IGE EPITOPE-LIKE PEPTIDES AND USES THEREOF

Abstract

The present invention relates to IgE epitope-like peptides which have ability to bind to allergen specific IgE paratopes. Said allergen specific IgEs are bound to effector cells of allergic patients. The IgE epitope-like peptides of the invention cover the paratopes of said IgE bound on effector cells, prevent biding of causative allergen on said IgE on effector cells, and thereby prevent degranulation and secretion of mediators of allergic inflammation from effector cells, after contact with the causative allergen. The present invention relates to the methods of using such IgE epitope-like peptides for therapy of allergic reaction. Said allergic reaction is caused by exposure to the causative allergen.

Claims

1. An IgE epitope-like peptide having the following structure:
Z-X.sup.1-His-Pro-Arg-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-U  (Structure I) wherein X.sup.1 is a polar or charged amino acid, preferably Asp or Asn; X.sup.2 is a hydrophobic, aromatic amino acid, preferably Phe or Tyr; X.sup.3 may or may not be present, but if present it is a polar or non-polar amino acid, preferably Asn, Ala, Gly, Ser or Pro; X.sup.4 may or may not be present, but if present it can be any amino acid, preferably Asp, Arg, Tyr, Pro, Thr, Glu, Leu or Phe; X.sup.5 may or may not be present, but if present it is preferably a charged or polar amino acid, and more preferably Ser, Asp or Val; X.sup.6 may or may not be present, but if present it is a polar amino acid, preferably Tyr, Ser or Asn; X.sup.7 may or may not be present, but if present it is a polar or charged amino acid, preferably Gln, Asn or Asp; X.sup.8 may or may not be present, but if present it is a non-polar or polar amino acid, preferably Val, Pro or Ser; X.sup.9 may or may not be present, but if present it is a non-polar amino acid, preferably Ala, Trp or Pro; Z is a hydrogen atom replacing the N-terminal amino group or an N-terminal amino group, or a stability enhancing moiety, bound either to the N-terminal amino group or directly to the alpha C-atom of the N-terminal residue; U is a hydrogen atom replacing the C-terminal carboxyl group or a C-terminal carboxyl group, or is a linker or a stability enhancing moiety, bound either to the C-terminal carbonyl group or directly to the alpha C-atom of the C-terminal residue.

2. The IgE epitope-like peptide of claim 1, wherein X.sup.1 is Asp or Asn.

3. The IgE epitope-like peptide of claim 1, wherein X.sup.2 is Phe or Tyr.

4. (canceled)

5. The IgE epitope-like peptide of claim 1, wherein X.sup.3 is present and is Asn, Ala, Gly, Ser or Pro.

6. (canceled)

7. (canceled)

8. The IgE epitope-like peptide of claim 1, wherein X.sup.4 is present and is Asp, Arg, Tyr, Pro, Thr, Glu, Leu or Phe.

9. (canceled)

10. (canceled)

11. The IgE epitope-like peptide of claim 1, wherein X.sup.5 is present and is Ser, Asp or Val.

12. (canceled)

13. (canceled)

14. The IgE epitope-like peptide of claim 1, wherein X.sup.6 is present and is Tyr, Ser or Asn.

15. (canceled)

16. (canceled)

17. The IgE epitope-like peptide of claim 1, wherein X.sup.7 is present and is Tyr, Ser or Asn.

18. (canceled)

19. (canceled)

20. The IgE epitope-like peptide of claim 1, wherein X.sup.8 is present and is Val, Pro or Ser.

21. (canceled)

22. (canceled)

23. The IgE epitope-like peptide of claim 1, wherein X.sup.9 is present and is Ala, Pro or Trp.

24. (canceled)

25. The IgE epitope-like peptide of claim 1, wherein one or more amino acids of said IgE epitope-like peptide are in L-form or D-form, or wherein the amino acids of said IgE epitope-like peptide are a combination of both L- and D-forms.

26. The IgE epitope-like peptide of claim 1, wherein the IgE epitope-like peptide comprises an amino acid sequence having at least 60% sequence identity with any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10.

