Discovery of human antibodies against the C5aR target using ... - DCU

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332 L. HUANG ET AL. reformatted clones were expressed as IgGs (5–15 mg/ml) in transiently transfected HEK293T cells. IgGs from these 16 clones were subsequently purified, and tested in cell binding assays. Testing reformatted IgGs for cell binding activities Since synthetic peptides lack post-translational modifications such as glycosylation and may adopt conformations different to the same sequence in the native protein, antibodies discovered from selection on peptide targets need to be further evaluated in cell-based assays. Reformatted IgGs were thus tested by flow cytometric analysis for their cell binding activities on C5aR-expressing cells. C5aR was induced in differentiated promonocytic U-937 cells (Gerard and Gerard, 1990). U-937 cells were terminally differentiated by treatment with 0.5 mM Bt 2 cAMP for 3 days, and then stained with IgGs by indirect immunofluorescence. Cells were first incubated with reformatted IgGs and control human IgG (hIgG) in staining buffer. S5/1, amAb that recognizes the C5aR N-terminal region (Oppermann et al., 1993) was used as a positive control. Flow cytometric analysis demonstrated that six reformatted IgGs (IgG-15, 28, 31, 33, 38 and 43) showed good binding to differentiated U-937 cells, with percentages of positive cells ranging from 11–30% (Fig. 3). The positive control antibody S5/1 also showed binding to cells with the percentage of positive cells being approximately 17%. The other 10 reformatted IgGs (IgG-4, 11, 12, 16, 17, 18, 25, 29, 32 and 37) did not show significant binding to cells, indicating that they were not cell binders. Consistent with the C5aR expression profile that this GPCR was not significantly expressed on undifferentiated U-937 cells but was induced in differentiated U-937 cells, one representative positive cell binder IgG-28 showed binding in differentiated U-937 cells but not in undifferentiated cells (Fig. 4), suggesting that IgG-28 is specific to differentiated U-937 cells. Figure 5. Specificity of positive cell binders to C5aR. Competition ELISA was carried out as described in the Experimental Procedures. The target wells were sequentially coated with streptavidin and biotinylated DX-1186 peptide (white and striped bars), and the background wells were coated just with streptavidin (black bars). Prior to the addition of hIgG control and positive IgGs, target wells were incubated with 10 mIgG (white bars) or 10 S5/1 (striped bars). Plotted in the histograms are ELISA signals as measured by A630. Testing the specificities of positive cell binding IgGs To further determine if the positive cell binding IgGs were specific to C5aR, the positive IgGs (IgG-15, 28, 31, 33, 38 and 43) were tested in a peptide-based ELISA. Each of the 6 IgGs bound to DX-1186 (Fig. 5). Competition ELISA with mAb S5/1 known to bind the same C5aR N-terminal region showed that all six IgGs were competed with by S5/1 (Fig. 5), indicating that these positive IgG cell binders were specific to C5aR. This was further confirmed by FACS analysis of C5aR-expressing cells showing that the IgG binding to cells was competed by S5/1 (data not shown). DISCUSSION Figure 4. Specific binding of positive IgG binders to differentiated U-937 cells. U-937 cells treated with Bt 2 cAMP at 0.5 mM for 3 days were used as differentiated cells, and untreated cells were used as undifferentiated cells. Both differentiated and undifferentiated cells were stained by indirect immunofluorescence and analyzed by flow cytometry. Shown here are histograms of representative positive IgG binder, IgG-28. We have used phage display technology to identify human antibodies to the chemoattractant receptor C5aR, a member of the GPCR superfamily. GPCRs have proved successful drug targets and a large proportion of existing drugs target these receptors. However, owing to the complex structures of these seven transmembrane domain-based proteins, GPCRs are often considered difficult targets for antibodybased therapeutics and targeting. We have designed a strategy using a synthetic peptide encompassing the N- terminal region of C5aR as the selection target. After three rounds of selection with a Fab phage library, we were able to obtain very high hit rates (50% of isolates with ELISA signals greater than 5-fold over background levels) utilizing two selection strategies: liquid binding and bead binding. In liquid binding selection, the input phage were incubated with the biotin-conjugated target in solution before capture Copyright # 2005 John Wiley & Sons, Ltd. J. Mol. Recognit. 2005; 18: 327–333
