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*
Institute of Immunology and Allergology, University of Bern, Inselspital, Bern, Switzerland; and
Novartis Forschungsinstitut, Vienna, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and Methods ResultsDiscussionReferences |
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-derived peptides
of variable length was unsuccessful. However, by screening random
peptide phage display libraries we isolated circular nona- and
octapeptides specifically recognized by BSW17. These constrained
peptides mimic at least a part of a conformational epitope and are thus
called mimotopes. These mimotopes, either phage displayed or
synthetically synthesized, did not react with any other anti-human
IgE antibody tested, but efficiently inhibited the binding of human IgE
to BSW17 only. The use of Rhodol-Green-labeled free cyclic peptide
proved that these interactions were not carrier dependent. Immunization
of rabbits with phage clones displaying the specific peptides on the
surface induced an anti-human IgE response specific for the epitope
of BSW17. Therefore, we conclude that such mimotopes or
mimotope-derived peptides might be used for vaccination to induce in
vivo a beneficial anti-IgE response as a novel immunotherapy. |
Introduction |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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Certain murine monoclonal anti-IgE Abs have been shown to inhibit
IgE production in vitro (12, 13, 14, 15) and the binding of IgE to the low
affinity receptor CD23 (FcRII) (16), or to prevent binding of
IgE within immune complexes to the high affinity receptor FcRI (17).
Thus we intended to define the corresponding epitope of a murine
monoclonal anti-human IgE Ab (BSW17) (18) that possesses all these
activities. Peptides representing the recognized epitope of this Ab may
be capable of inducing autoantibodies in vivo.
Peptide libraries displayed on filamentous phages have proven to be a powerful tool to define specific epitopes for mAbs, polyclonal sera, or receptor molecules on various cell types (19, 20, 21, 22). Constrained peptide libraries exist that can be used for the definition of conformational or discontinuous epitopes. In these libraries the peptides are flanked by cysteine residues that form a disulfide bridge (23, 24), constraining the structure of the peptide. Phage display-derived peptides isolated by affinity selection from linear or circular libraries are often called mimotopes, since they mimic the structure of the original epitope (25, 26). Upon immunization, mimotopes have been shown to induce an Ag-specific immune response directed against the epitope recognized by the mAb used for the affinity selection of phage clones (27, 28). Mimotope immunizations could therefore be a new way to induce epitope-specific Ab responses in vivo for cases where the complete Ag would be harmful (e.g., toxins) or induce undesired Ab specificities. Indeed, immunization with intact IgE does not necessarily induce exclusively Abs of the desired specificity (BSW17).
To identify the epitope specific for BSW17 we used two different
approaches. First, we constructed and then screened a random
peptide phage library, displaying Fc-derived peptides of varying
length. Because this approach did not generate peptides binding to
BSW17, we screened two random nonapeptide libraries (23, 29),
displaying the nonapeptides in either a linear or a circular form. In
this report we describe the identification and characterization of 13
clones from the circular nonapeptide library that were not recognized
by other available anti-human IgE Abs. No clones were identified
from the linear library. The clones from the circular library
efficiently inhibited the binding of IgE to BSW17. This finding was
also reproduced using synthetic peptides either coupled to a carrier
protein or as free peptide. Rabbits immunized with phage particles
displaying the peptides produced Abs directed against human IgE,
inhibiting the binding of BSW17 to human IgE. Thus, such peptides might
be used for vaccination to induce an anti-IgE response in humans,
resulting eventually in the generation of human autoantibodies of the
same specificity and therefore with the same beneficial properties as
the mouse mAb BSW17.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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Goat anti-rabbit IgG (Fc and 
/
)-conjugated peroxidase
(POX)4 was purchased
from Nordic Immunology (Tilburg, The Netherlands). Polyclonal sheep
anti-human IgE-conjugated POX was purchased from The Binding Site
Ltd. (Birmingham, U.K.). Mouse monoclonal anti-human IgE antibodies
Le27 and BSW17 were described previously (17). Human myeloma IgE-PS (a
gift from Drs. K. and T. Ishizaka, La Jolla, CA), IgE-Savazal (a gift
from Dr. V. Savazal, Pilsen, Czech Republique), IgE-WT (a gift from Dr.
