U.S. patent application number 14/810457 was filed with the patent office on 2016-02-04 for tolerogenic synthetic nanocarriers for allergy therapy.
This patent application is currently assigned to Selecta Biosciences, Inc.. The applicant listed for this patent is Selecta Biosciences, Inc.. Invention is credited to Christopher Fraser, Takashi Kei Kishimoto, Roberto A. Maldonado.
Application Number | 20160030554 14/810457 |
Document ID | / |
Family ID | 47068065 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160030554 |
Kind Code |
A1 |
Kishimoto; Takashi Kei ; et
al. |
February 4, 2016 |
TOLEROGENIC SYNTHETIC NANOCARRIERS FOR ALLERGY THERAPY
Abstract
Disclosed are synthetic nanocarrier compositions, and related
methods, comprising immunosuppressants and MHC Class II-restricted
epitopes of an allergen that provide tolerogenic immune responses
specific to the allergen.
Inventors: |
Kishimoto; Takashi Kei;
(Lexington, MA) ; Fraser; Christopher; (Arlington,
MA) ; Maldonado; Roberto A.; (Jamaica Plain,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Selecta Biosciences, Inc. |
Watertown |
MA |
US |
|
|
Assignee: |
Selecta Biosciences, Inc.
Watertown
MA
|
Family ID: |
47068065 |
Appl. No.: |
14/810457 |
Filed: |
July 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13457977 |
Apr 27, 2012 |
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14810457 |
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61531147 |
Sep 6, 2011 |
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61531153 |
Sep 6, 2011 |
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61531164 |
Sep 6, 2011 |
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61531168 |
Sep 6, 2011 |
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61531175 |
Sep 6, 2011 |
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61531180 |
Sep 6, 2011 |
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61531194 |
Sep 6, 2011 |
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61531204 |
Sep 6, 2011 |
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61531209 |
Sep 6, 2011 |
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61531215 |
Sep 6, 2011 |
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61480946 |
Apr 29, 2011 |
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61513514 |
Jul 29, 2011 |
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Current U.S.
Class: |
424/193.1 |
Current CPC
Class: |
A61K 39/385 20130101;
A61P 29/00 20180101; A61P 17/00 20180101; A61K 47/643 20170801;
A61P 43/00 20180101; A61P 37/06 20180101; A61K 2039/55555 20130101;
A61K 47/50 20170801; A61K 47/593 20170801; A61P 35/00 20180101;
A61K 47/544 20170801; B82Y 5/00 20130101; A61K 2039/5154 20130101;
G01N 2333/7051 20130101; G01N 2333/70514 20130101; A61K 9/14
20130101; A61P 37/04 20180101; G01N 33/56972 20130101; Y02A 50/30
20180101; A61P 1/16 20180101; A61K 9/5115 20130101; A61K 9/5146
20130101; A61P 11/02 20180101; A61P 37/00 20180101; G01N 33/505
20130101; A61K 38/38 20130101; A61P 41/00 20180101; A61P 37/02
20180101; A61K 9/51 20130101; A61K 2039/577 20130101; A61K 9/127
20130101; A61P 15/00 20180101; A61K 31/192 20130101; A61K 39/35
20130101; A61K 47/6937 20170801; A61K 38/1816 20130101; A61K 39/36
20130101; A61K 47/69 20170801; B82Y 40/00 20130101; A61K 39/00
20130101; A61K 47/6923 20170801; A61P 7/06 20180101; A61P 37/08
20180101; G01N 2333/70517 20130101; A61K 9/5153 20130101; A61K
39/001 20130101; A61K 47/52 20170801; A61K 2039/55511 20130101;
A61K 38/13 20130101; A61P 11/06 20180101; A61K 39/0008 20130101;
A61K 47/6929 20170801; A61K 39/36 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 39/385 20060101
A61K039/385; A61K 31/192 20060101 A61K031/192; A61K 47/48 20060101
A61K047/48; A61K 39/35 20060101 A61K039/35; A61K 31/436 20060101
A61K031/436 |
Claims
1. A composition comprising: (i) a first population of synthetic
nanocarriers that are coupled to immunosuppressants, and (ii) MHC
Class II-restricted epitopes of an allergen, wherein the
composition comprises substantially no B cell epitopes of the
allergen.
