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Published as doi: 10.1096/fj.08-117069.
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(The FASEB Journal. 2009;23:575-585.)
© 2009 FASEB

Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human Fc{gamma}RI

Stephane Bonetto*, Loredana Spadola{dagger}, Andrew G. Buchanan*, Lutz Jermutus*,1 and John Lund*,1

* MedImmune, Research, Granta Park, Cambridge, UK; and

{dagger} AstraZeneca R&D, DECS Global Compound Sciences, Mölndal, Sweden

1Correspondence: MedImmune, Research, Granta Park, Milstein Bldg., Cambridge, CB21 6GH, UK. E-mail: J.L., lundj{at}medimmune.com; or L.J., jermutusl{at}medimmune.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of short, structured peptides able to mimic potently protein-protein interfaces remains a challenge in drug discovery. We report here the use of a naive cyclic peptide phage display library to identify peptide ligands able to recognize and mimic IgG1-Fc functions with Fc{gamma}RI. Selection by competing off binders to Fc{gamma}RI with IgG1 allowed the isolation of a family of peptides sharing the common consensus sequence TX2CXX{theta}PXLLGC{Phi}XE ({theta} represents a hydrophobic residue, {Phi} is usually an acidic residue, and X is any residue) and able to inhibit IgG1 binding to Fc{gamma}RI. In soluble form, these peptides antagonize superoxide generation mediated by IgG1. In complexed form, they trigger phagocytosis and a superoxide burst. Unlike IgG, these peptides are strictly Fc{gamma}RI-specific among the Fc{gamma}Rs. Molecular modeling studies suggest that these peptides can adopt 2 distinct and complementary conformers, each able to mimic the discontinuous interface contacts constituted by the C{gamma}2-A and -B chains of Fc for Fc{gamma}RI. In addition, by covalent homodimerization, we engineered a synthetic bivalent 37-mer peptide that retains the ability to trigger effector functions. We demonstrate here that it is feasible to maintain IgG-Fc function within a small structured peptide. These peptides represent a new format for modulation of effector functions.—Bonetto, S., Spadola, L., Buchanan, A. G., Jermutus, L. Lund, J. Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human Fc{gamma}RI.


Key Words: effector functions • phage display • single-chain homodimer • molecular modeling • discontinuous interface • consensus sequence


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE GENERATION OF SMALL PROTEIN domains or peptides that are able to mimic protein-protein contact sites and show potent agonism or antagonism both in vitro and in vivo is a challenge in biological discovery. Although binding sites on enzyme surfaces and small ligand binding sites on receptor surfaces typically consist of a concave cleft shape, extracellular protein-protein interactions often involve large and relatively flat contact surfaces, lacking deep cavities and pockets that might provide compact binding sites for small molecules (1 , 2) . However, it may not be necessary for a small ligand to cover the entire protein-binding interface. Of these many intermolecular contacts, a very limited number of residues, clustered in a centralized region, may account for up to 85% of the free energy of binding, hence contributing predominantly to the generation of high-affinity interactions (3) . Many proteins are recognized by multiple partners. An important point is that these proteins tend to use the same binding "hot spots," which are recognized in specific spatial orientations. Although in theory, potential ligands could bind to a protein anywhere on its solvent-exposed surface, most peptides recognize localized sites that appear to coincide with natural ligand binding sites, and consequently can function as agonists or antagonists (4) . So, if proteins generally interact through compact functional epitopes, the task of identifying and designing small ligands may be achievable.

Since construction of a peptide phage library was described initially (5) , over 1000 articles related to this strategy have been reported. Phage display libraries are commonly used to identify small ligands able to mimic natural binding partners with desired functional properties (6) .

We used this technology to isolate cyclic peptides able to target the IgG-Fc (immunoglobulin G-fragment crystallizable) binding site on human Fc{gamma}RI and mimic human IgG1 triggering of function. IgG antibodies are the predominant isotype in serum and interstitial fluids and are the format used almost exclusively for therapeutic antibodies. Antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and superoxide generation are mediated through interaction of ICs (immune complexes) with Fc{gamma}Rs expressed on the surface of leukocytes, whereas CDC (complement-dependent cytotoxicity) occurs by interaction of ICs with the soluble complement system. Among the three classes of Fc{gamma} receptor, Fc{gamma}RI is a "high-affinity" receptor that interacts with monomeric, as well as complexed, human IgG1 or IgG3 (7) . The physiological role of Fc{gamma}RI is unclear, not least because Fc{gamma}RI is always present in combination with other receptor classes. In order to signal, Fc receptors need to be colocated at the cell surface (8) . Understanding the molecular basis of the Fc{gamma}RI-Fc interaction has been the subject of detailed investigations largely based on comparative binding (9) , activity studies of chimeric IgG (10) , Ig-Fc modified by site-directed mutagenesis (11) , and Fc peptide fragments (12) . More recently, a peptide display library selection has successfully identified a specific ligand C6–2 to Fc{gamma}RI able to promote receptor-mediated internalization (13) . Unlike phage-derived peptides identified on the other classes of receptors (14 15 16 17) , peptide C6–2 recognizes Fc{gamma}RI at a site unrelated to IgG binding. Following crystallization of the human Fc{gamma}RIII-Fc complex (18 19 20) and NMR spectroscopy studies of a mouse Fc{gamma}RII-IgG2b complex (21) , predominant interaction sites were identified within the CH2 domains and shared by all Fc{gamma}Rs, including the lower hinge sequence L234LGGPS239 from C{gamma}2-A and C{gamma}2-B chains and residues Leu328 to Pro329 from the C{gamma}2-A chain.

In the present study, we report the identification of an Fc-like peptide family that behaves as IgG, except it displays a strict specificity for Fc{gamma}RI. To our knowledge, these are the first peptides able to antagonize effector functions mediated by Fc{gamma}RI. Furthermore, we engineered a 37-residue single-chain homodimer peptide able to trigger a superoxide burst in a monocyte-like cell line. Therefore, we show that dimerization of monomeric Fc-mimetic peptides is sufficient to colocate Fc{gamma}RI on the cell surface. The results bring new molecular and structural insights to bear on the mechanism of IgG-receptor interaction and Fc{gamma}R colocation, and above all show that it is feasible to engineer a miniature Fc-like format able to mimic IgG-Fc mediation of effector function. These peptides may constitute the basis for the design of small ligands for the therapeutic treatment of cancer and inflammatory disorders, and suggest new opportunities for engineered antibodies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning procedures, construction libraries, and phage library synthesis
Three cyclic phage-peptide libraries, X2CX10CX2, X3CX8CX3, and X5CX4X5, were constructed by site-directed mutagenesis (22) using 3 oligonucleotides, 5'-GCTAAACAACTTTCAACAGTTTCTGCGGCCGC(SNN)2ACA(SNN)10ACA(SNN)2CTGTGCACTGTGAGAATAGAAGG-3'; 5'-GCTAAACAACTTTCAACAGTTTCTGCGGCCGC(SNN)3ACA(SNN)8ACA(SNN)3CTGTGCACTGTGAGAATAGAAGG-3'; and 5'-GCTAAACAACTTTCAACAGTTTCTGCGGCCGC(SNN)5ACA(SNN)4ACA(SNN)5CTGTGCACTGTGAGAATAGAAGG-3', where n = T, C, G, or A, and S = G or C, as described previously (23) . Briefly, Fd-tet-Dog1 phage vector was used to transform Escherichia coli strain CJ236 cells (New England BioLabs, Beverly, MA, USA), and phage supernatant from a single colony was precipitated with 0.15 vol of solution of 20% (w/v) polyethylene glycol (PEG-8000) and 2.5 M NaCl. dU-ssDNA template was extracted with Qiaprep Spin M13 kit (Qiagen, Hilden, Germany), and 20 µg was annealed with 0.7 µg of each phosphorylated oligonucleotide (90°C for 2 min, 50°C for 3 min, 20°C for 5 min). Following elongation with T4 DNA ligase (30 U) and T7 DNA polymerase (30 U) (20°C, 3 h), cccDNA was purified with a Roche DNA purification kit (Roche Diagnostics GmbH, Mannheim, Germany) and transfected into E. coli strain TG1 by electroporation (2.5 kV, 200 {Omega}, 25 µF in Bio-Rad micropulser; Bio-Rad, Hercules, CA, USA). Cells were plated on a large 2x TY plate (12.5 µg tetracycline/ml) and incubated for 16 h at 31°C. Phage libraries were prepared by PEG-8000, NaCl precipitation, and purified by CsCl gradient ultracentrifugation. The diversity of each library was estimated to be between 1 and 2.0 x 109 different clones, and their functionality was confirmed by biopanning on human serum albumin (HSA).