27. The IgE epitope-like peptide of claim 1, wherein the IgE epitope-like peptide comprises any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10.

28. The IgE epitope-like peptide of claim 1, wherein the IgE epitope-like peptide consists of any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10.

29. A peptides of claim 1, wherein said peptide is an IgE epitope-like peptide and is characterized by: a) Binding to peanut or Ara h 2 specific IgEs of peanut allergic patients; b) Ability to prevent cross-linking of said IgE on effector cells of peanut allergic patients; and/or c) Ability to prevent degranulation and secretion of mediators of allergic inflammation from effector cells of peanut allergic patients, after contact with the peanut or Ara h 2 allergen.

30. A retro-inverso analogue of the IgE epitope-like peptide of claim 1.

31-34. (canceled)

35. A pharmaceutical composition comprising at least one of the IgE epitope-like peptide of claim 1, or the retro-inverso analogue of the IgE epitope-like peptide, and optionally at least one pharmaceutically acceptable excipient.

36. The pharmaceutical composition of claim 35, wherein said composition is adapted for any of the following routes of administration: oral administration, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, intravenous infusion.

37. The IgE epitope-like peptide of claim 1, wherein X.sup.1 is Asp or Asn; X.sup.2 is Phe or Tyr; X.sup.3 is Asn, Ala, Gly, Ser or Pro; X.sup.4 is Asp, Arg, Tyr, Pro, Thr, Glu, Leu or Phe; X.sup.5 may or may not be present, but if present it is Ser, Asp or Val; X.sup.6 may or may not be present, but if present it is Tyr, Ser or Asn; X.sup.7 may or may not be present, but if present it is Gln, Asn or Asp; X.sup.8 may or may not be present, but if present it is Val, Pro or Ser; and X.sup.9 may or may not be present, but if present it is Ala, Trp or Pro.

38. The IgE epitope-like peptide of claim 1, wherein X.sup.1 is Asp or Asn; X.sup.2 is Phe or Tyr; X.sup.3 is Asn, Ala, Gly, Ser or Pro; X.sup.4 is Asp, Arg, Tyr, Pro, Thr, Glu, Leu or Phe; X.sup.5 is Ser, Asp or Val; X.sup.6 is Tyr, Ser or Asn; X.sup.7 is Gln, Asn or Asp; X.sup.8 is Val, Pro or Ser; and X.sup.9 is Ala, Trp or Pro.

39. A method for treatment of peanut allergy, for prevention of systemic adverse events during peanut immunotherapy or for prevention of allergic reaction during medical interventions with peanut allergens or peanut extracts, said method comprising administering a therapeutic effective amount of the pharmaceutical composition according to claim 35 to a patient in need thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0143] FIG. 1: Schematic presentation of IgE epitope-like peptides of Ara h 2

[0144] FIG. 2: A. Binding of IgE epitope-like peptides (ELP 1 to 10) to rabbit Ara h 2 antiserum compared to control wild-type phage. Average and standard deviations from duplicate experiments are shown. B. Individual IgE epitope-like peptides expressed as fusion proteins with pIII protein on the surface of filamentous phage compete for the same binding site with allergen. Black bars: IgE epitope-like peptides expressed on the surface of filamentous phage (5×10.sup.10 pfu/well) and Ara h 2 (0.25 μg/well). White bars: IgE epitope-like peptides expressed on the surface of filamentous phage (5×10.sup.10 pfu/well).

[0145] FIG. 3: Recognition of three (ELP 6, ELP 2 and ELP 5) IgE epitope-like peptides by IgE from the serum of five peanut-allergic patients. Labels C1 and C2 correspond to pIII fusions of target-unrelated peptides with an affinity for streptavidin and human leptin, respectively. Healthy control serum is also included.

[0146] FIG. 4: Basophil CD63 response after stimulation with peanut extract, Ara h 2 or specific IgE epitope-like peptides L12-N14, L7-N40 and L7-N48 in five peanut allergic patients. Data are presented as median and IQR.