2-32. (canceled)
33. A dosage form comprising the composition of claim 1.
34. A method comprising administering the composition of claim 1 to
a subject.
35. (canceled)
36. A method comprising: administering to a subject a composition
comprising: (i) a first population of synthetic nanocarriers that
are coupled to immunosuppressants, and (ii) MHC Class II-restricted
epitopes of an allergen, wherein the composition comprises
substantially no B cell epitopes of the allergen, wherein the
composition is in an amount effective to reduce an undesired immune
response to the allergen in the subject, and wherein the subject is
experiencing or is at risk of experiencing the undesired immune
response to the allergen.
37-78. (canceled)
79. A method comprising: (i) producing a first population of
synthetic nanocarriers that are coupled to immunosuppressants, (ii)
producing or obtaining MHC Class II-restricted epitopes of an
allergen, and (iii) ensuring there are substantially no B cell
epitopes of the allergen.
80-87. (canceled)
88. A composition of claim 1 for use in therapy or prophylaxis.
89. (canceled)
90. Use of the composition of claim 1 for the manufacture of a
medicament for use in a method of reducing an undesired immune
response to the allergen in a subject, the treatment or prophylaxis
of allergy.
91. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/457,977, filed Apr. 27, 2012, pending,
which claims the benefit under 35 U.S.C. .sctn.119 of U.S.
provisional application 61/480,946, filed Apr. 29, 2011,
61/513,514, filed Jul. 29, 2011, 61/531,147, filed Sep. 6, 2011,
61/531,153, filed Sep. 6, 2011, 61/531,164, filed Sep. 6, 2011,
61/531,168, filed Sep. 6, 2011, 61/531,175, filed Sep. 6, 2011,
61/531,180, filed Sep. 6, 2011, 61/531,194, filed Sep. 6, 2011,
61/531,204, filed Sep. 6, 2011, 61/531,209, filed Sep. 6, 2011, and
61/531,215, filed Sep. 6, 2011, the entire contents of each of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to synthetic nanocarrier compositions
with antigens that comprise MHC Class II-restricted epitopes of an
allergen and immunosuppressants, and related methods. The
compositions and methods allow for efficient and preferential
uptake by APCs to shift the immune response in favor of tolerogenic
immune response development specific to the allergen. The
compositions and methods provided, therefore, can be used to
generate a tolerogenic immune response in a subject that is
suffering or is expected to suffer from an allergic response to an
allergen.
BACKGROUND OF THE INVENTION
[0003] Allergic responses in a subject are generally exaggerated
and undesired but may be reduced through the use of
immunosuppressant drugs. Conventional immunosuppressant drugs,
however, are broad-acting. Additionally, in order to maintain
immunosuppression, immunosuppressant drug therapy is generally a
life-long proposition. Unfortunately, the use of broad-acting
immunosuppressants are associated with a risk of severe side
effects, such as tumors, infections, nephrotoxicity and metabolic
disorders. Accordingly, new immunosuppressant therapies would be
beneficial.
SUMMARY OF THE INVENTION
[0004] In one aspect, a composition comprising (i) a first
population of synthetic nanocarriers that are coupled to
immunosuppressants, and (ii) a second population of synthetic
nanocarriers that are coupled to MHC Class II-restricted epitopes
of an allergen, wherein the composition comprises substantially no
B cell epitopes of the allergen is provided. In another embodiment,
the first population of synthetic nanocarriers are also coupled to
MHC Class I-restricted epitopes of the allergen.
[0005] In one embodiment, the first population and second
population are the same population. In another embodiment, the
first population and second population are different
populations.
[0006] In yet another embodiment, the immunosuppressants comprise a
statin, an mTOR inhibitor, a TGF-.beta. signaling agent, a
corticosteroid, an inhibitor of mitochondrial function, a P38
inhibitor, an NF-.kappa..beta. inhibitor, an adenosine receptor
agonist, a prostaglandin E2 agonist, a phosphodiesterasse 4
inhibitor, an HDAC inhibitor or a proteasome inhibitor. In still
another embodiment, the mTOR inhibitor is rapamycin or an analog
thereof.