Stanworth, Birmingham, U.K.) were affinity purified as described
previously (30). Chimeric IgE-JW8 (31) was purified from supernatants
of the productive cell line by affinity chromatography using
anti-IgE mAb Le27 bound to affinity adsorbents (Boehringer
Mannheim, Mannheim, Germany). IgE-SUS11 was purified from supernatant
of an in-house produced hybridoma (32). The anti-IgE Abs 11-1,
11-3, 11-4, and 11-5 were obtained from Dr. R. Vasilov (NPO
Biotechnologia, Moscow, Russia), and 4F4, 4F12, 5D4, 2B9, 5B9, IE10,
4F1, and ICI were obtained from Drs. M. P. Samoilovich and V.
B. Klimovich (Hybridoma Technology Laboratory, Central Research
Institute for Roentgeno-Radiology, St. Petersburg, Russia). AbIT is a
commercial Ab from Immunotech International (Marseille, France).
POX (horseradish POX; Sigma Chemcial Co., St. Louis, MO)-coupled Abs (Le27-POX, BSW17-POX) were prepared by the periodate method as described previously (33). Anti-IgE-POX was used at a dilution of 1/1000 in PBS containing 0.15% casein (Fluka Chemie AG, Buchs, Switzerland).
Construction of a random peptide library
Four micrograms of cDNA encoding Fc constant domains 1 to 4
was ligated with 10 U of T4 DNA ligase (Boehringer) at 16°C
overnight, phenol extracted, and digested with 1 mU of DNase I
(Boehringer) in 50 mM Tris and 10 mM MnCl2, pH 7.4, at
16°C for 40 min. After extraction twice with phenol, samples were
heated to 65°C for 10 min and cooled on ice; then 0.05 mM dNTP
(Boehringer, Germany) was added, and the mixture was incubated with 5 U
of T4 DNA polymerase (Boehringer) and 5 U of Escherichia
coli DNA polymerase (Boehringer) in 5 mM MgCl2 for 15
min. After phenol extraction, 1 µg of DNA was ligated into the
EcoRV site of pComb8 vector (34) in ligase buffer
(Boehringer) containing 1.2% polyethylene glycol 8000 (Fluka) and 2 U
of T4 DNA ligase (Boehringer) at 16°C overnight. After phenol
extraction, DNA was introduced by electroporation into 200 µl of
electrocompetent E. coli XL-I blue (Stratagene, La Jolla,
CA) using a Bio-Rad Gene pulser (Bio-Rad, Richmond, CA) according to
the manufacturer’s instructions. Transfected bacteria were grown in SB
medium (35) containing 10 µg/ml tetracycline (Boehringer) and 20
µg/ml carbenicillin (Fluka) at 37°C for 2 h with vigorous
shaking. One hundred milliliters of SB containing 10 µg/ml
tetracycline and 50 µg/ml carbenicillin was added, and incubation was
continued for 1 h. VCS M13 helper phage (1012 CFU;
Stratagene) were added; after 2 h at 37°C, kanamycin
(Boehringer) was added to a final concentration of 70 µg/ml, and
incubation was continued overnight. Phage particles were precipitated
by centrifugation at 4,000 x g at 4°C for 20 min,
supernatant was mixed with 38 ml of an ice-cold, sterile-filtered
solution of 12% NaCl/16% polyethylene glycol 8000, cooled on ice for
30 min, and centrifuged for 30 min at 10,000 x g at
4°C. The phage pellet was resuspended in 2 ml of PBS/0.15% casein
and stored at 4°C.
Phage amplification and biopanning
For amplification of phage libraries, 2 ml of a liquid culture of E. coli XL-I blue grown in SB to OD600 = 1.0 was incubated with eluted phages from biopanning or 1010 CFU from the amplified library was incubated for 15 min at room temperature. Ten milliliters of SB containing 10 µg/ml tetracycline and 20 µg/ml carbenicillin was added, and 10, 1, and 0.1 µl were plated on LB plates (36) containing 100 µg/ml carbenicillin. The culture was incubated at 37°C for 1 h, then 100 ml of SB medium containing 10 µg/ml tetracycline and 50 µg/ml carbenicillin was added, and incubation was continued for 1 h. VCS M13 helper phage (1012 CFU) were added, and after 2 h at 37°C, kanamycin was added to a final concentration of 70 µg/ml, and incubation continued overnight. Phage precipitation was performed as described above.