[0147] FIG. 5: Inhibition of basophil CD63 response to Ara h 2 stimulation after the treatment with specific IgE epitope-like peptides L12-N14, L7-N40 and L7-N48 in three peanut allergic patients.

DETAILED DESCRIPTION OF THE INVENTION

[0148] The “IgE epitope-like peptides” as used herein are peptides that mimic epitopes on allergens onto which IgE antibodies are binding to. The IgE epitope-like peptides bind to antigen-binding site of allergen specific IgEs, do not cross-link allergen specific IgEs on effector cells of allergic patients and do not induce activation and release of mediators of allergic inflammation from effector cells.

[0149] The invention further provides IgE epitope-like peptides, discovered by screening of biological peptide libraries. The IgE epitope-like peptides of the invention are binding to peanut and Ara h 2 specific IgEs of peanut allergic patients. They do not cross-link IgEs on basophil and mast cells of the allergic patient and consequently not do induce activation and release of mediators of allergic inflammation from said effector cells. They have the ability to prevent peanut and Ara h 2 allergen IgE cross-linking and thus inhibit mast cell and basophil activation and degranulation in peanut allergic patients.

[0150] The IgE epitope-like peptide according to the present invention is a peptide with short chain length, containing a sequence of amino acids or an analogous sequence. Written from amino terminus to carboxy terminus, the peptide of the present invention has the general structure:

Z-X.sup.1-His-Pro-Arg-X.sup.2-X.sup.3-X.sup.4-X.sup.5-X.sup.6-X.sup.7-X.sup.8-X.sup.9-U

[0151] wherein X.sup.1 is a polar or charged amino acid, preferably Asp or Asn; X.sup.2 s a hydrophobic, aromatic amino acid, preferably Phe or Tyr; X.sup.3 may or may not be present, but if present it is a polar or non-polar amino acid, preferably Asn, Ala, Gly, Ser or Pro; X.sup.4 may or may not be present, but if present it can be any amino acid, preferably Asp, Arg, Tyr, Pro, Thr, Glu, Leu or Phe; X.sup.5 may or may not be present, but if present it is preferably a charged or polar amino acid, and more preferably Ser, Asp or Val; X.sup.6 may or may not be present, but if present it is a polar amino acid, preferably Tyr, Ser or Asn; X.sup.7 may or may not be present, but if present it is a polar or charged amino acid, preferably Gln, Asn or Asp; X.sup.8 may or may not be present, but if present it is a non-polar or polar amino acid, preferably Val, Pro or Ser; X.sup.9 may or may not be present, but if present it is a non-polar amino acid, preferably Ala, Trp or Pro; Z is a hydrogen atom replacing the N-terminal amino group or an N-terminal amino group, or a stability enhancing moiety, bound either to the N-terminal amino group or directly to the alpha C-atom of the N-terminal residue; U is a hydrogen atom replacing the C-terminal carboxyl group or a C-terminal carboxyl group, or is a linker or a stability enhancing moiety, bound either to the C-terminal carbonyl group or directly to the alpha C-atom of the C-terminal residue.

[0152] According to some embodiments X.sup.1 is Asp or Asn.

[0153] According to some embodiments X.sup.1 is Asp.

[0154] According to some embodiments X.sup.1 is Asn.

[0155] According to some embodiments X.sup.2 is Phe or Tyr.

[0156] According to some embodiments X.sup.2 is Phe.

[0157] According to some embodiments X.sup.2 is Tyr.

[0158] According to some embodiments X.sup.3 is not present.

[0159] According to some embodiments X.sup.3 is present.

[0160] According to some embodiments X.sup.3 is Asn, Ala, Gly, Ser or Pro.

[0161] According to some embodiments X.sup.3 is Asn.

[0162] According to some embodiments X.sup.3 is Ala.

[0163] According to some embodiments X.sup.3 is Gly.

[0164] According to some embodiments X.sup.3 is Ser.

[0165] According to some embodiments X.sup.3 is Pro.

[0166] According to some embodiments X.sup.4 is not present.

[0167] According to some embodiments X.sup.4 is present.