[0007] In a further embodiment, the allergen induces, or is
expected to induce, an undesired immune response in a subject. In
one embodiment, the undesired immune response is allergen-specific
antibody production. In another embodiment, the undesired immune
response is allergen specific CD4+ T cell proliferation and/or
activity. In still another embodiment, the undesired immune
response is allergen-specific B cell proliferation and/or activity.
In yet a further embodiment, the allergen comprises an asthma
antigen, a hay fever antigen, a hives antigen, an eczema antigen, a
plant allergen, an insect sting allergen, an insect allergen, an
animal allergen, a fungal allergen, a drug allergen, haptens, small
chemicals, a pet allergen, a latex allergen, a mold allergen, a
cosmetic allergen or a food allergen. In still a further
embodiment, the food allergen comprises a milk allergen, an egg
allergen, a nut allergen, a fish allergen, a shellfish allergen, a
soy allergen, a legume allergen, a seed allergen or a wheat
allergen. In another embodiment, the nut allergen is a peanut
allergen or a tree nut allergen. In another embodiment, the plant
allergen is a ragweed allergen. In yet another embodiment, the
allergen is associated with hay fever or allergic asthma.
[0008] In one embodiment, the composition is in an amount effective
to reduce the undesired immune response to the allergen when
administered to a subject. In another embodiment, the subject has
or is at risk of having an allergy. In another embodiment, the
allergy is allergic asthma, hay fever, hives, eczema, a plant
allergy, a pet allergy, a latex allergy, a mold allergy, a cosmetic
allergy, a food allergy, an insect sting allergy, an insect
allergy, an animal allergy, a fungal allergy, a drug allergy or an
allergy to a hapten or small chemical. In yet another embodiment,
the food allergy is a milk allergy, an egg allergy, a nut allergy,
a fish allergy, a shellfish allergy, a soy allergy, a legume
allergy, a seed allergy or a wheat allergy. In still another
embodiment, the nut allergy is a peanut allergy or a tree nut
allergy. In yet another embodiment, the allergy is hay fever or a
ragweed allergy.
[0009] In one embodiment, the load of the immunosuppressants and/or
epitopes on average across the first and/or second population of
synthetic nanocarriers is between 0.0001% and 50%. In another
embodiment, the load of the immunosuppressant and/or epitopes on
average across the first and/or second population of synthetic
nanocarriers is between 0.1% and 10%.
[0010] In a further embodiment, the synthetic nanocarriers of the
first population and/or second population comprise lipid
nanoparticles, polymeric nanoparticles, metallic nanoparticles,
surfactant-based emulsions, dendrimers, buckyballs, nanowires,
virus-like particles or peptide or protein particles. In yet a
further embodiment, the synthetic nanocarriers of the first
population and/or second population comprise lipid nanoparticles.
In still a further embodiment, the synthetic nanocarriers of the
first population and/or second population comprise liposomes. In
another embodiment, the synthetic nanocarriers of the first
population and/or second population comprise metallic
nanoparticles. In yet another embodiment, the metallic
nanoparticles comprise gold nanoparticles. In still another
embodiment, the synthetic nanocarriers of the first population
and/or second population comprise polymeric nanoparticles. In a
further embodiment, the polymeric nanoparticles comprise polymer
that is a non-methoxy-terminated, pluronic polymer. In one
embodiment, the polymeric nanoparticles comprise a polyester, a
polyester coupled to a polyether, polyamino acid, polycarbonate,
polyacetal, polyketal, polysaccharide, polyethyloxazoline or
polyethyleneimine. In another embodiment, the polyester comprises a
poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic
acid) or polycaprolactone. In still another embodiment, the
polymeric nanoparticles comprise a polyester and a polyester
coupled to a polyether. In a further embodiment, the polyether
comprises polyethylene glycol or polypropylene glycol.
[0011] In another embodiment, the mean of a particle size
distribution obtained using dynamic light scattering of the
synthetic nanocarriers of the first and/or second population is a
diameter greater than 100 nm. In one embodiment, the diameter is
greater than 150 nm. In another embodiment, the diameter is greater
than 200 nm. In still another embodiment, the diameter is greater
than 250 nm. In yet another embodiment, the diameter is greater
than 300 nm.