For biopanning, Costar RIA plates A/2 (Costar, Cambridge, MA) were coated overnight at 4°C with 20 µg/ml BSW17 in 0.1 M carbonate buffer, pH 9.6, and later blocked with PBS/0.15% casein. Phage (2 x 1011 CFU) were added and incubated at 37°C for 2 h, then washed 10 times with PBS/0.1% Tween-20. The wells were rinsed with water, and the bound phage were eluted with a total of 200 µl of 0.1 M HCl, pH 2.2, for 10 min. Eluted phages were neutralized with 2 M Tris base and amplified as described.
Selection of positive clones
Fifty microliters of an E. coli XL-I blue liquid culture grown in SB to OD600 = 1.0 was incubated with 1 µl of a 10-8 dilution of amplified phages after the third round of biopanning for 15 min at room temperature and afterward plated on LB plates containing 100 µg/ml carbenicillin and grown overnight. Colonies were randomly picked and plated on LB plates containing 100 µg/ml carbenicillin. After 4 h at 37°C, nitrocellulose filters soaked with 10 mM isopropyl-ß-D-thiogalactopyranoside (Biofinex Wallisellen, Switzerland) were placed on top of the plates, and incubation was continued overnight at 32°C. Filters were removed and incubated at 37°C for 30 min in a chloroform atmosphere. Bacterial debris was removed by incubating filters in 50 mM Tris (pH 8), 150 mM NaCl, 5 mM MgCl2, 3% BSA, 100 U of DNase I, and 40 mg of lysozyme (Boehringer) per 100 ml for 1 h, blocked in TBS/1.5% casein, and incubated overnight with BSW17-POX in PBS/0.15% casein. Filters were sequentially washed with TBS, TBS/0.05% Tween-20, and TBS for 10 min each. Filters were incubated in TBS containing 600 µg/ml 4-chloro-1-naphtol (Merck, Darmstadt, Germany) and 0.042% hydrogen peroxide for staining.
IgE binding to solid phase anti-IgE mAb BSW17 for RIA
Soft microtiter plates (Dynatech, Chantilly, VA) were coated with 20 µg/ml BSW17 in 0.1 M carbonate buffer, pH 9.6, overnight at 4°C and subsequently blocked with PBS/0.15% casein. IgE-SUS11 was 125I labeled using the chloramine-T method (37), and 100,000 cpm/well was used. All experiments were performed at room temperature; plates were washed three times with 0.9% NaCl/0.05% Tween-20 and cut in pieces, then each well was measured individually in a gamma counter (LKB, Zürich, Switzerland) for 1 min.
Immunodot assays with phage particles
Anti-IgE mAbs were diluted in PBS to a concentration of 500 µg/ml, and 1 µl was dotted onto nitrocellulose (HWG 304 FO, Schleicher and Schuell, Riehen, Switzerland). Nitrocellulose was blocked in PBS/0.15% casein for 1 h. Strips were incubated overnight at room temperature with phage supernatants diluted 1/10 in PBS/0.15% casein. Washing was performed sequentially in PBS, PBS/0.05% Tween-20, and PBS for 10 min each. Developing rabbit anti-phage-POX was diluted 1/1000 in PBS/0.15% casein and incubated for 4 h. Washing and staining were performed as described and read with a hand-held reflection densitometer (Gretag Ltd., Regensdorf, Switzerland).
Immunodot assays with rabbit serum
IgE mAbs were diluted in PBS to a concentration of 500 µg/ml,
and 1 µl was dotted onto nitrocellulose. Nitrocellulose was blocked
in PBS/0.15% casein for 1 h. Rabbit serum was diluted 1/200 in
PBS/0.15% casein and incubated overnight. Washing was performed
sequentially in PBS, PBS/0.05% Tween-20, and PBS for 10 min each. Goat
anti-rabbit IgG (Fc and /)-conjugated POX was diluted 1/1000
in PBS/0.15% casein and incubated for 4 h. Washing and staining
were performed as described, and staining was read with a hand-held
reflection densitometer.
Inhibition of binding of BSW17 to IgE
Strips were prepared as described above and incubated with rabbit serum at a dilution of 1/50 in PBS/0.15% casein overnight. After washing, mAb BSW17-POX was added in a 1/50,000 dilution in PBS/0.15% casein for 4 h. Subsequent washing and staining were performed as described.