[0168] According to some embodiments X.sup.4 is Asp, Arg, Tyr, Pro, Thr, Glu, Leu or Phe.

[0169] According to some embodiments X.sup.4 is Asp.

[0170] According to some embodiments X.sup.4 is Arg.

[0171] According to some embodiments X.sup.4 is Tyr.

[0172] According to some embodiments X.sup.4 is Pro.

[0173] According to some embodiments X.sup.4 is Thr.

[0174] According to some embodiments X.sup.4 is Glu.

[0175] According to some embodiments X.sup.4 is Leu.

[0176] According to some embodiments X.sup.4 is Phe.

[0177] According to some embodiments X.sup.5 is not present.

[0178] According to some embodiments X.sup.5 is present.

[0179] According to some embodiments X.sup.5 is Ser, Asp or Val.

[0180] According to some embodiments X.sup.5 is Ser.

[0181] According to some embodiments X.sup.5 is Asp.

[0182] According to some embodiments X.sup.5 is Val.

[0183] According to some embodiments X.sup.6 is not present.

[0184] According to some embodiments X.sup.6 is present.

[0185] According to some embodiments X.sup.6 is Tyr, Ser or Asn.

[0186] According to some embodiments X.sup.6 is Tyr.

[0187] According to some embodiments X.sup.6 is Ser.

[0188] According to some embodiments X.sup.6 is Asn.

[0189] According to some embodiments X.sup.7 is not present.

[0190] According to some embodiments X.sup.7 is present.

[0191] According to some embodiments X.sup.7 is Gln, Asn or Asp.

[0192] According to some embodiments X.sup.7 is Gln.

[0193] According to some embodiments X.sup.7 is Asn.

[0194] According to some embodiments X.sup.7 is Asp.

[0195] According to some embodiments X.sup.8 is not present.

[0196] According to some embodiments X.sup.8 is present.

[0197] According to some embodiments X.sup.8 is Val, Pro or Ser.

[0198] According to some embodiments X.sup.8 is Val.

[0199] According to some embodiments X.sup.8 is Pro.

[0200] According to some embodiments X.sup.8 is Ser.

[0201] According to some embodiments X.sup.9 is not present.

[0202] According to some embodiments X.sup.9 is present.

[0203] According to some embodiments X.sup.9 is Ala, Pro or Trp.

[0204] According to some embodiments X.sup.9 is Ala.

[0205] According to some embodiments X.sup.9 is Pro.

[0206] According to some embodiments X.sup.9 is Trp.

[0207] According to some embodiments Z is a hydrogen atom replacing the N-terminal amino group.

[0208] According to some embodiments Z is an N-terminal amino group.

[0209] According to some embodiments Z is a stability enhancing moiety, bound either to the N-terminal amino group or directly to the alpha C-atom of the N-terminal residue.

[0210] According to some embodiments U is a hydrogen atom replacing the C-terminal carboxyl group.

[0211] According to some embodiments U is a C-terminal carboxyl group.

[0212] According to some embodiments U is a stability enhancing moiety, bound either to the C-terminal carbonyl group or directly to the alpha C-atom of the C-terminal residue.

[0213] The amino acids of said peptide may be in L-form (L-enantiomer), D-form (D-enantiomer), D-form with reverted order (i.e., retro-inverso peptide), combination of both enantiomers or in the form of alpha N-substituted glycine residues forming the corresponding peptoid derivative.

[0214] The peptide of the present invention may also be in the form of a peptidomimetic containing non-standard (non-proteinogenic) amino acids or the phosphonate, amidate, carbamate ester or sulphonamide backbone linkages replacing the peptide backbone but retaining the sequences of side chains of the present invention.

[0215] The specific examples of the IgE epitope-like peptide of this invention include, but are not limited to, those listed in Table 1.

TABLE-US-00001 TABLE 1  Specific examples of the IgE epitope-like peptides SEQ ID NO Amino acid sequence 1 DHPRFNDSYNSP 2 DHPRFNRDNDVA 3 DHPRFNYVSQPW 4 DHPRFAP 5 DHPRYGP 6 DHPRFST 7 DHPRFAE 8 DHPRFPL 9 DHPRFSF 10 NHPRFNL

[0216] An IgE epitope-like peptide may comprise an amino acid sequence having at least 60%, such as at least 70%, sequence identity with any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10.