[0012] In yet a further embodiment, the aspect ratio of the
synthetic nanocarriers of the first population and/or second
population is greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or
1:10.
[0013] In another embodiment, the composition further comprises a
pharmaceutically acceptable excipient.
[0014] In another aspect, a dosage form comprising any of the
compositions provided herein is provided.
[0015] In yet another aspect, any of the compositions or dosage
forms provided can be administered to a subject. In one embodiment,
the subject has or is at risk of having an allergy. In another
embodiment, the subject has or is at risk of having an undesired
immune response against an allergen. In still another embodiment,
an undesired immune response to an allergen is reduced in the
subject with the composition or dosage form. In one embodiment, the
undesired immune response is allergen-specific antibody production.
In another embodiment, the undesired immune response is
allergen-specific CD4+ T cell proliferation and/or activity. In
still another embodiment, the undesired immune response is
allergen-specific B cell proliferation and/or activity.
[0016] In yet another aspect, a method comprising administering to
a subject a composition comprising (i) a first population of
synthetic nanocarriers that are coupled to immunosuppressants, and
(ii) a second population of synthetic nanocarriers that are coupled
to MHC Class II-restricted epitopes of an allergen, wherein the
composition comprises substantially no B cell epitopes of the
allergen, wherein the composition is in an amount effective to
reduce an undesired immune response to the allergen in the subject,
and wherein the subject is experiencing or is at risk of
experiencing the undesired immune response to the allergen is
provided. In still another aspect a method comprising reducing an
undesired immune response to an allergen in a subject by
administering a composition comprising (i) a first population of
synthetic nanocarriers that are coupled to immunosuppressants, and
(ii) a second population of synthetic nanocarriers that are coupled
to MHC Class II-restricted epitopes of the allergen, wherein the
composition comprises substantially no B cell epitopes of the
allergen, wherein the composition is in an amount effective to
reduce the undesired immune response to the allergen in the
subject, and wherein the subject is experiencing or is at risk of
experiencing the undesired immune response to the allergen is
provided. In a further aspect, a method comprising administering a
composition to a subject according to a protocol that was
previously shown to reduce an undesired immune response to an
allergen in one or more test subjects;
wherein the composition comprises (i) a first population of
synthetic nanocarriers that are coupled to immunosuppressants, and
(ii) a second population of synthetic nanocarriers that are coupled
to MHC Class II-restricted epitopes of the allergen, wherein the
composition comprises substantially no B cell epitopes of the
allergen, wherein the composition is in an amount effective to
reduce the undesired immune response to the allergen in the
subject, and wherein the subject is experiencing or is at risk of
experiencing the undesired immune response to the allergen is
provided.
[0017] In one embodiment, the first population and second
population are the same population. In another embodiment, the
first population and second population are different
populations.
[0018] In yet another embodiment, the method further comprises
providing or identifying the subject.
[0019] In still another embodiment, the immunosuppressants comprise
a statin, an mTOR inhibitor, a TGF-.beta. signaling agent, a
corticosteroid, an inhibitor of mitochondrial function, a P38
inhibitor, an NF-.kappa..beta. inhibitor, an adenosine receptor
agonist, a prostaglandin E2 agonist, a phosphodiesterasse 4
inhibitor, an HDAC inhibitor or a proteasome inhibitor. In a
further embodiment, the mTOR inhibitor is rapamycin or an analog
thereof.
[0020] In one embodiment, the allergen induces or is expected to
induce an undesired immune response in the subject. In one
embodiment, the undesired immune response is allergen-specific
antibody production. In another embodiment, the undesired immune
response is allergen-specific CD4+ T cell proliferation and/or
activity. In still another embodiment, the undesired immune
response is allergen-specific B cell proliferation and/or activity.
In another embodiment, the allergen comprises an asthma antigen, a
hay fever antigen, a hives antigen, an eczema antigen, a plant
allergen, an insect sting allergen, an insect allergen, an animal
allergen, a fungal allergen, a drug allergen, a pet allergen, a
latex allergen, a mold allergen, a cosmetic allergen or a food
allergen. In yet another embodiment, the food allergen comprises a
milk allergen, an egg allergen, a nut allergen, a fish allergen, a
shellfish allergen, a soy allergen, a legume allergen, a seed
allergen or a wheat allergen. In still another embodiment, the nut
allergen is a peanut allergen or a tree nut allergen. In yet
another embodiment, the plant allergen is a ragweed allergen.