Inhibition of binding of BSW17 to IgE on sensitized CHO-FcRI
transfectants
CHO cells (2 x 105) transfected with
human FcRI 
-chain (17) were sensitized with 20 U of IgE-SUS11-IgE
for 15 min. After washing, FITC-labeled BSW17 that had been complexed
with different amounts of synthetically synthesized KLH-coupled BSW17
mimotope (pepBSW.29-8-KLH; for synthesis details see next paragraph)
for 30 min was incubated with the cells for 15 min. After washing,
cells were analyzed by FACS (EPICS Profile II, Coulter Electronics,
Hialeah, FL)
Synthesis of N-Rhodol Green (RG)-labeled pepBSW.29-8
The peptide corresponding to PhBSW.29-8 was prepared on a Biolynx 4170 automated peptide synthesizer (LKB Pharmacia, Uppsala, Sweden). All acylation reactions were conducted using a fivefold excess of F-moc amino acids activated with 1 equivalent of benzotriazole-1-yl-oxy-rs-pyrrolidino-phosphonium in the presence of 1 equivalent of hydroxybenzotriazole and 2 equivalents of di-isopropylethylamine. All reactions were conducted in dimethylformamide. A coupling time of 45 min was used throughout. The cleavage of the peptide from the resin and side chain deprotection were conducted by treatment of the peptide resin with trifluoroacetic acid/ethanedithiole/water (94/5/1) for 2.5 h. After precipitation of the crude peptide with ether, lyophilization was performed in a water/t-butanol (1:1) mixture. The peptide was checked for purity by HPLC and mass spectrometry (m.w. = 1924.15).
The cyclization of the freshly reduced peptide was conducted on Ekathiox Resin (Peptides International, Inc., Louisville, KY, catalogue no. RSH-1299-PI). The reaction was checked with Ellman’s reagent. After separation from the resin, the peptide was purified by RP-HPLC. The cyclic peptide was checked by capillary electrophoresis and mass spectrometry (MW = 1922.10 C85 H116 N24 O24 S2).
The peptide was labeled with RG carboxylic acid succinimidyl ester (R-6108, Molecular Probes, Eugene, OR) and purified by RP-HPLC to a purity >98%. Pooled fractions were lyophilized and stored at -80°C.
Binding of RG-labeled mimotope (pepBSW.29-8-RG) to BSW17
Steady state fluorescence measurements were performed on a SLM 8000C spectrofluorometer equipped with JD-490 photomultipliers and a 450-watt xenon Arclamp (SLM Instruments, Urbana, IL). Spectral band widths were set at 8 and 16 nm for excitation and emission, respectively. Measurements were performed at 25°C in a 750Tl quartz cuvette (10-mm optical path length). PepBSW.29-8-RG at a concentration of 30 nM in 150 mM sodium phosphate, pH 6.8, was incubated with increasing concentrations of BSW17 from a 15.33-µM stock solution in the same buffer. Titrations were performed with the excitation wavelength set at 500 nm, and the emission wavelength set at 533 nm. The spectra were corrected for Raman scattering from the buffer solution, for dilution, and for inner filter effects due to BSW17 additions. The signal was adjusted for lamp intensity changes by ratio mode detection with a metal scatter in the reference channel. An OG 515 emission cut-off filter was used for stray light reduction. For each addition of BSW17, 10 intensity measurements were taken with an integration time of 10 s each. For data analysis the two points with the highest and lowest values each were dropped, and the average was calculated automatically by a hand-written program, ANIS3, which also provides for all possible individual corrections to judge the quality of the titration.
For data analysis, the percent enhancement of the integrated fluorescence intensity of the pepBSW.29-8-RG/BSW17 complex was plotted as function of the total concentration of BSW17 and fit to the equation F = Fmin + (Fmax - FMin) * (1/[pepBSW.29-8-RG]) * (([pepBSW.29-8-RG] + n*[BSW17] + Kd)/2 - ([pepBSW.29-8-RG] + n*[BSW17] + Kd)2/4 - (4n*[pepBSW.29-8-RG] * [BSW17])1/2).
The Marquardt algorithm in the program GraFit 3.0 Erithacus Software Ltd, Staines, U.K. was used to optimize the maximal fluorescence intensity, Fmax, and Kd, the intrinsic dissociation constants for the pepBSW.29-8-RG interaction with BSW17, assuming a 1:2 stochiometry with two independent and equal binding sites.