Phage-peptide panning selection
Each phage library was subject to 3 rounds of panning selection according to 3 different conditions (cond. 1, cond. 2, and cond. 3). Maxisorp microplates (Nunc A/S, Roskilde, Denmark) were coated with human Fc{gamma}RIa (0.3 µM in 0.1 M NaHCO3, pH 8.6) (R&D Systems Inc., Minneapolis, MN, USA) for the first round, and for subsequent rounds at 0.3 µM for cond. 1, 30 nM for cond. 2, and 0.1 µM for cond. 3. The plates were blocked at each selection round with 0.3 nM Fc{gamma}RI, supplemented alternatively with either 4% (w/v) powdered milk/0.1 M NaHCO3 or 0.5% BSA (w/v)/0.1 M NaHCO3 for 2 h at 4°C. Phage-peptide libraries [3.0x1011 plaque-forming units (PFU)/well] were equilibrated in PBS/4% powdered milk or 0.5% BSA for 1 h at 20°C and incubated on plates for 3 h at 24°C. After washing 10 times with PBS/0.1% Tween-20 and 10 times with PBS in cond. 1, 20 times with PBS/0.1% Tween-20 and 20 times with PBS in cond. 2, and 5 times with PBS/0.1% Tween-20 and 5 times with PBS in cond. 3, retained phage-peptides were eluted by competition with human IgG1 (2 µM) for 1 h. The second round of selection was performed as previously, but using 5.0 x 1010 PFU in cond. 1, and 2.5 x 1010 in cond. 2 and 3. The third round was performed from 2.0 x 1010 PFU in cond. 1, 5.0 x 109 in cond. 2, and 1.0 x 1010 in cond. 3. After each selection round, the pools of eluted phage were amplified overnight at 37°C and then purified by precipitation for the next round of affinity selection.

Biotinylation of IgG1 antibody
Biotinylation was conducted in 200 µl of 100 mM NaHCO3 (pH 8.2) with 60 µM of IgG1 and EZ-link NHS-LC-Biotin (Perbio/Pierce, Rockford, IL, USA) at a protein:biotin molar ratio of 1:4 for 15 min at 24°C. Biotinylation efficiency was ascertained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis (Ciphergen Biosystems Ltd, Guilford, UK). The reaction was stopped by gel filtration over a PD-10 column (Amersham Biosciences, Uppsala, Sweden) and the biotinylated protein concentration was determined by spectrophotometry at 280 nm ({epsilon}280=1.4 mg–1 ml–1).

Peptide synthesis and dimerization
Synthetic 18-mer peptides were produced in 1–3 mg amounts and characterized by analytical HPLC and LC/MS (Pepscan Systems, Lelytad, The Netherlands). Peptides were synthesized with a free N terminus and a biotinylated C terminus in the general form NH2-AQX3CX8CX3K-biot in an oxidized state. Cyclic hinge-CH2 peptide (sequence AQTAPCAPA PELLGCPSV) corresponding to the cyclic hinge sequence of native IgG1 constructed according to the format for cyclic octa-peptides, and peptide 22 (peptide 22 S4S13) with Ser substitutions replacing both Cys residues were also synthesized as described previously. The single-chain dimeric peptides were synthesized and produced in 10-mg amounts (AMS Biotechnology, Oxon, England) using a standard solid-phase peptide synthesis on Wang resin with fluorenylmethoxycarbonyl (Fmoc) -protected amino acids. Briefly, homodimer peptides were synthesized as linear dimers, trityl (Trt) was used as a side-chain protecting group for the first pair of Cys residues on the first monomer, and S-acetamidomethyl aminoacetal (Acm) as a side-chain protecting group for the second pair of Cys residues on the second monomer. The resin and the protecting Trt groups were then cleaved by trifluoroacetic acid (TFA), and electrospray ionization-mass spectrometry (ESI-MS) analysis was used to confirm the correct structure of the peptide with Cys (Acm) intact. The unprotected Cys residues were air oxidized to form the first intramolecular disulfide bridge, and then purified by HPLC to achieve separation of high purity peptide containing the disulfide bridge. The correct mass was verified by ESI-MS analysis and lyophilized. The second intramolecular disulfide bridge was formed using iodine, which also directly removes the Acm protection group from the Cys side chain. After ESI-MS analysis to check for successful oxidation, the peptide was column purified and lyophilized. Finally, the peptide was analyzed for purity and the mass value of the correct bicyclic dimer was verified. For the assays, peptides were solubilized in either deionized water or in 2.5–50% CH3CN or dimethylformamide (DMF) to a final concentration of 5 mM. Molecular masses of single-chain dimeric peptides were assessed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS) analysis according to the manufacturer’s instructions (Ciphergen Biosystems Ltd). Briefly, peptides were diluted to 1 pmol/µl in ProteinChip EAM-1 matrix (Bio-Rad) solubilized in 0.5% (v/v) TFA and 50% (v/v) acetonitrile. One picomole of this solution was spotted onto a ProteinChip NP20 array (Bio-Rad) and left to air-dry. The samples were analyzed in the positive ionization mode on a SELDI-TOF mass spectrometer (Ciphergen Biosystems). The m/z spectrum was calibrated using bovine ubiquitin standard (8564.8 Da) (Bio-Rad).

Production of soluble extracellular domains of human Fc{gamma}Rs
Human Fc{gamma}RI extracellular domain was obtained from a commercial source (R&D Systems Inc.), which was expressed in the mouse NS0 cell line fused to the signal peptide of human CD33 and to a polyhistidine tag. To express the other human Fc{gamma}R in soluble form, DNA was purchased that encoded human Fc{gamma}RIIa, Fc{gamma}RIIb, and Fc{gamma}RIIIa extracellular domains (Geneservice, Cambridge, UK), cloned in fusion with a Flag-His-tag DNA sequence in pDEST12.2 (Invitrogen, Paisley, UK) and transfected into HEK-293 EBNA cells (6x106 cells). Cells were grown on serum-free CD-CHO medium. The supernatant was harvested from 2–8 days and secreted protein was purified on an ÄKTA Explorer system (Amersham Pharmacia Biotech, Uppsala, Sweden) using a 5 ml HisTrap HP column (Amersham Biosciences). Protein was eluted with a linear gradient of imidazole (40–400 mM), and fractions were analyzed by SDS-PAGE on NuPAGE Novex 4–12% Bis-Tris gels (Invitrogen). Pooled samples were concentrated using an Amicon Ultra-15 30-kDa filter (Amicon Millipore, Watford, UK), loaded onto a Superdex S75 HR 10/30 gel Filtration Column (Amersham Pharmacia Biotech) and purified using an ÄKTA Explorer system. Fractions were analyzed by SDS-PAGE, pooled, and concentrated, and the concentration estimated spectrophotometrically (280 nm). The molecular mass was confirmed by MALDI-TOF/MS (Cyphergen).