[0021] In a further embodiment, the first population of synthetic
nanocarriers are also coupled to MHC Class I-restricted epitopes of
the allergen.
[0022] In one embodiment, the load of the immunosuppressants and/or
epitopes on average across the first and/or second population of
synthetic nanocarriers is between 0.0001% and 50%. In another
embodiment, the load of the immunosuppressants and/or epitopes on
average across the first and/or second population of synthetic
nanocarriers is between 0.1% and 10%.
[0023] In a further embodiment, the synthetic nanocarriers of the
first population and/or second population comprise lipid
nanoparticles, polymeric nanoparticles, metallic nanoparticles,
surfactant-based emulsions, dendrimers, buckyballs, nanowires,
virus-like particles or peptide or protein particles. In yet a
further embodiment, the synthetic nanocarriers of the first
population and/or second population comprise lipid nanoparticles.
In still a further embodiment, the synthetic nanocarriers of the
first population and/or second population comprise liposomes. In
another embodiment, the synthetic nanocarriers of the first
population and/or second population comprise metallic
nanoparticles. In yet another embodiment, the metallic
nanoparticles comprise gold nanoparticles. In still another
embodiment, the synthetic nanocarriers of the first population
and/or second population comprise polymeric nanoparticles. In one
embodiment, the polymeric nanoparticles comprise
non-methoxy-terminated, pluronic polymer. In one embodiment, the
polymeric nanoparticles comprise a polyester, a polyester coupled
to a polyether, polyamino acid, polycarbonate, polyacetal,
polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine.
In another embodiment, the polyester comprises a poly(lactic acid),
poly(glycolic acid), poly(lactic-co-glycolic acid) or
polycaprolactone. In still another embodiment, the polymeric
nanoparticles comprise a polyester and a polyester coupled to a
polyether. In a further embodiment, the polyether comprises
polyethylene glycol or polypropylene glycol.
[0024] In another embodiment, the mean of a particle size
distribution obtained using dynamic light scattering of the
synthetic nanocarriers of the first and/or second population is a
diameter greater than 100 nm. In one embodiment, the diameter is
greater than 150 nm. In another embodiment, the diameter is greater
than 200 nm. In still another embodiment, the diameter is greater
than 250 nm. In yet another embodiment, the diameter is greater
than 300 nm.
[0025] In yet a further embodiment, the aspect ratio of the
synthetic nanocarriers of the first population and/or second
population is greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7 or
1:10.
[0026] In one embodiment, one or more maintenance doses of the
composition comprising the first population and second population
of synthetic nanocarriers is administered to the subject. In
another embodiment, the method further comprises assessing the
undesired immune response to the allergen in the subject prior to
and/or after the administration of the composition comprising the
first population and second population of synthetic nanocarriers.
In one embodiment, the undesired immune response is
allergen-specific antibody production. In another embodiment, the
undesired immune response if allergen-specific CD4+ T cell
proliferation and/or activity. In still another embodiment, the
undesired immune response is allergen-specific B cell proliferation
and/or activity.
[0027] In one embodiment, the subject has or is at risk of having
an allergy. In another embodiment, the allergy is allergic asthma,
hay fever, hives, eczema, a plant allergy, an insect sting allergy,
an insect allergy, an animal allergy, a fungal allergy, a drug
allergy, a pet allergy, a latex allergy, a mold allergy, a cosmetic
allergy or a food allergy. In yet another embodiment, the food
allergy is a milk allergy, an egg allergy, a nut allergy, a fish
allergy, a shellfish allergy, a soy allergy, a legume allergy, a
seed allergy or a wheat allergy. In still another embodiment, the
nut allergy is a peanut allergy or a tree nut allergy. In another
embodiment, the plant allergy is a ragweed allergy.
[0028] In a further embodiment, the administering is by
intravenous, intraperitoneal, transmucosal, oral, subcutaneous,
pulmonary, intranasal, intradermal or intramuscular administration.