Immunization
Freshly prepared phage particles (1012 CFU) were dialyzed against PBS at 4°C. One milliliter was emulsified with CFA for the first immunization or with 1 ml of IFA for boosting. Immunization was repeated s.c. every 14 days. After the third boost, 12 ml of blood was taken and clotted for 4 h at room temperature in glass vials, centrifuged for 10 min at 2000 x g. The supernatant was frozen at -20°C.
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Results |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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peptide library and selection of
mAb-specific epitopes by biopanning of phage display libraries
In a first attempt to characterize the epitope of the anti-IgE
mAb BSW17, we chose an M13-based bacteriophage system as described
previously (34, 35). Pilot experiments using the vector pComb8 showed
that we were able to express single as well as multiple domains
along the surface of the phage body that were recognized by different
anti-IgE mAbs (data not shown).
To obtain smaller parts of the Fc constant domains, possibly
containing only the epitope of BSW17, cDNA for the Fc part was
digested with DNase I, resulting in random fragments of varying lengths
(data not shown). After ligation into the phagemid vector pComb8, the
phage display library resulted in 3 x 106
transformants. This represents 4 times the theoretical number of
fragments longer than 12 bp, assuming that 8 x 105
12-mer or longer fragments can be generated from the full-length cDNA
of 1284 bp. The transformed bacterial cells produced phage particles
displaying this random library as fusion proteins with the major
coat protein pVIII on the phage surface. Affinity selection of the
phage display library with BSW17 was performed by biopanning as
described previously. For a control we used another mouse monoclonal
anti-human IgE Ab, termed Le27, recognizing a heat-resistant
epitope within the fourth constant domain. However, our attempt to
select epitope displaying phages specific for mAb BSW17 after four
rounds of biopanning was clearly negative (Fig. 1
A).
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B).
After the third round the library was analyzed, and clones were
isolated displaying peptides shorter than 35 amino acids, as judged by
DNA analysis on agarose gels (data not shown). In contrast, BSW17
recognized none of these clones, but some of the longer peptides (>50
amino acids in length) were recognized by a polyclonal sheep
anti-human IgE Ab (data not shown). Thus, we assumed that no short
linear primary amino acid sequence had been displayed by the phages in
a conformation that could be recognized by the mAb BSW17. Selection of BSW17-specific mimotopes and sequence analysis
To search for a possible conformational epitope, we screened two
random peptide libraries (23, 29). One of the libraries displays linear
peptides of nine amino acids in length, of a random sequence, and fused
to the major coat protein pVIII. The second, circular library is
similar to the first, but has additionally two cysteins flanking the
random nonapeptide sequence, giving a constraint to the peptide by
allowing the formation of a disulfide bridge. After three rounds of
biopanning with the two libraries using BSW17 as a target, we observed
an approximately 6000-fold increase in the number of eluted phages, but
only in the case of the circular library (Fig. 1A).
From the third round of biopanning on the mAb BSW17, we randomly picked
82 clones and tested them for binding of POX-labeled BSW17. By means of
this assay we arbitrarily selected 13 clones (16% of the screened
clones) reacting strongly with BSW17. These clones were negative on
other available mouse mAb anti-human IgE as shown in (Table I).
This result indicated that the
peptides were recognized by the binding site of BSW17. DNA sequence
analysis of the 13 binding phage clones revealed that the displayed
peptides can be grouped into four classes according to the deduced
amino acid sequence (Table II). None of
the peptide sequences showed homology to the primary IgE amino acid
sequence (EMBL accession no. V00555; L00022). Surprisingly three clones
contained a peptide of eight amino acids instead of nine. Moreover, the
phage clones displaying the octapeptide bound stronger to BSW17
compared with the other nonapeptide clones and determined by immunodot
assays (Tables I and II, clones 18, 29, and 36). The postulated
structure common to all the clones consists of a core of seven amino
acids, where the two amino acids toward the amino terminus have a polar
charge, followed by three polar, but uncharged, and two nonpolar amino
acids (Table II). All clones contained a tryptophan and a valine at the
carboxyl terminus of the nonapeptide and a glycine in the middle.