Competitive ELISA to Fc{gamma}RI
Microtiter plates (Maxisorp; Nunc) were coated with Fc{gamma}RI (30 nM) in 0.1 M NaHCO3 (pH 8.6) at 4°C for 16 h. Subsequently, plates were blocked for 2 h at 4°C using 0.7 nM Fc{gamma}RI, 4% (w/v) powdered milk, 0.1 M NaHCO3. Following washing with PBS, IgG1 (1.3 µM) was incubated with peptide at 50, 17, 6, or 2 µM for 2 h at 24°C. After washing, IgG1 binding was determined with a horseradish peroxidase-conjugated F(ab')2 fragment goat {alpha}-IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) (1:5000) for 1h and revealed with 3,3',5,5'-tetramethylbenzidine as substrate (Sigma-Aldrich Corp., St Louis, MO, USA). After quenching by addition of 50 µl of 0.5 M sulfuric acid, the absorbance was measured at 450 nm. The absence of competitor was equivalent to 100% binding, such that the absorbance measured in the presence of peptide gave the percentage of inhibition.

Peptide binding to Fc{gamma}Rs by AlphaScreen assay
The AlphaScreen technology (Perkin-Elmer BioSignal, Montreal, QC, Canada) was used to measure interaction between biotinylated peptides 22, 29, 30, and 33 (0.4 nM to 100 µM) bound to streptavidin donor beads and Fc{gamma} receptors (20 nM) (Fc{gamma}RI and Fc{gamma}RIIIb (R&D Systems Inc.), as well as Fc{gamma}RIIa, Fc{gamma}RIIb, and Fc{gamma}RIIIa bound to nickel chelate acceptor beads. A control measurement with biotinylated IgG1 (0.19 nM to 137 nM) was performed in parallel. The reaction mixtures were incubated for 2 h in the dark and subsequently the luminescence was monitored on a Fusion-{alpha} microplate analyzer with excitation at 680 nm and emission at 600 nm (Perkin-Elmer BioSignal). The 3 greatest signals due to peptide binding were normalized relative to the 3 greatest signals due to IgG1 binding for each Fc{gamma}R.

Superoxide burst activation and inhibition assays
To generate multimeric complexes, an excess of 5 nmol of peptide was incubated with 1 mg of streptavidin-coated paramagnetic beads (Dynabeads M-280 Streptavidin; Invitrogen) in PBS for 30 min at 24°C. Beads complexed with 0.5 nmol of biotinylated-IgG1 were also prepared. Superoxide bursts were measured as lucigenin-enhanced chemiluminescence, as described previously (24) . U937 cells (100 µl) preincubated with {gamma}-IFN, 10 µl of lucigenin (2.5 mM), and 10 µl of bead-peptide complexes (130 pM to 1.0 µM) or IgG1-bead complexes (10 pM to 11 nM), or single-chain dimeric peptides (70 nM to 170 µM) were incubated for 5 min at 37°C to initiate superoxide measurements. Chemiluminescence was monitored in 96-well plate format over a 60-min period at 37°C using a Berthold LB940 Luminometer (Berthold Technologies, Bad Wildbad, Germany). For the inhibition assay, either 10 µl of SRBCs (109 cells/ml) derivatized using NIP-caproate-o-succinimide (0.23 mM) (Pierce) in borate buffered saline (100 mM boric acid, 25 mM sodium tetraborate, 75 mM NaCl, pH 8.1) for 1 h at 24°C and sensitized (1x108 NIP-RBCs) with 450 nM {alpha}-NIP IgG1 for 1 h at 37°C or 10 µl of a suboptimal concentration of peptide 33 complexed on beads or homodimeric peptide 33 were incubated with 100 µl of cells and 10 µl of serially diluted soluble peptides (10 nM to 300 µM) or IgG1 (3 pM to 100 nM). Chemiluminescence was measured as described previously.

Rosetting assay
Binding of bead-peptide complexes to Fc{gamma}Rs expressed on U937 (previously stimulated with {gamma}-IFN at 1000 U/ml), K562, Daudi, and primary NK cells was determined by rosette formation. All human leukocyte cell lines were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA). The cell lines were incubated in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C. NK cells were purified from blood packs by centrifugation on Ficoll gradients (Histopaque-1077; Sigma-Aldrich Company Ltd, Dorset, UK) and by negative selection using a magnetic bead isolation kit (human NK isolation kit II; Miltenyi Biotech Ltd., Surrey, UK). Bead complexes (6x108 beads/ml) described in the previous section were incubated with each cell type (2x106 cells/ml) at a ratio of 100:1 of beads to cells in a final volume of 100 µl of PBS. After 20 min at 24°C, 10 µl of acridine orange (66 µM) was added, and rosette formation was assessed under UV/visible illumination (Olympus BX61; Olympus, Tokyo, Japan). The ability of the cells to bind complexes was expressed as the percentage of cells that each bound 5 or more beads. For each condition 200 cells were counted in 3 replicates.

Phagocytosis and flow cytometry
U937 cells were preincubated with {gamma}-IFN (1000 U/ml) for 48 h in RPMI 1640 medium supplemented with 10% FBS depleted of IgG, 100 U/ml penicillin, and 100 µg/ml streptomycin, and resuspended at 2 x 106 cells/ml in HBSS/BSA. 5 nmoles of peptides or 500 nmole of biotinylated-IgG1 were incubated in PBS for 30 min at 24°C with 1 mg of streptavidin-coated paramagnetic beads and 0.5 or 50 nmoles of fluorescein-biotin respectively (Invitrogen). 100 µl of U937 cells were then incubated with 10 µl of fluorescent bead complexes for 2 h at 37°C. Parallel incubations were also performed in the presence of 10 µl of IgG1 (67 µM). Trypan blue (0.4%) was added to each sample for 1 h at 4°C to quench the extracellular fluorescence. The proportion of cells loaded with fluorescent bead complexes was determined by using a FACSCalibur flow cytometer (BD Biosciences, Oxford, UK), and the data were processed by CellQuest Pro Software. The relative fluorescence intensity of 10,000 cells was measured for each sample. In addition, internalization of bead-peptide complexes was visualized by Olympus BX61 epifluorescence microscopy.