[0217] An IgE epitope-like peptide may comprise an amino acid sequence having at least 80%, such as at least 90%, sequence identity with any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10.

[0218] An IgE epitope-like peptide may comprise an amino acid sequence having at least 90%, such as at least 95%, sequence identity with any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10.

[0219] An IgE epitope-like peptide of the present invention may be a variant of a peptide comprising any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10, wherein one or more (such as one, two, three, four or five) amino acids in the reference sequence SEQ ID NO: 1 to SEQ ID NO: 10 are substituted by conservative substitutions.

[0220] An IgE epitope-like peptide of the present invention may be a variant of a peptide comprising any one of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10, wherein one to five (such as one to three) amino acids in the reference sequence SEQ ID NO: 1 to SEQ ID NO: 10 are substituted by conservative substitutions.

[0221] The IgE epitope-like peptides of the present invention bind to peanut and Ara h 2 specific IgEs of peanut allergic patients that are bound on the surface of effector cells. The IgE epitope-like peptides prevent cross-linking of said IgE and thereby prevent degranulation and secretion of mediators of allergic inflammation from said effector cells, after contact with the causative allergen.

[0222] The present invention further relates to the pharmaceutical composition comprising an IgE epitope-like peptide of the present invention, and optionally at least one pharmaceutically acceptable excipient.

[0223] The at least one pharmaceutically acceptable excipient may be any suitable pharmaceutically acceptable excipient known in the art.

[0224] The pharmaceutical composition may be adapted for any suitable route of administration, but preferably is adapted for any of the following routes of administration: oral administration, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, intravenous infusion.

[0225] The present invention further provides methods for treatment of peanut allergy, for prevention of systemic adverse events during peanut immunotherapy and/or for prevention of allergic reaction during medical interventions with peanut allergen or peanut extracts, said methods comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a patient in need thereof.

[0226] The present invention further provides methods for treatment of peanut cross-reactive allergies, for prevention of systemic adverse events during cross-reactive nut immunotherapy and/or for prevention of allergic reaction during medical interventions with cross reactive allergens or allergen extracts, said methods comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a patient in need thereof.

[0227] Additionally the invention provides a general method of using IgE epitope-like peptides in the treatment of clinically relevant allergies, for prevention of systemic adverse reactions during specific immunotherapy and/or for prevention of allergic reaction during medical interventions, where IgE epitope-like peptides bind to antigen-binding site of allergen specific IgEs, do not cross-link allergen specific IgEs on effector cells of allergic patients and do not induce activation and release of mediators of allergic inflammation from said effector cells. By binding to antigen-binding site IgE epitope-like peptides prevent the IgE cross-linking by the causative allergen and thus prevent the allergen induced effector cell activation and release of mediators of allergic inflammation in allergic patients.

EXAMPLES

Example 1

[0228] IgE Epitope-Like Peptides

[0229] Affinity-purified rabbit IgG specific to Ara h 2 was immobilized onto protein G or protein A coupled to Dynabeads (Thermo Fisher Scientific), incubated in 0.5% BSA (Sigma-Aldrich, St. Louis, Mo., USA) in PBST for 30 min and used as a target in biopanning. Linear dodecamer, linear heptamer and cyclic heptamer random peptide phage libraries (New England Biolabs, Ipswich, Mass., USA) were screened according to the manufacturer's instructions. Briefly, 10.sup.11 plaque forming units were incubated with 15 μL of immobilized Dynabeads for 1 h at room temperature. Unbound phages were washed with PBST. Bound phages were eluted either specifically with Ara h 2 or non-specifically with 0.1 M glycine buffer with pH 2.2 and amplified in the Escherichia coli K12 ER2738 to be used in the next rounds of biopanning. After the third round, E. coli were infected with the eluted phages and grown on LB plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside and isopropyl β-D-1-thiogalactopyranoside. The plates were incubated overnight at 37° C. Individual plaques were picked for each type of phage library used and amplified. The DNA from the phage clones were isolated and sequenced to determine the displayed peptide primary structure. SEQ ID, name and amino acid sequence of representative IgE epitope-like peptides identified by screening peptide libraries are shown in FIG. 1 A. General structure of IgE epitope-like peptides is shown on FIG. 1B.