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For further studies we selected two phage clones: one displaying
the nonapeptide (PhBSW.6-9), and one displaying the octapeptide
(PhBSW.29-8) as shown in Table II. Both phage clones were used at a
concentration of 1011 CFU/ml and inhibited the binding
of 125I-labeled IgE to BSW17 (Fig. 2). To quantitate this inhibition, we
determined the amount of cold IgE necessary for the same reduction in
binding of [125I]IgE-SUS11. The inhibition achieved with
1011 CFU/ml of phage clone PhBSW.6-9 corresponded to a
concentration of 1 µg/ml IgE-SUS11 (corresponding to 6 x
1012 epitopes that can theoretically be recognized by BSW17
in the IgE molecule, based on a 188 kDa molecular mass for human IgE),
and that for clone PhBSW.29-8 corresponded to a concentration of 1.7
µg/ml IgE-SUS11 (corresponding to 1 x 1013
epitopes). Assuming that both peptides have comparable affinities for
BSW17, we estimated that 64 or 109 binding sites are blocked per
PhBSW.6–9 or PhBSW.29-8 phage particle, respectively.
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RI
transfectants
To further elucidate the specificity of the selected peptides, the
mimotope of phage clone PhBSW.29-8 was synthesized chemically and
coupled to KLH (pepBSW.29-8-KLH). We used this construct to inhibit the
interaction of BSW17 with IgE bound to FcRI-transfected CHO cells.
As shown in Figure 3, preincubation of
BSW17 with KLH-coupled peptide (0.05, 0.5, 5, and 50 µg) inhibited
binding to CHO-bound IgE in a dose-dependent manner (87, 86, 40, and
48% inhibition, respectively), whereas KLH alone did not inhibit this
interaction. No binding to the untransfected parental cell line was
observed.
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To prove that the cyclic mimotopes not only bind to BSW17
when associated with phage particles or a carrier protein,
the PhBSW.29-8 mimotope was synthetically synthesized and
labeled with the rhodamine derivative RG (pepBSW.29-8-RG) at the
N-terminus. A titration of this fluorescent derivative at a
concentration of 30 nM with BSW17 resulted in an approximately 20%
increase in RG fluorescence intensity (Fig. 4) at saturation. The affinity of this
construct to BSW17 was determined by a nonlinear, least squares fit to
the quadratic equation describing the relative increase in RG
fluorescence emission intensity enhancement as a function of the total
BSW17 concentration. Two equivalent, independent binding sites for
pepBSW.29-8-RG on BSW17 were assumed. The resulting intrinsic
dissociation constant was 78.1 ± 25.1 nM.
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Induction of an epitope-specific immune reaction by immunization of rabbits with mimotope-displaying phage clones
To investigate whether the mimotopes represent an immunogenic
structure corresponding to the natural epitope on the IgE molecule,
rabbits were immunized with phage particles, either with phage clone
PhBSW.6-9, clone PhBSW.29-8, or with the helper phage VCS M13. After
immunization and two boosts, sera from the rabbits were collected and
tested for anti-human IgE Abs (Table III). The sera showed an anti-IgE
response; meanwhile, the control rabbit immunized with M13 phage alone
showed no anti-IgE reactivity.
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, the serum of the control rabbit,
immunized with M13 helper phage, did not inhibit binding of BSW17 to
IgE. In contrast, sera from the two rabbits immunized with BSW17
mimotope reduced the signal up to 65%. In an additional control
experiment we used Le27 at a concentration of 10 µg/ml instead of
serum. This anti-IgE Ab did not influence the binding of BSW17 to
IgE (data not shown). These experiments clearly demonstrate the
presence of an elicited specific anti-IgE response directed against
the epitope of BSW17 in the rabbit.
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Discussion |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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RI -chain emerged several
years ago (5, 6, 7, 8, 9, 13, 15). At present, two humanized anti-IgE Abs
are in clinical trials (8, 9, 10, 11). Both Abs are nonanaphylactogenic, since
they recognize an epitope in the vicinity of or in the binding site for
FcRI and as a consequence are unable to recognize
receptor-bound IgE.
In contrast, the nonanaphylactogenic mouse monoclonal anti-human
IgE Ab BSW17 recognized receptor-bound IgE (17). Moreover, free immune
complexes of IgE and BSW17 did not bind to FcRI, probably due to a
conformational change induced by BSW17 within the Fc part.
Therefore, BSW17 was able to form immune complexes with receptor-bound
IgE, thereby shifting the thermodynamic balance of bound vs unbound
immune complexes toward the unbound by preventing the reassociation
with the receptor (17). These findings indicated that the part of the
IgE molecule containing the epitope for BSW17 may be crucial for the
biologic activity.