Molecular modeling of the Fc mimetic peptide
A sequence similarity search was carried out for peptide 33 in the PDB database using the algorithm BLAST2. The most similar cyclic peptide (code 1s6w) corresponded to the synthetic hepcidin peptide from hybrid white striped bass (25) . The NMR structure of this peptide was used for building a homology model using the program PRIME (Schrödinger, LLC, Portland, OR, USA). After the initial model was built the program suite AMBER8 was used to perform a short molecular dynamics simulation in explicit water. The system was solvated by adding a cubic box of pre-equilibrated TIP3P water, and then minimized (conjugate gradient) and equilibrated using the Langevin temperature equilibration scheme and SHAKE constraints on hydrogen atoms. During the simulation temperature and pressure were maintained constant at 300 K and 1 atm. The simulation was run for a total of 2 ns, and the structure reached stability after 1.3 ns. The average structure over the final 700 ps was calculated and minimized. In parallel, a homology model of Fc{gamma}RI was built and refined using the program PRIME and based on the sequence and structure of the receptor within the hFc{gamma}RIII-Fc complex (18) . As a starting point to modeling Fc{gamma}RI-peptide complexes, residues Leu10 to Gly12 of the peptide were brought into proximity with Fc{gamma}RI as per the motif Leu234-Gly237 of the B-chain of the Fc fragment in complex with Fc{gamma}RIII. Similarly, residues Leu11 and Pro8 of the peptide were introduced to Fc{gamma}RI as per the motif residue Leu235 and Pro239 of the A-chain of the Fc fragment in complex with Fc{gamma}RIII. Subsequently, Fc{gamma}RI-peptide complexes were optimized using standard molecular mechanics techniques (as implemented in MacroModel) to ensure consistent placement of side chain and to eliminate physically unreasonable steric interactions between them.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selection of Fc{gamma}RI binding phage-peptides
Unlike linear peptides, constrained peptides have restricted conformational freedom that often generates ligands with increased affinity and specificity (26) . Phage display technology can introduce structural constraints by formation of covalent disulfide bonds between cysteine residues (27) . Three naive 16-mer monocyclic libraries (CPEP-4, CPEP-8, and CPEP-10) were fused at the NH2-terminal end of the pIII mature protein and displayed in polyvalent format on the M13 bacteriophage surface. To modify phage-peptide selection stringency, the receptor concentration, the phage input, and the number of washing cycles were varied according to three sets of conditions (conditions 1, 2, and 3). In addition, to select phage-peptides specific for the IgG1-Fc binding site, elution with IgG1 was performed. Following biopanning a significant increase in the number of eluted phage-peptides was observed only with the CPEP-8 library (~2000-fold after round 2 and under the first set of conditions). The phage-peptide sequences revealed seventeen unique sequences that shared strong homology and fitted the general consensus sequence TX2CXX{theta}PXLLGC{Phi}XE ({theta} represents a hydrophobic residue which is often L or W, and {Phi} is usually an acidic amino acid residue) (Table 1 ). The peptide sequences varied according to selection conditions, which revealed that each biopanning condition was unique. Close to 70% of amino acid residues enclosed between the cystine residues display hydrophobic side chains. The C-terminal tripeptide sequence flanking the cyclic ring comprises one or more acidic residues. The linear mapping of the consensus onto the IgG1 primary sequence showed a strong similarity with the fragment 232–236 (EU numbering) overlapping the lower hinge at the CH2 domain N terminus (Table 1) .


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  		Table 1. Amino acid sequence and frequency of phage-peptides obtained from biopanning and amplification with library CPEP-8

Binding to Fc{gamma}RI and activation of receptor-mediated functions
Initially we examined whether selected peptides could antagonize IgG1 binding for Fc{gamma}RI. Thirteen of 17 peptides were able to compete significantly with IgG1 for binding to Fc{gamma}RI in a dose-dependent way (Fig. 1 ). Inactive peptides were either uncyclized (peptides 35 and 37) or did not exhibit the dipeptide motif Leu10-Leu11 in the hydrophobic core (peptide 34). It is likely that the absence of both proline residues in the core and/or charged C-terminal end could contribute to their inactivity as well. The control peptide corresponding to the linear hinge-CH2 sequence 227–244 from IgG1 (p hinge-CH2) did not inhibit the binding of IgG1 to the receptor over the concentration range tested. Similar observations had already been reported from studies carried out on human Fc{gamma}RI with peptides G233LLGGPYG240 (28) , E233LLGGPSVF241 (12) , and C233LLGGLGC240 (13) .


Figure 1
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  		Figure 1. Ability of peptides to compete with IgG1 for binding to Fc{gamma}RI. Fc{gamma}RI coated onto microwells was incubated with 1.3 µM human IgG1 and 3-fold serial dilutions of soluble peptide from 50 µM (hatched bars), 16.6 µM (filled bars), 5.53 µM (gray bars), and 1.84 µM (stippled bars). Without peptide as inhibitor, the absorbance due to binding of IgG1 was normalized to 100%. IgG1 binding was detected by competition ELISA. The corresponding sequence of each peptide is given in Table 1 . p hinge-CH2, linear hinge-CH2 peptide (sequence PPCPAPELLGGPSVFLFP); cp, cyclic peptide; p 37, linear peptide 37. Results shown are the means of 4 separate experiments.

Receptor clustering induced by binding of IgG-antigen complexes triggers is known to trigger effector functions on leukocytes. Thus, we investigated the ability of peptide complexed on beads to induce both the release of reactive oxygen intermediates, and phagocytosis from IFN-{gamma} stimulated U937 cells (29) . Bead-IgG1 complexes were effective at inducing superoxide production (Fig. 2 ). Among the 17 bead-peptide complexes, 1 group (peptides 22, 30, and 33) showed a strong agonist activity and 2 groups exhibited reduced agonist activity (peptides 24, 29, and 31 and peptides 23, 25, and 32). All the other peptides tested, as exemplified by peptides 21, 27, and the linear hinge peptide, failed to trigger a superoxide burst. Interestingly, the cyclic hinge-CH2 peptide (cp hinge-CH2) lacked any functional properties. To examine the structural contribution of the disulfide bond, the covalent bridge in peptide 22 was omitted (peptide 22 S4S13). This double substitution caused complete loss of activity for peptide 22 (data not shown).


Figure 2
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  		Figure 2. Ability of peptides to activate superoxide production from {gamma}-IFN stimulated U937 cells. Cells (2x105) were activated with serial dilutions of either bead-peptide or bead-IgG complexes. After incubation with 2.5 mM lucigenin, luminescence was measured over a period of 60 min. RLU, relative luminescence units. Results shown are the means of 5 separate experiments.

Phagocytic activity of complexes was examined by flow cytometry after quenching of external fluorescence (Fig. 3A, B ), although internalized fluorescein may be underestimated at acidic pH. The internalized fluorescence of bead-peptide 22 complexes approached within 2-fold of that seen for bead-IgG1 complexes and was 10-fold greater than that detected with uncomplexed beads or beads complexed with an irrelevant peptide (Fig. 3C ). In addition, in the presence of IgG1, the bead-peptide 22 signal intensity could be reduced to background (Fig. 3D ). The results obtained with beads coupled with peptides 29, 30, or 33 were similar to those described for peptide 22 (data not shown).


Figure 3
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  		Figure 3. Phagocytosis of fluorescent bead-peptide complexes by {gamma}-IFN stimulated U937 cells. Cells (2x105) were incubated with FITC-labeled beads saturated with peptide or IgG. Cells were then treated with trypan blue to quench the extracellular fluorescence. The ingested fluorescence was monitored by epifluorescence microscopy and using a FACScalibur flow cytometer. A, B) Same cell under nonfluorescent (A) and fluorescent (B) conditions. Internalization of fluorescent beads complexed with peptide 22 can be seen clearly; view x400. C, D) Flow cytometric profiles showing the fluorescence intensity of bead complexes without or with IgG1 (67 µM) respectively. C) Profile A, beads not complexed (blue trace) and beads complexed with irrelevant peptide (sequence AQTHFDTCSWMYCWDGWW) (red trace). Profile B, beads complexed with peptide 22 (green trace). Profile C, beads complexed with IgG1 (yellow trace). D) Profile D, beads not complexed (blue trace), beads complexed with irrelevant peptide (red trace), and beads complexed with peptide 22 (green trace). Profile E, beads complexed with IgG1 (yellow trace). Results shown are representative of 3 separate experiments.

