U.S. patent application number 16/231149 was filed with the patent office on 2019-05-09 for method for the production of hydrolyzed allergens.
This patent application is currently assigned to BIOTECH TOOLS S.A.. The applicant listed for this patent is BIOTECH TOOLS S.A.. Invention is credited to Marie-Ange Benoit, Laetitia Frisch, Thierry Legon, Gael Placier.
Application Number | 20190134199 16/231149 |
Document ID | / |
Family ID | 44881614 |
Filed Date | 2019-05-09 |
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United States Patent
Application |
20190134199 |
Kind Code |
A1 |
Placier; Gael ; et
al. |
May 9, 2019 |
METHOD FOR THE PRODUCTION OF HYDROLYZED ALLERGENS
Abstract
A pharmaceutical composition of hydrolyzed allergens and its use
for treating, curing or preventing allergic reactions. The
hydrolyzed allergens are prepared by: a) extracting a source of
allergens comprising allergenic proteins to form an extract, b)
purifying the extract to remove non-protein components to form a
purified extract, c) denaturing the purified extract with a first
denaturing agent to form a purified denatured extract, d) refining
the purified denatured extract to remove impurities to form a
refined denatured extract, e) denaturing the refined denatured
extract with a second denaturing agent to form denatured allergen
mixture, and f) hydrolyzing the denatured allergen mixture to form
the hydrolyzed allergens.
Inventors: |
Placier; Gael; (Brussels,
BE) ; Frisch; Laetitia; (Pecq, BE) ; Legon;
Thierry; (Korbeek Lo, BE) ; Benoit; Marie-Ange;
(Vedrin, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOTECH TOOLS S.A. |
Brussels |
|
BE |
|
|
Assignee: |
BIOTECH TOOLS S.A.
Brussels
BE
|
Family ID: |
44881614 |
Appl. No.: |
16/231149 |
Filed: |
December 21, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14125036 |
Apr 16, 2014 |
10195276 |
|
|
PCT/EP2012/061404 |
Jun 15, 2012 |
|
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16231149 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/04 20180101;
C07K 14/415 20130101; C12P 21/06 20130101; A61K 45/06 20130101;
C07K 14/43531 20130101; A61K 39/35 20130101; A61P 37/08 20180101;
A61P 37/02 20180101; A61K 39/00 20130101 |
International
Class: |
A61K 45/06 20060101
A61K045/06; A61K 39/35 20060101 A61K039/35; C07K 14/435 20060101
C07K014/435; A61K 39/00 20060101 A61K039/00; C07K 14/415 20060101
C07K014/415; C12P 21/06 20060101 C12P021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 15, 2011 |
EP |
11170031.6 |
Claims
1. A pharmaceutical composition comprising hydrolyzed allergens
obtained by a) extracting a source of allergens comprising
allergenic proteins to form an extract, b) purifying the extract to
remove non-protein components to form a purified extract, c)
denaturing the purified extract with a first denaturing agent at a
pH of 7.0 to 11.0 to form a purified denatured extract, d) refining
the purified denatured extract to remove impurities to form a
refined denatured extract, e) denaturing the refined denatured
extract with a second denaturing agent at a pH of 1.0 to 7.0 to
form denatured allergen mixture, f) hydrolyzing the denatured
allergen mixture to form the hydrolyzed allergens, and g) purifying
the hydrolyzed allergens to remove peptides with molecular weights
above 10,000 Da and below 1,000 Da, wherein 70% or more of the
peptides are between 10,000 Da and 1,000 Dawherein the first
denaturing agent is a mixture of a chaotropic agent and a reducing
agent and wherein the second denaturing agent is a mixture of a
chaotropic agent and a reducing agent.
2. The pharmaceutical composition of claim 1, further additionally
at least one substance selected from the group of nucleoside
triphosphates, nucleoside diphosphates, nucleoside monophosphates,
nucleic acids, peptide nucleic acids, nucleosides or analogs
thereof, immunosuppressive cytokines, compounds inducing expression
of immunoproteasomes, 1,25-dihydroxyvitamin D3 or analogs thereof,
lipopolysaccharides, endotoxins, heat shock proteins, thioredoxin
with either NADPH or NADP-thioredoxin reductase, reducing agent,
dithiothreitol, adrenergic receptor agonists such as salbutanol,
adrenergic receptor antagonists such as butoxamine, compounds that
regulate the expression of the adhesion molecule ICAM-1,
N-acetyl-L-cysteine, y-L-glutamyl-L-cysteinyl-glycine (reduced
L-glutathione), alpha-2-macroglobulins, inducers for Foxp3 gene
expression, flavonoids, isoflavonoids, pterocarpanoids, stilbenes
such as resveratrol, tachykinin receptor antagonists, chymase
inhibitors, vaccine adjuvant or immunomodulators like CpG, aluminum
hydroxide, calcium phosphate, TLR-4 agonists (i.e. MPL) and TLR-9
agonists or tolerogenic adjuvant like zymosan, beta1,3-glucan,
regulatory T-cell inducer, a muco-adhesive agent for attaching the
particle to the intestinal mucosal lining such as a plant lectin,
zinc, zinc salts, polysaccharides, vitamins and bacterial lysates
or particles displaying surface linked antibodies.
3. The pharmaceutical composition according to claim 1, for oral,
subcutaneous, nasal, epicutaneous or intralymphatic
administrations, for sublingual drug delivery, or for enteric drug
delivery.
4. The pharmaceutical composition of claim 1, wherein extracting is
performed in a solution comprising no salt or a salt selected from
carbonate, bicarbonate, phosphate, acetate, TRIS and HEPES, wherein
extracting is performed with an extraction medium.
5. The pharmaceutical composition of claim 1, wherein the purifying
and/or refining comprises one or more of an ion exchange
chromatography step, a gel filtration or size exclusion
chromatography step, a hydrophobic interaction chromatography step,
a pseudo affinity or affinity chromatography step.