In the case of rat IgE, synthetic peptides corresponding to the sequence of the native Ag induced Abs in vivo (38). Furthermore, immunization with mimotopes also induced an epitope-specific immune response in vivo (28, 39). More importantly, even protective immune reactions elicited by mimotope immunizations have been shown for several infectious agents (27, 40, 41). Therefore, peptide mimotopes might be a valuable approach to develop vaccines for the induction of a defined Ab response to complex Ags.
In our case, using the mouse monoclonal anti-human IgE Ab BSW17, we identified phage clones displaying peptides mimicking a conformational epitope of the IgE molecule. The isolation of these peptides for BSW17 was only possible using a constrained random nonapeptide library, in which peptides are displayed as circular loops held together by a disulfide bridge. The amino acid sequences of the BSW17-specific mimotopes were not present in the primary amino acid sequence of the IgE molecule, and degradation of the disulfide bond destroyed the mimotope for BSW17 (data not shown). Therefore, we assume that the epitope for BSW17 is of a discontinuous nature.
Inhibition and immunization experiments documented the specificity of
the mimotopes for the mAb BSW17, excluding a nonspecific binding via
framework sequences or the Fc
constant part of BSW17. Additionally,
the mimotopes were specifically recognized by BSW17 in the context of
another carrier protein (Fig. 3) and also without carrier (Fig. 4),
indicating that the mimotope alone is responsible for the interaction
with BSW17 without involvement of structures originating from the phage
particle. A computer-generated model of the three-dimensional structure
of our BSW17 mimotopes as displayed on the phage protein pVIII showed
the heptapeptide motif pointing away from the phage pVIII protein,
rendering it accessible for an Ab (data not shown). The described
charge motif might be enough to mimic the structure of the natural
epitope, as can be speculated from the sequence alignment of the
mimotopes. A detailed analysis of the three-dimensional structure of
the mimotope will be published elsewhere.
Despite the great number of independent clones in the libraries used, they cover only a small percentage of all the theoretically possible nonapeptides (5 x 1011). It is possible that the linear or constrained nonapeptide library does not contain peptides mimicking the most optimal structure of the native epitope. Moreover, it has been shown that Ab-specific mimotopes derived from different combinatorial libraries varied among the different libraries used for screening (42). Finally, the intrinsic dissociation constant measured for pepBSW.29-8-RG binding to BSW17 (78.1 ± 25.1 nM) was more than 1 log higher than the dissociation constant known for the BSW17-IgE interaction (1.2 nM) (our unpublished observation). This further indicated that the mimotope may not represent the optimally fitting structure. Therefore, we are presently screening several phage libraries, displaying peptides of different lengths, to study whether additional mimotopes for BSW17 can be found.
Since our mimotopes are displayed as fusion proteins with the major coat protein (up to 60–100 copies along the filamentous phage particle), it might be possible that avidity rather than affinity played a role in selecting the mimotopes. Nevertheless, it was reported for streptavidin that screening of conformationally constrained peptide libraries revealed clones of much higher affinities than those of linear peptide libraries (43).
In summary, this report describes the isolation of mimotopes corresponding to the epitope of the monoclonal anti-IgE Ab BSW17. Binding and inhibition studies either with mimotope-displaying phage particles or with synthetic peptides proved the specificity of the isolated mimotopes. Finally, immunization of rabbits with mimotope induced anti-IgE Abs whose binding to IgE was inhibited by BSW17. Similar products may serve as agents for the prevention of allergic disease.
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Acknowledgments |
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Footnotes |
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2 Current address: Loyola University Medical Center, Oncology Institute, 2160 South First Ave., Maywood, IL 60153.
3 Address correspondence and reprint requests to Prof. B. M. Stadler, Institute of Immunology and Allergology, University of Bern, Inselspital, CH-3010 Bern. E-mail address:
4 Abbreviations used in this paper: POX, peroxidase; SB, super broth; LB, Luria broth; TBS, Tris-buffered saline; KLH, keyhole limpet hemocyanin; RG, Rhodol Green; RP-HPLC, reverse phase high performance liquid chromatography.
Received for publication May 5, 1997. Accepted for publication December 9, 1997.
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References |
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TopAbstractIntroductionMaterials and Methods ResultsDiscussionReferences |
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