Inhibition of superoxide burst triggered by IgG1 or bead-peptide 33 complexes
Fc{gamma}RI recognition by the peptides was assessed by their ability to inhibit superoxide generation triggered by using IgG1-sensitized RBCs for the 4 strong agonist peptides (Fig. 4A ) and alternatively by using peptide 33-sensitized beads for the nonagonist peptides (Fig. 4B ) as a simpler and more controllable assay system where the response to peptide 33 as an inhibitor was equivalent in both assays (Fig. 4A, B ). The 4 strong peptide agonists could inhibit superoxide generation in a dose-dependent manner, but were 3 orders of magnitude less effective as monomers relative to monomeric IgG1 (Fig. 4A ). Thus, the apparent IC50 values were between 1 to 2 µM for peptides 22, 30, and 33, and 20 µM for peptide 29. Interestingly, in addition to competing IgG1 for binding to Fc{gamma}RI (Fig. 1 ), some nonagonist peptides, especially peptides 21 and 27, could inhibit the activity of multimeric peptide 33 (Fig. 4B ), with IC50 values of 15 and 7 µM, respectively. In addition, cyclic and linear hinge-CH2 peptides and peptide 22 S4S13 were only weakly able or unable to prevent receptor-mediated functions triggered by multimeric peptide 33. Taken together, these data indicate that unlike the hinge-CH2 control peptides, some cyclic peptides can mimic the interaction of IgG1 with Fc{gamma}RI by triggering or inhibiting Fc{gamma}RI-mediated effector functions in complexed or soluble form, respectively. However, there was no strict correlation between the ability to trigger or inhibit effector functions for a given peptide.


Figure 4
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  		Figure 4. Peptide inhibition of immune or multimeric complex-mediated superoxide bursts by peptides from {gamma}-IFN stimulated U937 cells. A) Cells (2x105) were activated by NIP-derivatized RBCs sensitized with {alpha}-NIP IgG1 and incubated with serial dilutions of IgG1 or soluble peptides. B) Cells were activated using a submaximal concentration of multimeric peptide 33 complexes and incubated with serial dilution of soluble peptides. Apparent peptide IC50 values are defined as the peptide concentration required to inhibit 50% of the signal seen with the IgG immune complexes (A) or the multimeric peptide 33 complexes (B). cp hinge-CH2, cyclic hinge-CH2 peptide (sequence AQTAPCAPAPELLGCPSV); p hinge-CH2, linear hinge-CH2 peptide (sequence PPCPAPELLGGPSVFLFP); p 22 S4S13, linear S4S13 peptide 22 (sequence AQTDTSLMLPLLGSDEE). Results shown are the means of 4 separate experiments.

Binding of peptides to Fc{gamma}Rs
The affinity of monomeric IgG1 for Fc{gamma}RI is high (KD=10–8–10–9 M) relative to Fc{gamma}RII and Fc{gamma}RIII (KD=10–5–10–7 M) (30) . Under physiological conditions, complexed IgG1 binds effectively to all Fc{gamma}Rs. The binding of peptides 22, 29, 30, and 33 to Fc{gamma}RIIa, Fc{gamma}RIIb, and Fc{gamma}RIIIa was investigated using AlphaScreen technology. Peptide binding to each receptor was compared with IgG1 binding (equivalent to 100%) (Fig. 5A ). The binding assays showed clearly that the 4 peptides interacted efficiently with Fc{gamma}RI but did not cross-react with the other receptor classes (while only the binding to Fc{gamma}RIIIa is shown, the results were similar for the other low affinity receptors). Additionally, no significant binding to any Fc{gamma}Rs was detected for the linear or cyclic hinge-CH2 peptides (data not shown). To corroborate these findings, binding specificity was also assessed by rosette-formation using different human cell lines (28) (Fig. 5B, C ). Bead-IgG1 complexes set the standard (100% rosettes) for each cell type. The 4 peptide complexes were potent at rosette-formation mediated specifically via Fc{gamma}RI. For the other cell lines the percentage rosetting was minimal, and did not exceed background levels.


Figure 5
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  		Figure 5. Peptide binding specificity for Fc{gamma}Rs assessed by AlphaScreen and rosette formation on Fc{gamma}R bearing cells. A) Each biotinylated peptide was incubated with streptavidin donor beads, while the Fc{gamma}Rs were preincubated with nickel chelate acceptor beads. Signal due to peptide complexes added over a range of concentrations of 37, 111, and 333 nM was normalized relative to the signal due to IgG1 binding (equivalent to 100%) at 5, 15, and 46 nM for Fc{gamma}RI, Fc{gamma}RIIa, and Fc{gamma}RIIb; and 15, 46, and 137 nM for Fc{gamma}RIIIa and Fc{gamma}RIIIb. Graph shows binding results for Fc{gamma}RI, with results for Fc{gamma}RIIIa shown as an example. Results shown were means of 4 independent experiments. B) Microbeads were sensitized with 10 µM peptide (22, 29, 30 or 33) or 400 nM IgG1 and incubated with 2 x 105 primary NK, Daudi, K562, or U937 cells at a ratio of 100:1 (molecule-bead complexes:leukocyte cells). Peptide or IgG-bead complex binding to cells was measured as rosette formation using a fluorescence microscope. Rosettes were defined as at least 5 beads clustered around 1 cell. Percentage rosetting indicates the ratio of rosetted cells to total cells counted. Control beads were not conjugated with any peptide. Results shown are 1 of 5 comparable experiments. C) Rosette formation with peptide 22 and a U937 cell visualized by light microscopy (x400).

Modeling of the Fc{gamma}RI-peptide complex
The Fc{gamma}R family shares a high degree of homology (50–96%) in the sequence and the structure of its ectodomains (8) . Based on the crystal structure of human Fc{gamma}1 complexed to Fc{gamma}RIII (19) , a docked complex of peptide with Fc{gamma}RI was generated. Peptide 33 was modeled on the structure of a referenced cyclic peptide and subsequently superimposed onto IgG-Fc residues predominant for recognition. As a result of alignment, energy minimization, and molecular dynamic simulation, 2 slightly different models for A-chain and B-chain peptide conformers were obtained (Fig. 6A, B , and Supplemental Fig. 6E). Pro8 residue of the A-chain conformer makes multiple hydrophobic contacts with residues Trp87 and Trp110 from Fc{gamma}RI, resembling the interaction between Pro329 of C{gamma}2-A with these tryptophan residues in both the Fc{gamma}RIII/Fc complex and the Fc{gamma}RI/Fc model of Sonderman (19) (Fig. 6C ). In addition, the hydrophobic interactions of Leu11 with Leu114 and Tyr116 resemble the interaction reported between Leu235 of the C{gamma}2-A lower hinge with Leu114 in the Fc{gamma}RI/Fc model (19) . The lack of favorable interactions that Leu11 makes with Fc{gamma}RIII would contribute to the lack of binding of peptide 33 with Fc{gamma}RIII (data not shown). In the B-chain conformer, residue Leu11 makes hydrophobic interactions with Tyr116, His131, and Trp132 of Fc{gamma}RI (Fig. 6D ). The hydrophobic region of Fc{gamma}RIII defined by a cluster of histidine residues 116, 131, and 132 has been reported to interact closely with Leu235 of C{gamma}2-B (19) . Additional potential hydrogen bonds are found between the backbone of Pro8 and Leu10 of the B-chain conformer and Arg159 and Asn117 of Fc{gamma}RI, respectively.