6. The pharmaceutical composition of claim 1, wherein denaturing is
performed with a denaturing agent selected from the group of
chaotropic agents, reducing agents and mixtures thereof.
7. The pharmaceutical composition according to claim 6, wherein the
first and/or second denaturing agent comprises urea at a
concentration of more than 4 M, and guanidinium chloride at a
concentration above 3 M.
8. The pharmaceutical composition of claim 1, wherein hydrolyzing
is performed with an enzyme.
9. The pharmaceutical composition of claim 1, wherein the removal
of the peptides is performed by size exclusion chromatography
and/or by ultrafiltration, wherein the size exclusion
chromatography is performed in the presence of chaotropic
agents.
10. The pharmaceutical composition of claim 1, wherein the sources
of allergens are peanuts.
11. The pharmaceutical composition of claim 8, wherein the
hydrolyzing is performed in the presence of a chaotropic agent and
a reducing reagent.
12. The pharmaceutical composition of claim 10, wherein the source
of allergens is a mixture of at least two
species/subspecies/varieities/hybrids and/or transgenic
peanuts.
13. The pharmaceutical composition of claim 12, wherein the peanuts
are selected from the Arachis genus.
14. The pharmaceutical composition of claim 13, wherein the peanuts
selected from the Arachanis genus are hypogaea or fastigiata.
15. The pharmaceutical composition of claim 1, wherein denaturing
is performed with a denaturing agent selected from the group of
urea, guanidinium chloride, dithiotreitol, thiglycerol,
.beta.-mercaptoethanol, tris (2-carboxyethyl) phosphine (TCEP), and
mixtures thereof.
16. The pharmaceutical composition of claim 1, wherein extracting
is performed in a solution comprising phosphate, purifying or
refining includes a precipitation step, the precipitation step is
performed with a solution comprising trichloroacetic acid,
denaturing is performed with a combination of urea and
dithiotreitol, the hydrolyzing is performed with pepsin, the
removal is performed by size exclusion chromatography in the
presence of chaotropic agents, and the source of allergens are
peanuts from Arachis hypogaea.
17. The pharmaceutical composition of claim 1, wherein the second
denaturing agent is tris(2-carboxyethyl)-phosphine (TCEP).
18. A method for the treatment, cure or prevention of allergic
reactions comprising administering the pharmaceutical composition
of claim 1.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 14/125,036, filed Apr. 16, 2014, which is a national stage
entry of International Application No. PCT/EP2012/061404, filed
Jun. 15, 2012, which claims priority to European Application No.
11170031.6, filed Jun. 15, 2011, each of which is incorporated by
reference in its entirety.
[0002] The present invention is related to a method for the
production of hydrolyzed allergens, more precisely to hydrolyzed
allergens with a reduced allergenicity.
[0003] A general method for the production of hydrolyzed allergens
is known from WO 2008/000783 A1. The method disclosed therein
comprises the steps of extracting, purifying, denaturing and
hydrolyzing of different natural materials like milk, venom, egg,
weed, grass, tree, shrub, flower, vegetable, grain, fungi, etc.
[0004] However, advanced experimental studies by using the known
method show that hydrolyzing of allergen proteins is complex and
requires further procedural steps in order to hydrolyze the
allergen proteins completely.
[0005] It is an object of the present invention to overcome at
least some of the drawbacks of prior art, especially to provide
antigens from natural allergens with a significantly reduced
capability to trigger allergenic reactions compared to the crude
allergen extract, but able to stimulate B-cells and T-cells.
[0006] The object is solved by a method for the production of
hydrolyzed allergens from allergens comprising the steps of: [0007]
a) extracting a source of allergens comprising allergenic proteins
to form an extract, [0008] b) purifying the extract to remove
non-protein components to form a purified extract, [0009] c)
denaturing the purified extract with a first denaturing agent to
form a purified denatured extract, [0010] d) refining the purified
denatured extract to remove impurities to form a refined denatured
extract, [0011] e) denaturing the refined denatured extract with a
second denaturing agent to form denatured allergen mixture, and
[0012] f) hydrolyzing the denatured allergen mixture to form the
hydrolyzed allergens.
[0013] Surprisingly, it could be shown that applying two steps of
denaturation, allergen proteins can be completely hydrolyzed.
[0014] "Extracting" as used herein is a treatment of an allergen
source with an extraction medium including water, buffer or organic
solvents to separate soluble ingredients from a non-soluble
residue. The use of aqueous systems (comprising at least 50%
H.sub.2O) is preferred.
[0015] "Denaturating" as used herein is a process in which the
proteins lose their quaternary, tertiary and secondary structure,
especially this term refers to the treatment with one or several
denaturing agents.
[0016] Step a) is an extracting step.
[0017] Extraction is preferably performed with aqueous solutions.
Suitable salts are salts such as, but not restricted to disodium
hydrogen phosphate, carbonate, bicarbonate, phosphate, acetate,
TRIS and HEPES.
[0018] Also in contrast to many other extraction methods, it is
preferred that the amount of extraction medium is comparatively
large, i.e. at least 20 times the weight of the source of
allergens, preferably 100 times the weight or more.
[0019] The extract is designated in the figures and examples as
crude protein extract.
[0020] Step b) is a purification step.
[0021] After extraction of the source of allergens, i.e. step a),
the extract is purified (step b) to remove non-protein components
such as sugars, lipids, nucleic acids and the like.
[0022] Purifying of the extract may be performed by one or more of
the following: [0023] ion exchange chromatography steps (including
anion exchange chromatography and cation exchange chromatography),
[0024] size exclusion chromatography steps (also called gel
filtration), [0025] precipitation steps, [0026] hydrophobic
interaction chromatography steps, [0027] pseudo-affinity and
affinity chromatographies and/or [0028] diafiltration.
