Discovery of human antibodies against the C5aR target using phage display technology

• Published in: Journal of molecular recognition Vol. 18; no. 4; pp. 327 - 333
• Main Authors: Huang, Lili, Sato, Aaron K., Sachdeva, Meena, Fleming, Tony, Townsend, Susan, Dransfield, Daniel T.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01-07-2005
• Subjects: Amino Acid Sequence; antibodies; C5aR; Cell Differentiation - drug effects; GPCRs; Humans; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - isolation & purification; Immunoglobulin Fab Fragments - pharmacology; Immunoglobulin G - genetics; Immunoglobulin G - isolation & purification; Immunoglobulin G - pharmacology; Membrane Proteins - immunology; Molecular Sequence Data; Peptide Library; phage display; Receptor, Anaphylatoxin C5a; Receptors, Complement - immunology; U937 Cells
• ISSN: 0952-3499; 1099-1352
• DOI: 10.1002/jmr.735

Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane‐associated as well as to activated forms of enzymes. We have applied this approach to G‐protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N‐terminal region (residues 1–31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR‐expressing cells, the differentiated U‐937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N‐terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs. Copyright © 2005 John Wiley & Sons, Ltd.