Figure 6
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  		Figure 6. Molecular modeling of cyclic peptide 33 superimposed onto Fc{gamma}1 and complexed to Fc{gamma}RI. A) Overlaid ribbon structure of 2 peptide models obtained after docking into Fc{gamma}RI. Red ribbon represents the main chain atoms of the A-chain conformer aligned with the Fc-A chain; blue ribbon represents the main chain atoms of the B-chain conformer aligned with the Fc-B chain. L10, L11, G12, and P8 from the cyclic peptide are indicated; disulfide bridge C4:C13 is yellow, nitrogen atoms are blue, and oxygen atoms are red. B) Superimposition ribbon structure of 2 conformers with the Fc{gamma}1 fragment taken from the complex with Fc{gamma}RIII. A-chain conformer is red, B-chain conformer is blue, Fc{gamma}1 is green. C{gamma}2 carbohydrate chains have been omitted deliberately for clarity. C, D) Close-up view of the interaction surfaces of the A-chain conformer (C) and B-chain conformer (D) with the Fc{gamma}RI model. Interface residues between conformers and the receptor are described. Tryptophan-proline sandwich formed by W87 and W110 (C) and hydrophobic area constituted by Y116, H131, and W132 (D) are maintained in these models.

Biological activity of single-chain homodimer peptide
Clustering of cell surface receptors is a powerful and flexible regulatory device in many signal transduction mechanisms, and particularly in the immune responses. Probably the most widely studied example is the cross-linking of Fc{epsilon}RI on mast cells (31) . We investigated whether a covalent peptide homodimer could serve as the minimal unit able to induce release of reactive oxygen intermediates. Peptides 22 and 33 were selected for synthesis as antiparallel homodimer (carboxy-amino linked) and parallel (carboxy-carboxyl linked) homodimer peptides. Among the 4 dimeric peptides, only the dimeric parallel peptide 33 (Fig. 7A ) was able to efficiently induce a superoxide burst with an apparent EC50 value around 2 µM (Fig. 7B ) Mass spectrometric analysis confirmed the presence of dimeric peptide 33 at the anticipated molecular mass (4494 Da) (data not shown). As expected, IgG1 and soluble peptide 33 were both able to inhibit effector functions mediated by dimeric peptide 33 (Fig. 7C ).


Figure 7
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  		Figure 7. Superoxide generation from {gamma}-IFN stimulated U937 cells triggered by single-chain homodimer peptide. A) Parallel homodimeric peptide 33 sequence. Peptide is depicted in single-letter amino acid code. Polylysine linker was chosen to optimize solubility. Bicyclic peptide was oxidized to generate 2 intramolecular disulfide bonds (Cys6-Cys15 and Cys27-Cys36) indicated by bold lines. B) Cells (2x105) were activated with serial dilutions of parallel dimeric peptide 33 or bead-peptide 33 complex. After incubation with 2.5 mM lucigenin, luminescence was measured over a period of 60 min. Results shown are means of 4 separate experiments. C) Cells were incubated using a nonsaturating concentration of dimeric peptide 33, and superoxide bursts were inhibited using a concentration range of IgG1 (inverted triangles) or soluble peptide 33 (solid squares). Incubation and measurements were performed and obtained as described above. Results shown are the means of 3 separate experiments. Key: cpDim 33, cyclic parallel homodimeric peptide 33; cp 33, cyclic peptide 33.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many proteins function by binding to multiple partners. These interactions tend to share common hot-spot contacts, which are adaptive and energetically preferred sites (4) . Structural analysis of IgG complexed with different binding partners has delineated several of these epitopes. Protein A, Protein G, rheumatoid factor, and neonatal receptor each have radically different scaffolds, yet bind an overlapping common site at the C{gamma}2/C{gamma}3 interface (32 33 34 35 36) . Fc{gamma}Rs and C1q recognize overlapping regions in the C{gamma}2 domain, within the f{gamma}2 β-strand, the b6 bend, and the lower hinge region, which are adjacent to each other in three-dimensional space (28 , 37) .

Characterization of molecular mechanisms by which Fc{gamma}Rs recognize IgG is critical to understand better the antibody-dependent mechanism of immunoregulation (38) and may facilitate design of IgG and small molecule drugs (39) . Within the last years, new and promising strategies have been undertaken to identify small "scaffolds" able to mimic FcR-Ig interactions. Using advanced techniques, including phage-peptide libraries (13 14 15 , 17) , combinatorial chemistry (40) or by compiling experimental data from the literature (12 , 41) , encouraging results have stimulated the development of small peptide ligands.

Our study identified a family of cyclic peptide able to mimic the functional epitope of Fc{gamma}1 for Fc{gamma}RI (Table 1) . The 17 phage-octapeptides share a strong sequence homology, such that approaching 90% of clones contain the tripeptide LLG within the hydrophobic core and the majority exhibit the LPXLLG motif. Most of them can inhibit IgG binding to Fc{gamma}RI (Fig. 1 ). Some can trigger Fc{gamma}RI-mediated effector functions efficiently (Figs. 2 and 3 ) and also inhibit IgG-mediated Fc{gamma}RI activation within a micromolar concentration range (Fig. 4A ). Conversely, some nonagonist peptides can also antagonize Fc{gamma}RI activation (Fig. 4B ). These latter peptides represent a class of "Fc nonmimetic peptides" that may lack sufficient avidity to trigger receptor-mediated effector function when in multimeric form, but can inhibit effector function at sufficiently high concentrations when in soluble form. Another explanation is that the Fc nonmimetic peptides may have a set of incomplete or unfavorable structural determinants, required to colocate Fc{gamma}RI molecules appropriately via the Fc-binding site.

Many studies based on single amino acid mutations or on chimeric antibodies have reported the involvement of the sequence E233LLGGP238 of IgG1 in the recognition of all Fc{gamma}R (42) , and especially residues Leu234 and Leu235 for Fc{gamma}RI (37) . The solved crystal structure of Fc{gamma}RIII-Fc{gamma}1 shows that 60% of the interface area involves the 4 lower hinge residues L234LGG237 (18 19 20) . This model also underlined the significant role of the loop at residues 327-333 and, more particularly, that of the Pro329 residue. In our study, the linear hinge-CH2 peptide corresponding to the hinge fragment sequence of IgG1, the cyclic hinge-CH2 peptide enclosing the hinge sequence according to the CPEP-8 format, and linear peptide 22 were unable to trigger a superoxide burst and showed only a weak ability to antagonize IgG-mediated effector functions. These observations suggest that the LLG motif is necessary but not sufficient to confer function. Consequently, we conclude that the peptide core sequence does not correspond simply to the lower hinge sequence P232ELLG236 of IgG1, but in contrast is comprised of several noncontiguous determinants folded closely into a scaffold of 8 amino acid residues that are required to make the peptide functional.

Modeling studies of the Fc{gamma}R1-peptide 33 complex reveal 2 complementary conformers able to mimic the functional interface of both Fc chains (Fig. 6A, B ). The character of this interaction is principally hydrophobic and includes contributions by Pro8 and Leu11 of the A-chain conformer (Fig. 6C ) and Leu11 of the B-chain conformer (Fig. 6D ). This model suggests that Pro8 in the PXLLG core motif does not match Pro232 from the lower hinge but rather Pro329 in the F/G loop of the CH2 domain. This model is consistent with the view that the cyclic peptide comprises at least 2 separate discrete elements that actively take part in multiple interactions with Fc{gamma}RI. However, to date we are not able to tell the relative contribution of each conformer to binding.

Furthermore, in contrast to IgG1, the cyclic peptides bind Fc{gamma}RI specifically among the Fc{gamma}R classes (Fig. 5 ), a result consistent with the modeling data where at least one of two conformers is disadvantaged for Fc{gamma}RIII recognition. Even though all the receptors recognize a conserved theme in the IgGs, the reactivity profiles revealed the receptor bound overlapping but nonidentical sites (11 , 18 , 28 , 37) . Preliminary experiments have identified some specifically binding peptides for the low affinity receptors (17) . Thus, a peptide consensus with the motif PPXXLG has been isolated by phage display on Fc{gamma}RIII (unpublished results).