[0029] In a preferred embodiment ion exchange chromatography is
used wherein in case of a cation exchanger the loading solution has
a pH between the pKa of the acidic function of the cation exchanger
and the pKa of the protein having the lowest pKa of the proteins in
the extract. In case of an anion exchanger the pH is between the
pKa of the basic function of the anion exchanger and the pKa of the
protein having the highest pKa of the proteins constituting the
extract.
[0030] Through this method all proteins bind to the ion exchanger
while the neutral impurities and the impurities with the same
charge as the ion exchange resin will be removed.
[0031] Alternatively, the allergen proteins can be precipitated by
the addition of at least 50% (w/v) ammonium sulphate, more
preferably of at least 90% (w/v) ammonium sulphate. In a preferred
embodiment, the precipitation can also be performed by the addition
of at least 2% (w/v) trichloroacetic acid (TCA), preferably 5%
(w/v), more preferably of at least 10% (w/v) of TCA.
[0032] Typically, several different proteins are present in the
protein fraction of the purified extract. The relative amounts of
the proteins in the purified extract can be easily measured using
methods like SDS-PAGE followed by densitometry.
[0033] Step c) is the first denaturation step.
[0034] As a next step (step c)) a denaturation is performed. The
denaturing agent is preferably a chaotropic agent, a reducing agent
or a mixture thereof. Suitable chaotropic agents are for example
urea and guanidinium chloride. Typical reducing agents are for
example dithiotriethol, .beta.-mercaptoethanol, thio-glycerol, tris
(2-carboxyethyl) phosphine (TCEP) and mixtures thereof.
[0035] A typical pH value is between 7.0 and 11.0, preferably
between 7.0 and 10.0, more preferably between 7.0 and 9.0 for the
first denaturation step.
[0036] Denaturing is preferably performed for at least 15 minutes,
preferably at least 30 minutes and more preferably at least 60
minutes at a temperature between 15 and 40.degree. C., preferably
between 20 and 37.degree. C.
[0037] In a preferred embodiment the reducing agent used for the
denaturation is DTT (Dithiothreitol).
[0038] A suitable concentration of urea is 3 M or more, preferably
4 M or more. A suitable concentration of guanidinium is preferably
2 M, preferably 3 M or more.
[0039] Step d) is a purification step.
[0040] As step d) a further purification is performed. In a
preferred embodiment gel filtration or diafiltration procedures
will be applied. Use of chromatography, especially size exclusion
chromatography is preferred. It is believed that by step d) further
purification impurities which were not covalently bound to the
protein components of the allergens and have been separated from
the proteins by step c) can now be removed from the
preparation.
[0041] Step e) is the second denaturation step.
[0042] After the step d), a second denaturing step is performed
(step e)). For this denaturing step a second denaturing agent is
used which can be the same or have different composition from step
c). In a preferred embodiment the reducing agent used for the
second denaturation step is TCEP.
[0043] It is preferred that the pH for the second denaturing step
is set between 1.5 and 9.0. Preferably the pH is close to the
optimum pH activity of the selected enzyme used in step f). In a
preferred embodiment the pH is lower than 7.0 or lower than 5.0 or
lower than 3.0 but preferably higher than 1.0. Denaturing is
preferably performed for at least 15 minutes, preferably at least
30 minutes and more preferably at least 60 minutes at a temperature
between 15 and 40.degree. C., preferably between 20 and 37.degree.
C.
[0044] The next step (step f)) is a hydrolyzing step.
[0045] The hydrolyzing step is typically performed with an enzyme.
Suitable enzymes are for example pepsin, trypsin, and/or
chymotrypsin. The hydrolyzing step can be performed in the presence
of a chaotropic agent, preferably urea or guanidinium chloride,
too. During hydrolyzing, the concentration of urea and guanidinium
chloride should be below 4 M, preferably below 3 M. The hydrolyzing
step can also be performed in presence of a reducing agent,
preferably TCEP. During hydrolysis, the concentration of TCEP is
preferably below 10 mM. Preferably, pepsin is used. More
preferably, pepsin at a pH range of 1.0-3.0 is used.
[0046] Step g) is a purification and selection of the hydrolyzed
allergens.
[0047] In a further step (step g), the hydrolyzed allergens can be
purified to form a purified hydrolysate, wherein specifically
fragments of peptides, i.e. typically fragments with molecular
weights below 1.000 Da, and, for instance, nonhydrolyzed fragments
of proteins and/or peptides with molecular weights above 10.000 Da
are removed. The peptides of the purified hydrolysate, therefore,
comprise peptides with molecular weights between 1.000 and 10.000
Da. Preferably, less than 10% of the peptides have a molecular
weight above 10.000 Da and less than 20% of the peptides have a
molecular weight below 1.000 Da so that 70%, or more preferably 80%
of the peptides are between 10.000 Da and 1.000 Da.
[0048] Suitable methods for removing large or small peptides are
ultrafiltration and size exclusion chromatography. Again this size
exclusion chromatography may be performed in the presence of
chaotropic agents, for example urea, guanidinium chloride, ethylene
glycol, isopropanol and mixtures thereof.
[0049] Preferably, the purification of the hydrolyzed peptides is
performed by size exclusion chromatography and/or by
ultrafiltration, wherein the size exclusion chromatography step is
preferably performed in the presence of chaotropic agents,
preferably selected among urea, guanidinium chloride, ethylene
glycol, isopropanol and mixtures thereof.
[0050] One advantage of the hydrolysate is that the peptides are
the digestion result of purified denatured proteins. They have a
reduced potency to induce immediate allergic reactions and
co-inflammatory reactions as well.
[0051] In a preferred embodiment of the present invention, the
source of allergens is a natural source comprising milk, venom,
egg, weed, grass, tree, shrub, flower, vegetable, grain, fungi,
fruit, berry, nut, seed, bean, fish, shellfish, seafood, meat,
spices, insect, mite, mould, animal, pigeon tick, worm, soft coral,
animal dander, nematode, Hevea brasiliensis and mixtures
thereof.