Example 2

[0230] Binding of the IgE Epitope-Like Peptides to Anti Ara h 2 Rabbit Antiserum

[0231] Individual IgE epitope-like peptides expressed as fusion proteins with pill protein on the surface of filamentous phage were evaluated for their binding to anti Ara h 2 rabbit antiserum (INDOOR Biotechnologies, Charlottesville, Va., USA) in duplicates with ELISA. The wells of the microtiter plates were coated overnight at 4° C. with Ara h 2 rabbit antiserum diluted to 1:1000 in 0.1 M NaHCO3 (pH 8.6). The wells were blocked with 5% skim milk in PBS and washed with PBST. Next, 100 μL equal amount (5×10.sup.10 pfu/well) of phages was added and incubated for 1 h at room temperature. For detection, mice anti-M13 monoclonal antibodies conjugated with HRP (GE Healthcare, Little Chalfont, UK) diluted to 1:5000 were incubated for 1 h at room temperature and the TMB Super Tracker (ImmunoO4) with added 0.006% H.sub.20.sub.2 was used as a substrate. The reaction was terminated with 2 M H.sub.2SO.sub.4, and the absorbance was measured at 450 nm with a microtiter plate reader. The results show, that IgE epitope-like peptides bind to anti Ara h 2 rabbit antiserum.

[0232] Purified phage clones (5×10.sup.10 pfu/well) and Ara h 2 (0.25 μg/well) were added to microtiter plate wells coated with Ara h 2 rabbit antiserum, blocked (as above) and allowed to compete for the target antibody binding site. After 1 h of incubation at room temperature, anti-M13 monoclonal antibodies conjugated with HRP (GE Healthcare) diluted to 1:5,000 were added. Detection was performed as above. The results show that individual IgE epitope-like peptides expressed as fusion proteins with pIII protein on the surface of filamentous phage compete for the same binding site with allergen.

Example 3

[0233] Binding of IgE Epitope-Like Peptides to IgE of Peanut Allergic Patients

[0234] Three selected IgE epitope-like peptides (ELP 2, ELP 5, ELP 6) were extracted as recombinant fusions with bacteriophage minor coat protein pIII from the periplasm of host bacteria with osmotic shock. Two target-unrelated control peptides also fused to protein pIII (binders to streptavidin and human leptin) were extracted in the same way and served as negative controls (C1, C2). Briefly, E. coli host bacteria were infected with individual phage clones and grown for 2 hours at 37° C. with agitation. Bacteria pellets were spun down at 5000×g for 10 min and resuspended in 1 ml of an ice-cold solution consisting of 20% sucrose, 200 mM Tris-HCl pH 8.0 and 1 mM EDTA supplemented with protease inhibitor cocktail (EZBlock™, BioVision, San Francisco, USA) at a dilution of 1:200. After 1 h incubation on ice with occasional stirring supernatants were harvested by centrifugation at 12000×g for 20 minutes at 4° C. Resulting periplasmic extracts containing peptide-pII fusion proteins were concentrated (4-5 fold) and sucrose buffer exchanged for PBS by ultrafiltration with 10 kDa cut-off membrane (Microsep Advance Centrifugal Device, Pall Corporation, New York, USA). For immunoprecipitation of each selected peptide, 20 μg of affinity purified rabbit IgG against Ara h 2 (INDOOR Biotechnologies, Charlottesville, Va., USA) were covalently coupled to 1 mg of Dynabeads M-280 Tosylactivated™ (Thermo Fisher Scientific) according to manufacturer's protocol. Control peptides were immunoprecipitated in the same way using 10 μg of anti-human leptin antibody (R and D Systems Cat #MAB398 RRID:AB_2136056) coupled to 1 mg of Dynabeads M-280 Tosylactivated™ in the case of peptide with the affinity toward human leptin and 0.5 mg of streptavidin-coupled beads (Dynabeads™ MyOne™ Streptavidin T1, Thermofisher Scientific) in the case of peptide with the affinity towards streptavidin. After the concentrated periplasmic extracts containing pill-fusion proteins were incubated with respective beads for 1 h at room temperature under agitation, the beads were washed three times with 0.01 M Na-phosphate (pH 7.4) and captured fusion proteins eluted in 25 μl glycine-HCl (pH 2.5) following immediate neutralization with 1 M Tris (pH 8.0).