Screening of random peptide libraries has already reported the isolation of potent peptides able to spontaneously form covalent or noncovalent homodimers in solution (43 , 44) . By bivalent interaction with two receptors, the peptides inhibited or promoted receptor dimerization, a prerequisite to initiate cell signal transduction. Fc{gamma}Rs clustering at the leukocyte cell surface trigger signal activation in the cell. This mechanism is driven by IgG-antigen complexes under physiological conditions. We report here the synthesis of a single-chain dimeric peptide constituted by 2 covalently linked identical monomers able to elicit a biological response from the U937 cell surface membrane (Fig. 7 ). While the concept of the "antigen-driven receptor cluster" is recognized, its mechanistic aspects are less well defined, although colocation of 2 receptors has been suggested (45) . Unlike IgG1 where the stoichiometry with Fc{gamma}RI is 1:1, it is plausible that the dimeric peptide binds Fc{gamma}RI with a ratio 1:2 and so can elicit functional cross-linking of 2 Fc{gamma}RI monomers.

Identification of such peptides could contribute to the characterization of the structural basis of the Fc{gamma}R1-IgG interaction and help to better define Fc{gamma}RI clustering mechanisms in their quantitative, structural, and mechanistic aspects. Possession of peptides specific for each Fc{gamma}R may help resolve the contribution of individual receptors to immune functions. Conjugated to monovalent or multivalent IgG fragments, these peptides could generate new antibody formats for the therapeutic treatment of tumors or infection, in addition to modulating specifically the activation-inhibition balance mediated by Fc{gamma}Rs (46 , 47) . In soluble form, they may be used as candidates to block Fc{gamma}R-mediated effector functions linked with inflammatory and autoimmune disorders. Conjugated to relevant IgG variable domains, the single-chain homodimer agonist peptide seems attractive when the clearance of univalent antigen is ineffective (48) . However, it is clear that optimization strategies including rational design and molecular modeling will be required to improve the pharmaceutical properties of these peptides, both for potency and for stability to proteases. After a recent publication detailing the minimization of the antigen binding site (49) we here show additionally that specific functionalities of the antibody Fc moiety can be engineered into a substantially smaller peptide. Together, these data demonstrate that Fc-like peptide mimetics of antibody function can be generated opening new options for production, mode of administration, and tissue penetration.


   ACKNOWLEDGMENTS
 
We thank Prof. Sir Aaron Klug (Laboratory of Molecular Biology, Cambridge, UK) for a critical review of the manuscript. We thank Dr. Gulin Güler-Gane (MedImmune, Cambridge, UK) for providing soluble human Fc{gamma}R samples, Dr. Jelena Jovanovic (MedImmune) for mass spectrometry measurements, and Jon Large (MedImmune) for preparation of graphics.