[0052] Preferred allergens used in this invention are especially
grass pollen, house dust mite, birch pollen and peanuts.
[0053] Alternatively, synthetic sources of allergens as starting
materials can be used. Synthetic sources of allergens means
biotechnological produced proteins like recombinant proteins and/or
genetically modified organisms.
[0054] In a more preferred embodiment of the present invention,
peanuts and House Dust Mites (purified mites) are the source of
allergens.
[0055] Preferably, the source comprises a mixture of allergens.
[0056] Preferably, the peanuts are selected among the Arachis
genus, preferably from hypogaea species, more preferably from
hypogaea and fastigiata. Sub-species comprise Virginia, Spanish,
Valencia varieties and/or hydrids such as Runner or even transgenic
peanuts obtained by genetic engineering.
[0057] Preferably, a mixture of at least 2, preferably 3
species/subspecies/varieties/hybrids and/or transgenic peanuts is
used. In a preferred embodiment the red seed coat (tegument) of the
peanuts has been removed.
[0058] The hydrolyzed allergens of the present invention can be
used for the preparation of a pharmaceutical composition and/or
food composition for inducing tolerance and desensitization.
Induction of tolerance can be used to cure or prevent allergic
reactions.
[0059] The allergic reaction to be treated or prevented depends on
the source of allergens, i.e. allergy to peanuts are prevented or
treated by using allergens from peanuts, whereas allergy to grass
pollen are treated with allergens from grass pollen.
[0060] A further embodiment of the present invention is a
pharmaceutical composition comprising the hydrolyzed allergens of
the present invention. Additionally, the pharmaceutical composition
may comprise one or more of the following substances: nucleoside
triphosphates, nucleoside diphosphates, nucleoside monophosphates,
nucleic acids, peptide nucleic acids, nucleosides or analogs
thereof, immunosuppressive cytokines, compounds inducing expression
of immunoproteasomes, 1,25-dihydroxyvitamin D3 or analogs thereof,
lipopolysaccharides, endotoxins, heat shock proteins, thioredoxin
with either NADPH or NADP-thioredoxin reductase, reducing agent,
dithiothreitol, adrenergic receptor agonists such as salbutanol,
adrenergic receptor antagonists such as butoxamine, compounds that
regulate the expression of the adhesion molecule ICAM-1,
N-acetyl-L-cysteine, y-Lglutamyl-L-cysteinyl-glycine (reduced
L-glutathione), alpha-2-macroglobulins, inducers for Foxp3 gene
expression, flavonoids, isoflavonoids, pterocarpanoids, stilbenes
such as resveratrol, tachykinin receptor antagonists, chymase
inhibitors, vaccine adjuvant or immunomodulators like CpG, aluminum
hydroxide, calcium phosphate, TLR-4 agonists (i.e. MPL) and TLR-9
agonists or tolerogenic adjuvant like zymosan, beta-1,3-glucan,
regulatory T-cell inducer, a muco-adhesive agent for attaching the
particle to the intestinal mucosal lining such as a plant lectin,
zinc, zinc salts, polysaccharides, vitamins and bacterial lysates
or particles displaying surface linked antibodies.
[0061] In a preferred embodiment, the pharmaceutical composition is
prepared for subcutaneous administration, nasal administration,
epicutaneous administration, intralymphatic administration, oral
administration, for sublingual drug delivery, or for enteric drug
delivery.
[0062] One further embodiment of the present invention are purified
hydrolyzed allergens obtainable by the method of the present
invention.
[0063] A further embodiment of the present invention is the use of
trichloroacetic acid as a precipitation means for the precipitation
of allergenic proteins.
DESCRIPTION OF THE FIGURES
[0064] FIG. 1: Protein profile of peanut crude extract by SDS-PAGE.
4 to 12% Bis-Tris gel. Lane 1-5: Molecular weight markers, lane 2:
crude protein extract of Runner type (13 .mu.g), lane 3: crude
protein extract of Virginia type (13 .mu.g), lane 4: crude protein
extract of Spanish type (13 .mu.g), lane 6: crude protein extract
of peanut mix (13 .mu.g). Staining performed with Coomassie
brilliant blue R-250. Allergens: allergen 1: .+-.60 kDa; allergen
2: .+-.17 kDa; allergen 3: 2 subunits .+-.20 kDa and .+-.40 kDa;
allergen 4: .+-.37 kDa.
[0065] FIG. 2: SEC G 25 elution profile. The ratio column
volume/sample volume was 7. The resin was equilibrated with 2 M
urea, 0.1 M Tris-HCl, pH 8.0 at a flow rate of 10 ml/min. The
elution was followed by the absorbance at 280 nm.
[0066] FIG. 3: Protein profile of purified allergen extract by
SDS-PAGE. 4-12% Bis-Tris gel. Lane 1: molecular weight markers,
lane 2: purified denatured extract (13 .mu.g). Staining performed
with Coomassie brilliant blue R-250. Allergen 1: .+-.60 kDa;
allergen 2: .+-.17 kDa; allergen 3: 2 subunits .+-.20 kDa and
.+-.40 kDa; allergen 4: .+-.37 kDa.
[0067] FIG. 4: Immuno reactivity by IgE western-blot. Lane 1:
molecular weight markers, lane 2: purified denatured extract (13
.mu.g). Membrane blocked by BSA 2% (w/v). Pool of 6 patient sera
diluted to 1/500. IgE binding detected by goat anti-human IgE
peroxidase conjugate diluted to 1/1.000 and revealed by TMB
substrate. Allergen 1: .+-.60 kDa; allergen 2: .+-.17 kDa; allergen
3: 2 subunits.+-.20 kDa and .+-.40 kDa; allergen 4: .+-.37 kDa.