[0235] Immunodot assay: 2 μl of each sample containing 200 μg of IgE epitope-like peptides (ELP 2, ELP 5, ELP 6)-pIII fusion proteins were spotted onto a 0.45 μm nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% skimmed milk in Tris-buffered saline/0.05% Tween 20 (TBST) for 3 hours at room temperature. Following washing, the membrane was incubated with a sera pool (patients 1-9) or with individual serum (patients 1 to 12) diluted to 1:10 in 0.05% TBST overnight at 4° C. Membranes were washed three times with 0.1% TBST and incubated with HRP-conjugated goat anti-human IgE antibodies (RRID: AB_2535570) diluted to 1:2000 in 1% BSA/0.1% TBST for 2 h at room temperature. The reactive dots were visualized with CCD image analysis system (G-Box, Syngene, United Kingdom) after 5 minutes incubation in SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). The results show that IgE epitope-like peptides (ELP 2, ELP 5, ELP 6) bind to patients' IgE (FIG. 3).

Example 4

[0236] IgE Epitope-Like Peptides Showing No Allergenic Activity

[0237] Basophil Activation Assay (BAT) was performed on the heparinized whole blood incubated with basophil stimulation buffer with IL-3 (Buhlmann, Switzerland) containing fMLP (50 μg/ml; Sigmal Aldrich, Germany), anti-FceRI mAbs (550 ng/ml; Buhlmann), peanut extract (33.3-0.333 ng/ml), Ara h2 (3.53-0.353×10-7 μg/ml; Indoor Biotechnologies, UK), and individual synthetic IgE epitope-like peptides (L12-N14, L7-N40 and L7-N48) (1-0.001 mg/ml; EZBiolab, CA USA) at 37° C. for 15 minutes. Degranulation was stopped by chilling on ice, after which anti-CD63, anti-CD123, and anti-HLA-DR mAb (BD Biosciences, USA) were added and incubated for 20 minutes. Finally, whole blood probes were lysed, washed, fixed, and analyzed within 2 hours on a FACSCanto II flow cytometer (BD Biosciences). IgE epitope-like peptides showed no allergenic activity, compared to peanut extract or Ara h 2 (FIG. 4).

Example 5

[0238] Blocking of Allergenic Activity of Ara h 2 by IgE Epitope-Like Peptides

[0239] Inhibition of Ara h 2 induced basophil activation by IgE epitope-like peptides. Basophil Activation Assay (BAT) was performed on the heparinized whole blood incubated with mixture of all three free synthetic IgE epitope-like peptides (L12-N14, L7-N40 and L7-N48) in the final concentration 50 μg/ml for 15 minutes at 37° C. Afterwards, Ara h2 in the final concentration of 3.5×10.sup.−2-3.5×10.sup.−5 μg/ml was added and samples were incubated for another 15 min at 37° C. Degranulation was stopped by chilling on ice, after anti-CD64/anti-CD123/anti-HLA-DR mAb were added and incubated for 20 min. Finally, whole blood probes were lysed, washed, fixed, and analyzed within 2 hours on a FACSCanto II flow cytometer (BD Biosciences). The results of BAT inhibition with IgE epitope-like peptides were compared to the results of BAT at same final concentrations of Ara h2. The results show that IgE epitope-like peptides markedly reduce allergenic activity of Ara h 2 (FIG. 5).

REFERENCES

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