HUMAN ANTIBODIES AGAINST THE C5aR TARGET 333 on streptavidin beads; in bead binding selection, the input phage were incubated with the target immobilized on beads. There are advantages and disadvantages for both selection strategies. In liquid binding selection, high-affinity binders may be enriched due to the lack of avidity effect, but the target concentration should be well controlled so that the target is well below the saturating concentration and potential binders will not be lost after immobilizing to beads. In bead binding selection, low-affinity binders also may be enriched due to avidity effect, but the target concentrations did not need to be well-controlled as in liquid binding selection. In this study, we included both selection strategies to ensure maximum success. Our results showed that both selections worked very well, resulting in similar hit rates. Representative Fab clones were reformatted into IgGs, and tested for cell binding activities by flow cytometry. Approximately 40% of the reformatted IgGs showed binding to differentiated U-937 cells in which C5aR expression was induced. Flow cytometric analysis of HepG2 cells known to express C5aR (Haviland et al., 1995) paralleled the results from U-937 cells: positive binders to differentiated U-937 cells were also positive binders to HepG2 cells, and negative binders to differentiated U-937 cells were also negative binders to HepG2 cells (data not shown). These positive cell binders were specific for C5aR since they bound to C5aR N-terminal peptide DX-1186 in peptide-based ELISA, and their binding was competed by S5/1, an anti-C5aR mAb known to bind the same C5aR N-terminal region (Oppermann et al., 1993). The cell binding activities of these positive binders also were competed by S5/1, suggesting specific binding to the C5aR N-terminal region. The library used here for selection is a Fab phage display library with combined natural and synthetic diversity (Hoet et al., 2004). The advantage of using a Fab phage library for selection is that there is no need to do phagemid rescue during selection and phage ELISA, thus greatly reducing the amount of time for selection and screening. The human Fab library has been used for selection with many soluble protein targets, yielding target-specific human antibodies with high affinity (Hoet et al., 2004). With the Fab library and the IgG reformatting procedure (Jostock et al., 2004), we were able to obtain specific human IgGs to a GPCR within a short period of time (8 weeks), demonstrating that our phage display approach has utility for rapid development of antibody therapeutic and imaging agents to GPCRs. Acknowledgments We thank Greg Conley for peptide synthesis and Gary Bassil for DNA sequencing. We also thank Albert Edge for helpful discussions and Clive Wood for critical reading of the manuscript. REFERENCES Azzazy HME, Highsmith WE Jr. 2002. Phage display technology: clinical applications and recent innovations. Clin. Biochem. 35: 425–445. Gerard C, Gerard NP. 1994. C5a anaphylatoxin and its seven transmembrane-segment receptor. A. Rev. Immunol. 12: 775–808. Gerard NP, Gerard C. 1990. The chemotactic receptor for human C5a anaphylatoxin. Nature 349: 614–617. Haviland DL, McCoy RL, Whitehead WT, Akama H, Molmenti EP, Brown A, Haviland JC, Parks WC, Perlmutter DH, Wetsel RA. 1995. Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of C5aR on nonmyeloid cells of the liver and lung. J. Immunol. 154: 1861–1869. Hoet RM, Cohen EH, Kent RB, Rookey K, Schoonbroodt S, Hogan S, Rem L, Frans N, Daukandt M, Pieters H, van Hegelsom R, Coolen-van Neer N, Nastri HG, Rondon IJ, Leeds JA, Hufton SE, Huang L, Kashin I, Devlin M, Kuang G, Steukers M, Viswanathan M, Nixon AE, Sexton DJ, Hoogenboom HR, Ladner RC. 2004. High affinity human antibodies from combined synthetic and captured diversity. Nat. Biotechnol. in press. Jostock T, Vanhove M, Brepoels E, van Gool R, Daukandt M, Wehnert A, van Hegelsom R, Dransfield D, Sexton D, Devlin M, Ley A, Mullberg J. 2004. Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries. J. Immunol. Meth. 289: 65–80. Makrides SC. 1998. Therapeutic inhibition of the complement system. Pharmac. Rev. 50: 59–87. Oppermann M, Raedt U, Hebell T, Schmidt B, Zimmermann B, Gotze O. 1993. Probing the human receptor for C5a anaphylatoxin with site-directed antibodies. Identification of a potential ligand binding site on the NH2-terminal domain. J. Immunol. 151: 3785–3794. Pellas TC, Wennogle LP. 1999. C5a receptor antagonists. Curr. Pharm. Des. 5: 737–755. Stockwin LH, Holmes S. 2003. Antibodies as therapeutic agents: vive la renaissance! Exp. Opin. Biol. Ther. 3: 1133–1152. Copyright # 2005 John Wiley & Sons, Ltd. J. Mol. Recognit. 2005; 18: 327–333
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