Received for publication July 29, 2008. Accepted for publication October 2, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ma, B., Elkayam, T., Wolfson, H., Nussinov, R. (2003) Protein-protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces. Proc. Natl. Acad. Sci. U. S. A. 100,5772-5777[Abstract/Free Full Text]
  2. Lo Conte, L., Chothia, C., Janin, J. (1999) The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285,2177-2198[CrossRef][Medline]
  3. Clackson, T., Wells, J. A. (1995) A hot spot of binding energy in a hormone-receptor interface. Science 267,383-386[Abstract/Free Full Text]
  4. DeLano, W. L., Ultsch, M. H., de Vos, A. M., Wells, J. A. (2000) Convergent solutions to binding at a protein-protein interface. Science 287,1279-1283[Abstract/Free Full Text]
  5. Scott, J. K., Smith, G. P. (1990) Searching for peptide ligands with an epitope library. Science 249,386-390[Abstract/Free Full Text]
  6. Mori, T. (2004) Cancer-specific ligands identified from screening of peptide-display libraries. Curr. Pharm. Des. 10,2335-2243[CrossRef][Medline]
  7. Gessner, J. E., Heiken, H., Tamm, A., Schmidt, R. E. (1998) The IgG Fc receptor family. Ann. Hematol. 76,231-248[CrossRef][Medline]
  8. Ravetch, J. V., Kinet, J. P. (1991) Fc receptors. Annu. Rev. Immunol. 9,457-492[Medline]
  9. Jefferis, R., Lund, J. (2002) Interaction sites on human IgG-Fc for Fc{gamma}R: current models. Immunol. Lett. 82,57-65[CrossRef][Medline]
  10. Chappel, M. S., Klein, M. H. (1991) Identification of the Fc{gamma} receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies. Proc. Natl. Acad. Sci. U. S. A. 88,9036-9040[Abstract/Free Full Text]
  11. Shields, R. L., Namenuk, A. K., Hong, K., Meng, Y. G., Rae, J., Briggs, J., Xie, D., Lai, J., Stadlen, A., Li, B., Fox, J. A., Presta, L. G. (2001) High resolution mapping of the binding site on human IgG1 for Fc{gamma}RI, Fc{gamma}RII, Fc{gamma}RIII, and FcRn and design of IgG1 variants with improved binding to the Fc{gamma}R. J. Biol. Chem. 276,6591-6604[Abstract/Free Full Text]
  12. Sheridan, J. M., Hayes, G. M., Austen, B. M. (1999) Solid-phase synthesis and cyclization of a large branched peptide from IgG Fc with affinity for Fc{gamma}RI. J. Pept. Sci. 5,555-562[CrossRef][Medline]
  13. Berntzen, G., Brekke, O. H., Mousavi, S. A., Andersen, J. T., Michaelsen, T. E., Berg, T., Sandlie, I., Lauvrak, V. (2006) Characterization of an Fc{gamma}RI-binding peptide selected by phage display. Protein Eng. Des. Sel. 19,121-128[Abstract/Free Full Text]
  14. Nakamura, G. R., Starovasnik, M. A., Reynolds, M. E., Lowman, H. B. (2001) A novel family of hairpin peptides that inhibit IgE activity by binding to the high-affinity IgE receptor. Biochemistry 40,9828-9835
  15. Nakamura, G. R., Reynolds, M. E., Chen, Y. M., Starovasnik, M. A., Lowman, H. B. (2002) Stable "zeta" peptides that act as potent antagonists of the high-affinity IgE receptor. Proc. Natl. Acad. Sci. U. S. A. 99,1303-1308[Abstract/Free Full Text]
  16. Stamos, J., Eigenbrot, C., Nakamura, G. R., Reynolds, M. E., Yin, J., Lowman, H. B., Fairbrother, W. J., Starovasnik, M. A. (2004) Convergent recognition of the IgE binding site on the high-affinity IgE receptor. Structure 12,1289-1301[Medline]
  17. Cendron, A. C., Wines, B. D., Brownlee, R. T., Ramsland, P. A., Pietersz, G. A., Hogarth, P. M. (2008) An Fc{gamma}RIIa-binding peptide that mimics the interaction between Fc{gamma}RIIa and IgG. Mol. Immunol. 45,307-319[Medline]
  18. Sondermann, P., Huber, R., Oosthuizen, V., Jacob, U. (2000) The 3.2-A crystal structure of the human IgG1 Fc fragment-Fc{gamma}-RIII complex. Nature 406,267-273[CrossRef][Medline]
  19. Sondermann, P., Kaiser, J., Jacob, U. (2001) Molecular basis for immune complex recognition: a comparison of Fc-receptor structures. J. Mol. Biol. 309,737-749[CrossRef][Medline]
  20. Radaev, S., Motyka, S., Fridman, W-H., Sautes-Fridman, C., Sun, P. D. (2001) The structure of a human type III Fc{gamma} receptor in complex with Fc. J. Biol. Chem. 276,16469-16477[Abstract/Free Full Text]
  21. Kato, K., Sautès-Fridman, C., Yamada, W., Kobayashi, K., Uchiyama, S., Kim, H., Enokizono, J., Galinha, A., Kobayashi, Y., Fridman, W. H., Arata, Y., Shimada, I. (2000) Structural basis of the interaction between IgG and Fc{gamma} receptors. J. Mol. Biol. 295,213-224[CrossRef][Medline]
  22. Kunkel, T. A., Roberts, J. D., Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154,367-382[Medline]
  23. Sidhu, S. S., Lowman, H. B., Cunningham, B. C., Wells, J. A. (2000) Phage display for selection of novel binding peptides. Methods Enzymol. 328,333-363[Medline]
  24. Pound, J. D., Lund, J., Jefferis, R. (1993) Aglycosylated chimaeric human IgG3 can trigger the human phagocyte respiratory burst. Mol. Immunol. 30,233-241[CrossRef][Medline]
  25. Lauth, X., Babon, J. J., Stannard, J. A., Singh, S., Nizet, V., Carlberg, J. M., Ostland, V. E., Pennington, M. W., Norton, R. S., Westerman, M. E. (2005) Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections. J. Biol. Chem. 280,9272-9282[Abstract/Free Full Text]
  26. Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O'Connell, M. P., Lazarus, R. A. (2000) Peptide exosite inhibitors of factor VIIa as anticoagulants. Nature 404,465-470[CrossRef][Medline]
  27. O'Neil, K. T., Hoess, R. H., Jackson, S. A., Ramachandran, N. S., Mousa, S. A., DeGrado, W. F. (1992) Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library. Proteins 14,509-515[CrossRef][Medline]
  28. Lund, J., Winter, G., Jones, P. T., Pound, J. D., Tanaka, T., Walker, M. R., Artymiuk, P. J., Arata, Y., Burton, D. R., Jefferis, R., Woof, J. M. (1991) Human Fc{gamma}RI and Fc{gamma}RII interact with distinct but overlapping site on human IgG. J. Immunol. 147,2657-2662[Abstract/Free Full Text]
  29. Lund, J., Pound, J. D., Jones, P. T., Duncan, A. R., Bentley, T., Goodall, M., Levine, B. A., Jefferis, R., Winter, G. (1992) Multiple binding sites on the CH2 domain of IgG for mouse Fc{gamma}RII. Mol. Immunol. 29,53-59[CrossRef][Medline]
  30. Hulett, M. D., Hogarth, P. M. (1994) Molecular basis of Fc receptor function. Adv. Immunol. 57,1-127[Medline]
  31. Schweitzer-Stenner, R., Pecht, I. (2005) Death of a dogma or enforcing the artificial: monomeric IgE binding may initiate mast cell response by inducing its receptor aggregation. J. Immunol. 174,4461-4464[Abstract/Free Full Text]
  32. Deisenhofer, J. (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20,2361-2370[CrossRef][Medline]
  33. Sarmay, G., Lund, J., Rozsnyay, Z., Gergely, J., Jefferis, R. (1992) Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc{gamma} receptor. Mol. Immunol. 29,633-639[CrossRef][Medline]
  34. Sauer-Eriksson, A. E., Kleywegt, G. J., Uhlen, M., Jones, T. A. (1995) Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. Structure 3,265-278[Medline]
  35. Burmeister, W. P., Huber, A. H., Bjorkman, P. J. (1994) Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372,379-383[CrossRef][Medline]
  36. Corper,, Sohi, A. L., , M.K., Bonagura, V. R., Steinitz, M., Jefferis, R., Feinstein, A., Beale, D., Taussig, M. J., Sutton, B. J. (1997) Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction. Nat. Struct. Biol. 4,374-381[CrossRef][Medline]
  37. Morgan, A., Jones, N. D., Nesbitt, A. M., Chaplin, L., Bodmer, M. W., Emtage, J. S. (1995) The N-terminal end of the CH2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc{gamma}RI and Fc{gamma}RIII binding. Immunology 86,319-324[Medline]
  38. Tortorella, D., Gewurz, B. E., Furman, M. H., Schust, D. J., Ploegh, H. L. (2000) Viral subversion of the immune system. Annu. Rev. Immunol. 18,861-926[CrossRef][Medline]
  39. Hazenbos, W. L., Gessner, J. E., Hofhuis, F. M., Kuipers, H., Meyer, D., Heijnen, I. A., Schmidt, R. E., Sandor, M., Capel, P. J., Daëron, M., van de Winkel, J. G., Verbeek, J. S. (1996) Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc{gamma}RIII (CD16) deficient mice. Immunity 5,181-188[CrossRef][Medline]
  40. Marino, M., Ruvo, M., De Falco, S., Fassina, G. (2000) Prevention of systemic lupus erythematosus in MRL/lrp mice by administration of an immunoglobulin-binding peptide. Nat. Biotechnol. 18,735-739[CrossRef][Medline]
  41. Radaev, S., Sun, P. D. (2001) Recognition of IgG by Fc{gamma} receptor. J. Biol. Chem. 276,16478-16483[Abstract/Free Full Text]
  42. Tamm, A., Schmidt, R. E. (1997) IgG binding sites in human Fc{gamma} receptors. Int. Rev. Immunol. 16,57-85[Medline]
  43. Livnah, O., Stura, E. A., Johnson, D. L., Middleton, S. A., Mulcahy, L. S., Wrighton, N. C., Dower, W. J, Jolliffe, L. K., Wilson, I. A. (1996) Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A. Science 273,464-471[Abstract]
  44. England, B. P., Balasubramanian, P., Uings, I., Bethell, S., Chen, M. J., Schatz, P. J., Yin, Q., Chen, Y. F., Whitehorn, E. A., Tsavaler, A., Martens, C. L., Barrett, R. W., McKinnon, M. (2000) A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor alpha chains. Proc. Natl. Acad. Sci. U. S. A. 97,6862-6867[Abstract/Free Full Text]
  45. Radaev, S., Sun, P. D. (2001) Recognition of immunoglobulins by Fc{gamma} receptor. Mol. Immunol. 38,1073-1083[CrossRef]
  46. Wong, C., Waibel, R., Sheets, M., Mach, J. P., Finnern, R. (2001) Human scFv antibody fragments specific for the epithelial tumour marker MUC-1, selected by phage display on living cells. Cancer Immunol. Immunother. 50,93-101[CrossRef][Medline]
  47. Lazar, G. A., Dang, W., Karki, S., Vafa, O., Peng, J. S., Hyun, L., Chan, C., Chung, H. S., Eivazi, A., Yoder, S. C., Vielmetter, J., Carmichael, D. F., Hayes, R. J., Dahiyat, B. I. (2006) Engineered antibody Fc variants with enhanced effector function. Proc. Natl. Acad. Sci. U. S. A. 103,4005-4010[Abstract/Free Full Text]
  48. Lu, Z. Y., Brochier, J., Wijdenes, J., Brailly, H., Bataille, R., Klein, B. (1992) High amounts of circulating interleukin (IL)-6 in the form of monomeric immune complexes during anti-IL-6 therapy. Towards a new methodology for measuring overall cytokine production in human in vivo. Eur. J. Immunol. 22,2819-2824[Medline]
  49. Qiu, X. Q., Wang, H., Cai, B., Wang, L. L., Yue, S. T. (2007) Small antibody mimetics comprising 2 complementarity-determining regions and a framework region for tumor targeting. Nat. Biotechnol. 25,921-929[Medline]




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