[0068] FIG. 5: Hydrolysis profile of proteins denatured twice by
SDS-PAGE. 4-12% Bis-Tris gel. Lane 1: molecular weight markers,
lane 2: crude protein extract (13 .mu.g), lane 3: denatured
allergen mixture (13 .mu.g), lane 4: hydrolyzed allergens (26
.mu.g). Staining performed with Coomassie brilliant blue R-250.
[0069] FIG. 6: Hydrolysis profile of proteins denatured once by
SDS-PAGE. 4-12% Bis-Tris gel. Lane 1: molecular weight markers,
lane 2: crude protein extract (13 .mu.g), lane 3: hydrolysate of
proteins denatured once (26 .mu.g), Staining performed with
Coomassie brilliant blue R-250.
[0070] FIG. 7: G 50 SEC elution profile. The column was
equilibrated with 2 M urea, 0.1 M Tris-HCl, pH 9.5 at a flow rate
of 10 ml/min. The ratio column volume/sample volume was 10. The
elution was followed by the absorbance at 280 nm.
[0071] FIG. 8: Comparison of the peptide profiles before and after
purification. The analysis was performed by SDS-PAGE. 4-12%
Bis-Tris gel. Lane 1: molecular weight markers, lane 2: crude
protein extract (13 .mu.g), lane 3: denatured allergen mixture (13
.mu.g), Lane 4: hydrolyzed allergens (26 .mu.g), lane 5: purified
hydrolysate (26 .mu.g). Staining performed with Coomassie brilliant
blue R-250.
[0072] FIG. 9: Calibration curve for HPLC analysis. 10 .mu.l of the
following standards (1 mg/ml) were injected onto the BioSep-SEC S
2000 column: CytoChrom c (12 kDa), Glucagon (3.5 kDa), 1 kDa
synthetic peptide.
[0073] FIG. 10: Size exclusion HPLC profile. Column: BioSep-SEC S
2000. Elution buffer: 50 mM Na.sub.2HPO.sub.4, 0.5% (w/v) SDS-pH
6.8. Flow rate 1 ml/min. Detection at 215 nm. 10 .mu.l of the
sample were injected.
[0074] FIG. 11: Profile of House Dust Mite proteins on 4 to 12%
Bis-Tris reducing SDS-Page at different steps of purification. Lane
1: Molecular weight markers; Lane 2: crude protein extract from
Dermatophagoides pteronyssinus; Lane 3: Protein profile obtained
after TCA precipitation; Lane 4: Purified denaturated extract
obtained after the second denaturation with TCEP; Lane 5: Profile
of denaturated proteins hydrolyzed with 0.4 Eu. Ph. U of pepsin/100
mg; Lane 6: Profile of denaturated proteins hydrolyzed with 4 Eu.
Ph. U of pepsin/100 mg. Lane 7: Profile of denaturated proteins
hydrolyzed with 16 Eu. Ph. U of pepsin/100 mg. Staining performed
with Coomassie brilliant blue R-250.
[0075] FIG. 12: Reduced allergenicity of peanut peptides. The
binding of IgE from pooled sera of peanut-allergic donors to peanut
allergens was more inhibited by preincubation with increasing
amounts of peanut intact allergens than by peanut allergen
peptides.
[0076] FIG. 13 a-d: Isotype profiles of the IgG antibody response
of Lewis rats immunized subcutaneously with peanut peptides (1
mg/injection at D0, D3, D7) emulsified with Incomplete Freund
Adjuvant (v/v). The results are expressed as the mean.+-.SD
(n=4).
[0077] FIG. 14: Splenocyte proliferation assay for Lewis rats
previously immunized subcutaneously with peanut proteins (100
.mu.g/injection) or peanut peptides (400 .mu.g or 1 mg/injection)
in response to increasing doses (from 6.25 to 100 .mu.g/ml) of
peanut peptides.
[0078] FIG. 15: Splenocyte proliferation assay for Lewis rats
previously immunized subcutaneously with peanut proteins (100
.mu.g/injection) or peanut peptides (400 .mu.g or 1 mg/injection)
in response to increasing doses (from 6.25 to 100 .mu.g/ml) of
peanut proteins.
[0079] FIG. 16: Competition ELISA for assessement of blocking
antibody activity. Peanut proteins were coated on microtitre plate.
The pooled serum of peanut allergic patients were mixed with serial
dilutions of rabbit antibodies generated to either peptides or
proteins of peanuts. After incubation, the IgE that bind to the
peanut proteins coated to the plate were detected using anti-human
IgE labeled peroxydase antibodies.
[0080] The invention is explained in more details by the following
examples.
Example 1: Peanut Allergens
Extraction of Peanut Allergens
[0081] A mix of three peanut types (Arachis hypogaea species
Runner, Virginia and Spanish were peeled, grinded and mixed. A 2%
(w/v) of the mix of peanuts was added to sodium phosphate (12.5 mM)
and incubated 1 h under stirring at room temperature. The solution
was then clarified and filtrated by adding Celite at 2% (w/v) and
passing through a 0.45 .mu.m filter. This sample constitutes the
crude protein extract.
[0082] The presence of allergens in the crude protein extract was
analyzed by Western-Blot using peanut allergic patient sera.
[0083] As shown in FIG. 1, there are four major allergens in the
crude protein extract (allergen 1, allergen 2, allergen 3 and
allergen 4).
Purification of Peanut Allergen Proteins
[0084] The allergen extract was purified by: [0085] Trichloroacetic
acid precipitation
[0086] This step was performed at room temperature (20 to
25.degree. C.).
[0087] 10% (w/v) trichloroacetic acid was added to the product
under stirring. Then, the precipitated extract was centrifuged
during 15 minutes at 10.000 g. The supernatant was carefully
discarded.
[0088] First Denaturation
[0089] The pellets were resuspended at 25 mg/ml in 8 M Urea, 0.1 M
Tris-HCl, pH 8.0 and 80 mM DTT were added. The solution was
incubated at 37.degree. C. for 1 h.
