunology
Subjects
Details
- Language :
- English
- ISSN :
- 1942-0870
- Volume :
- 6
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- MAbs
- Publication Type :
- Academic Journal
- Accession number :
- 25484064
- Full Text :
- https://doi.org/10.4161/mabs.34376