[0090] Size Exclusion Chromatography on a G25 Resin Column (Fine
Sephadex from GE Healthcare)
[0091] The purified denatured extract was immediately loaded on the
column and the proteins were eluted with 2 M Urea, 0.1 M Tris-HCl,
pH 8.0.
[0092] The presence of proteins was followed by the absorbance at
280 nm. The fractions of interest were pooled to constitute the
refined denatured extract.
[0093] FIG. 2 illustrates the SEC G25 elution profile followed by
the absorbance at 280 nm.
[0094] The refined denatured extract was further analyzed by
SDS-PAGE and by Western Blotting using peanut allergic patient
sera.
[0095] As shown in FIG. 3, the four major allergens visualized in
the extract by SDS-PAGE (cf. FIG. 1) are present in the purified
denatured extract.
[0096] FIG. 4 shows that all proteins and in particular the four
major allergens are recognized by peanut allergic patient sera and
then visualized with anti-human IgE antibodies.
[0097] Second Denaturation:
[0098] 8 M urea and 40 mM TCEP were added to the refined denatured
extract. Then, the pH was adjusted to 2.5. The solution was
incubated at 37.degree. C. for 1 h.
Hydrolysis of the Denatured Peanut Allergens
[0099] The denatured allergens were hydrolyzed using the following
protocol:
[0100] The denatured allergen mixture were diluted 4-fold with 10
mM HCl and acidified with HCl 6 N to pH 2.0. The protein hydrolysis
was performed with 16 Eu.Ph.U of pepsin for 100 mg of proteins at
37.degree. C., during 2 h. The hydrolysis was then stopped by
raising the pH to 10.0 with NaOH solution.
[0101] FIG. 5 shows a comparison between the crude protein extract
(lane 2), the denatured allergen mixture (lane 3) and the
hydrolyzed allergens (lane 4). It can be seen, proteins denatured
twice are almost totally hydrolyzed since only one residual peptide
band above 10 kDa is visualized on the profile.
[0102] In comparison thereto, FIG. 6, especially lane 3, shows the
case of proteins denatured once. Three residual proteins are
visualized on the profile of the corresponding hydrolysate. This
illustrates the benefit of the double denaturation since the
hydrolysis is less efficient when proteins are denatured only one
time.
Purification of Hydrolyzed Peanut Allergens
[0103] In order to eliminate the peptides with a MW.gtoreq.10.000
Da and MW.ltoreq.1.000 Da, the hydrolyzed allergens were purified
by: [0104] Size exclusion chromatography on G50 resin (fine
Sephadex from GE Healthcare). After increasing pH, the hydrolyzed
allergens were rapidly loaded on the G50 column. The peptides were
eluted with 2 M Urea, 0.1 M Tris-HCl, pH 9.5. The elution was
followed by the absorbance at 280 nm. The fractions containing the
peptides (MW.ltoreq.10 kDa) were pooled as shown in FIG. 7. [0105]
Diafiltration on 1 kDa membrane (ultrafiltration cassette Omega PES
from PALL). The peptides were concentrated 25-fold, diafiltrated
against 10 volumes of 50 mM sodium phosphate at pH 7.6 and finally
concentrated 2-fold. This sample constitutes the purified
hydrolysate.
[0106] The purified hydrolysate was analyzed by SDS-PAGE (see FIG.
8). The profile (lane 5) shows that there are no residual proteins
with molecular weights above 10 kDa.
[0107] The efficiency of the purification was controlled by size
exclusion HPLC. A BioSep-SEC S2000 column was equilibrated with 50
mM Na.sub.2HPO.sub.4, 0.5% (w/v) SDS, pH 6.8 at a flow rate of 1
ml/min. The peptides were detected at 215 nm.
[0108] The 10 kDa and 1 kDa limits were calculated from a
calibration curve as exemplified in FIG. 9.
[0109] As shown in FIG. 10, peptides with molecular weights between
1.000 Da and 10.000 Da represent about 80% of all peptides in the
purified hydrolysate.
Example 2: Allergens from House Dust Mite Dermatophagoides
Pteronvssinus
Protein Extraction of House Dust Mite
[0110] Proteins from House Dust Mite were extracted by incubation
in Phosphate Buffer Saline pH 7.4 during 1 h at room temperature
under stirring. The solution was clarified and filtrated by adding
Celite at 2% (w/v) and passing through a 0.45 .mu.m PVDF filter.
This sample constitutes the crude protein extract.
[0111] The crude protein extract seems to show the major allergens
(Derp1, Derp2) which can be localised according to their molecular
weight (25 kDa and 14 kDa respectively).
Purification of Allergen Proteins from House Dust Mite
[0112] The purification was performed by: [0113] Trichioracetic
Acid Precipitation
[0114] 10% (w/v) trichloracetic acid was added to the crude protein
extract under stirring for 5 min at room temperature. The proteins
were collected by centrifugation during 20 min at 10.000 g.
[0115] First Denaturation
[0116] After elimination of the supernatant, the pellet was
resuspended in 8 M urea, 0.1 M Tris pH 7-8. The solution was
incubated for 1 h at 37.degree. C. after pH adjustement to 7.5 and
addition of 80 mM DTT.
[0117] Size Exclusion Chromatography on G25 Resin Column
[0118] The proteins from the denaturated extract were loaded on the
column, and eluted with 2 M Urea, 0.1 M NaCl pH 9.0.
[0119] The presence of proteins was monitored by the measurement of
the absorbance at 280 nm.
[0120] Second Denaturation
[0121] The denaturation occurred by incubation at 37.degree. C. for
1 h in 4 M urea, 0.1 M NaCl and 40 mM TCEP with the pH adjusted to
2.5.
Hydrolysis of the Denaturated Allergens for House Dust Mite
[0122] The denaturated protein mixture was previously diluted
2-fold with 10 mM HCl and acidified with HCl 6N to pH 2.0. The
hydrolysis of proteins was conducted with 16 Eu.Ph.U of pepsin per
100 mg for 1 h at 37.degree. C.
[0123] FIG. 11 shows a profile of House Dust Mite proteins on 4 to
12% Bis-Tris reducing SDS-Page at different steps of purification.
[0124] Lane 1: Molecular weight markers; [0125] Lane 2: crude
protein extract from Dermatophagoides pteronyssinus; [0126] Lane 3:
Protein profile obtained after TCA precipitation; [0127] Lane 4:
Purified denaturated extract obtained after the second denaturation
with TCEP; [0128] Lane 5: Profile of denaturated proteins
hydrolyzed with 0.4 Eu.Ph.U of pepsin/100 mg; [0129] Lane 6:
Profile of denaturated proteins hydrolyzed with 4 Eu.Ph.U of
pepsin/100 mg. [0130] Lane 7: Profile of denaturated proteins
hydrolyzed with 16 Eu.Ph.U of pepsin/100 mg. Staining performed
with Coomassie brilliant blue R-250.
Example 3
[0131] With a method similar to example 2, hydrolyzed allergens of
grass and birch pollen were prepared.
Example 4: Peanut Peptides: Allergenicity, Immunogenicity and
Blocking Potential of Specific Antibodies
[0132] The allergenicity of peanut peptides was investigated by
analysis of their in vitro IgE-binding properties by an ELISA
inhibition assay. [0133] T cell proliferation, specific IgG-titres
following rat immunization were used to address their
immunogenicity. [0134] The blocking activity of the antibodies
generated to peanut peptides was assessed by a competition ELISA.
Allergenicity of Peanut Peptides: Reduced Binding of IgE from Serum
of Allergic Patients
[0135] Peanut peptides exhibit reduced allergenicity in vitro as
demonstrated by an ELISA inhibition assay.
[0136] The principle of the ELISA inhibition assay was to measure
the decrease of the binding to peanut proteins of IgE from serum of
allergic patients previously incubated with increasing amounts of
either peptides or proteins from peanuts.
[0137] Maxisorp 96 well microtitre plates were coated with 0.8
.mu.g/ml of peanut proteins in 0.1M carbonate-bicarbonate Buffer pH
9.6 overnight at 4.degree. C. After blocking for 1 h at 37.degree.
C., wells were incubated overnight at 4.degree. C. with 100 .mu.l
of mixtures of the serum pool (1/50 dilution) of peanut allergic
patients previously treated (1 h at 37.degree. C.) with serial
dilutions of peanut proteins (range: 1.25 .mu.g/ml to 63.5
.mu.g/ml) or peanut peptides (range: 50 .mu.g/ml to 20 ng/ml).
After washings, wells were incubated with peroxidase-labelled
anti-human IgE antibodies, and developped by incubating with 100
.mu.l of TMBS substrate. The reaction was stopped with 100 .mu.l of
1M H.sub.3PO.sub.4 and the optical density values were measured at
450-650 nm. The percentage of inhibition of IgE binding achieved by
preincubation in presence of peanut peptides or peanut proteins was
calculated as follow:
% inhibition=100-[[OD of the inhibed sample/mean OD of positive
controls].times.100]
[0138] Positive controls were 50 .mu.l of pooled human serum
diluted 1/25 mixed with 50 .mu.l of rabbit preimmune serum
(n=10).
[0139] FIG. 12 shows a reduced allergenicity of peanut peptides.
The binding of IgE from pooled sera of peanut-allergic donors to
peanut allergens was more inhibited by preincubation with
increasing amounts of peanut proteins than by peanut peptides.
Immunogenicity of Peanut Peptides: Induction of Specific Antibodies
in Rats
[0140] Peanut peptides are able to evoke humoral immune
responses.
[0141] Rats that have been immunized subcutaneously three times
with peanut peptides emulsified in Incomplete Freund Adjuvant
produce significant levels of IgG. The mixture of peanut peptides
induced IgG1, IgG2a and IgG2b.
[0142] At days 0, 3 and 7 (D0, D3, D7), a group of four seven
week-old female rats were immunized with 1 mg of peanut peptides
administered subcutaneously as an emulsion (v/v) with Incomplete
Freund Adjuvant.
[0143] Maxisorp 96 well microtitre plates were coated with 2
.mu.g/ml of peanut proteins in 0.1M carbonate-bicarbonate Buffer pH
9.6 overnight at 4.degree. C. After blocking for 1 h at 37.degree.
C., wells were incubated 1 h at 37.degree. C. with 100 .mu.l of
serial dilutions of serum (from 1/200 to 1/437.400 dilution) of
treated rats. Bound rat IgG were detected with 1/20.000 diluted
peroxidase-labeled anti-rat IgG. IgG1, IgG2a or IgG2b were detected
with biotin-labelled antibodies diluted respectively 1/1.500, 1/500
and 1/500. After incubation with peroxidase-streptavidin (1/200),
the color reaction was started by adding 100 .mu.l of TMBS
substrate. The reaction was stopped with 100 .mu.l of 1M
H.sub.3PO.sub.4 and optical density values were measured at 450-650
nm.
[0144] The results were expressed as titres. They were defined as
the maximal dilution of rat antisera that gave absorbances of
0.3.
[0145] FIG. 13 a-d shows isotype profiles of the IgG antibody
response of Lewis rats immunized subcutaneously with peanut
peptides (1 mg/injection at D0, D3, D7) emulsified with Incomplete
Freund Adjuvant (v/v). The results are expressed as the mean.+-.SD
(n=4).
Immunogenicity of Peanut Peptides: Stimulation of Cellular Immune
Responses
[0146] Peanut peptides are able to trigger the proliferation of T
cells isolated from spleen of rats previously immunized with peanut
peptides or peanut proteins.
[0147] Peanut peptides were compared to purified peanut proteins
for their capacity to stimulate T lymphocytes by using a cell
proliferation assay based on thymidine incorporation.
[0148] The study was conducted on seven week-old female Lewis
rats.
[0149] At D0, D3 and D7, four rats were immunized intraperitoneally
with 100 .mu.g of peanut proteins mixed with alum (v/v). Four rats
were treated with 400 .mu.g or 1 mg of peanut peptides administered
subcutaneously as an emulsion (v/v) with Incomplete Freund
Adjuvant.
[0150] At D21, the animals were sacrified and the splenocytes were
withdrawn. Cells were cultured in RPMI 1640 supplemented with 10%
(v/v) F tal Calf Serum, 1% non-essential amino-acids, 2 mM
L-glutamine, 1 mM pyruvate, 50 .mu.M .beta.-mercaptoethanol, 50
U/ml Penicillin, 100 .mu.g/ml streptomycin, 10 .mu.g/ml gentamicin,
1 mM HEPES (complete medium).
[0151] Cells were plated at a density of 2.times.10.sup.6/ml in
complete medium. Antigens, also in complete medium were used at the
following concentrations: 100, 50, 25, 12.5, 6.25 .mu.g/ml. All
cultures were carried out in a humidified incubator at 37.degree.
C. in 5% CO.sub.2. Proliferation assays were performed in
triplicate in 96-well round-bottom plates for 4 days with cells
being pulsed with 0.5 .mu.Ci of [.sup.3H]-thymidine/well at 72 h,
harvested 12 to 16 h later and counted for
.beta.-radioactivity.
[0152] The results were expressed as proliferation index calculated
as follow: [0153] Cells cultured in medium alone (non-stimulated
cells) were considered as 0% of proliferation.
[0153] Proliferation index=Cpm tested well(mean of triplicates)/cpm
(mean of triplicates) of medium-treated cells.
[0154] FIG. 14 shows a splenocyte proliferation assay for Lewis
rats previously immunized with peanut proteins (100
.mu.g/injection) or peanut peptides (400 .mu.g or 1 mg/injection)
in response to increasing doses (from 6.25 to 100 .mu.g/ml) of
peanut peptides.
[0155] Results are expressed as means.+-.SD of triplicate
stimulation index.
[0156] As shown on FIG. 14, peanut peptides were able to induce the
proliferation of T cells from rats previously immunized with peanut
proteins. Moreover, whatever the peanut peptide concentration in
the culture medium, the T cell proliferation was higher when rats
were previously immunized subcutaneously with peanut peptides in
presence of Incomplete Freund Adjuvant.
[0157] For each doses of peanut peptides, untreated rats did not
show any relevant proliferation.
[0158] FIG. 15 shows a splenocyte proliferation assay for Lewis
rats previously immunized with peanut proteins (100
.mu.g/injection) or peanut peptides (400 .mu.g or 1 mg/injection)
in response to increasing doses (from 6.25 to 100 .mu.g/ml) of
peanut proteins.
[0159] FIG. 15 displays the proliferation of T cells by increasing
doses of purified peanuts proteins. Albeit to a lower level when
compared to T cells from peanut protein immunized rats, the
purified peanut proteins also triggered a specific immune response
on T cells from rats immunized with peanut peptides administered
subcutaneously.
[0160] Untreated rats did not show any relevant proliferation
whatever the peanut protein concentration in the culture
medium.
[0161] Results are expressed as the means.+-.SD of triplicate
stimulation index.
[0162] These results demonstrated that peanut peptides have
conserved a biological activity linked to the presence of T cell
epitopes, capable of stimulating specific immune responses in
immunized animals.
Blocking Potential of the Antibodies Generated to Peanut
Peptides
[0163] Immunoglobulins induced by immunization of rabbits with
peanut peptides inhibit patient's IgE binding to protein
allergens.
[0164] The ability of rabbit anti-peanut peptides and anti-peanut
proteins to inhibit the binding of peanut-allergic patient's IgE
antibodies to allergens was examined by ELISA competition
experiments.
[0165] ELISA Maxisorp plates coated 2 h at 37.degree. C. with 0.8
.mu.g/ml of peanut proteins were incubated (v/v) with serial
twofold dilutions of rabbit antiserum to peanut peptides or peanut
proteins (from 1/2 to 1/1024 final dilution) in presence of pooled
human serum diluted 1/50 (final dilution). After overnight
incubation at 4.degree. C., bound IgE antibodies were detected with
1/8.000 diluted peroxidase-coupled polyclonal anti-human IgE
antibodies. The optical density values corresponding to bound IgE
were measured at 450-650 nm. The percentage of inhibition of IgE
binding achieved by preincubation in presence of serum from rabbit
(anti-peanut peptides or anti-peanut proteins) was calculated as
follow:
% inhibition=100-[[OD of the inhibed sample/mean OD of positive
controls].times.100]
[0166] Positive controls were 50 .mu.l of pooled human sera diluted
1/25 mixed with 50 .mu.l of rabbit preimmune serum (n=10).
[0167] FIG. 16 shows a competition ELISA for assessement of
blocking antibody activity. Peanut proteins were coated on
microtitre plate. The pooled serum of peanut allergic patients were
mixed with serial dilutions of rabbit antibodies generated to
either peptides or proteins of peanuts. After incubation, the IgE
that bind to the peanut proteins coated to the plate were detected
using anti-human IgE labeled peroxydase antibodies.
[0168] In conclusion, peanut peptides obtained by the present
invention exhibit features favourable for their use in allergy
immunotherapy. Indeed, they are characterized by reduced
allergenicity but preserved T-cell reactivity. Moreover, the
allergen-derived peptides are able to induce an immune response in
animals and generate IgG antibodies with blocking potential on
human IgE binding to the allergens.
* * * * *