In yet a further embodiment, the administering is by inhalation or
intravenous, subcutaneous or transmucosal administration.
[0029] In a further aspect, a method comprising (i) producing a
first population of synthetic nanocarriers that are coupled to
immunosuppressants, (ii) producing a second population of synthetic
nanocarriers that are coupled to MHC Class II-restricted epitopes
of an allergen, and (iii) ensuring the second population of
synthetic nanocarriers comprise substantially no B cell epitopes of
the allergen is provided.
[0030] In one embodiment, the first population and second
population are the same population. In another embodiment, the
first population and second population are different
populations.
[0031] In yet another embodiment, the method further comprises
making a dosage form comprising the first population and second
population of synthetic nanocarriers. In still another embodiment,
the method further comprises making a composition comprising the
first population and second population of synthetic nanocarriers or
a dosage form thereof available to a subject for
administration.
[0032] In another embodiment, the first population and second
population of synthetic nanocarriers that are produced are as
defined in any of the methods provided herein. In a further
embodiment, the method further comprises assessing an undesired
immune response to the allergen with a composition comprising the
first population and second population of synthetic nanocarriers.
In one embodiment, the undesired immune response in a subject is
assessed.
[0033] In yet a further aspect, a process for producing a
composition or dosage form comprising the steps of coupling a first
population of synthetic nanocarriers to immunosuppressants;
coupling a second population of synthetic nanocarriers to MHC Class
II-restricted epitopes of an allergen; and ensuring the second
population of synthetic nanocarriers comprise substantially no B
cell epitopes of the allergen is provided. In one embodiment, the
process comprises the steps of any of the methods provided
herein.
[0034] In another aspect, a composition or dosage form obtainable
by any of the methods or processes provided herein is provided.
[0035] In still another aspect, any of the compositions or dosage
forms provided herein may be for use in therapy or prophylaxis.
[0036] In yet another aspect, any of the compositions or dosage
forms provided herein may be for use in a method of reducing an
undesired immune response to an allergen in a subject, the
treatment or prophylaxis of allergy, or any of the methods provided
herein.
[0037] In a further aspect, a use of any of the compositions or
dosage forms provided herein for the manufacture of a medicament
for use in a method of reducing an undesired immune response to an
allergen in a subject, the treatment or prophylaxis of allergy, or
any of the methods provided herein is provided.
[0038] In yet a further aspect, a dosage form comprising any of the
compositions provided herein is provided.
[0039] In an embodiment of any of the compositions and methods
provided herein, antigens that are proteins that comprise the
aforementioned epitopes can be coupled to the synthetic
nanocarriers. In another embodiment, polypeptides or peptides that
comprise the aforementioned epitopes but additional amino acids
that flank one or both ends of the epitope(s) can be coupled to the
synthetic nanocarriers. In another embodiment, the epitopes
themselves are coupled to the synthetic nanocarriers.
BRIEF DESCRIPTION OF FIGURES
[0040] FIG. 1 shows results from a flow cytometric analysis of
Treg.
[0041] FIG. 2 shows an effect on the number of antigen-specific
effector T cells with synthetic nanocarriers of the invention
comprising immunosuppressant (rapamycin or simvastatin) (after a
single injection).
[0042] FIG. 3 shows a decrease in the number of popliteal lymph
node cells with synthetic nanocarriers of the invention comprising
immunosuppressant (rapamycin or simvastatin) (after multiple
injections).
[0043] FIG. 4 shows a reduction in antigen-specific IgG levels with
the administration of synthetic nanocarriers comprising ova peptide
and the immunosuppressant rapamycin.
[0044] FIG. 5 demonstrates a reduction in the number of
antigen-specific B cells with synthetic nanocarriers comprising ova
peptide and the immunosuppressant rapamycin.
[0045] FIG. 6 demonstrates an overall reduction in the number of
various immune cells in lavage samples from asthma model animal
subjects treated with synthetic nanocarriers comprising ova peptide
and immunosuppressant.
[0046] FIG. 7 demonstrates a reduction in the percentage of
dividing CD4+ T cells as a result of treatment with synthetic
nanocarriers comprising ova peptide and the immunosuppressant
rapamycin in asthma model animal subjects.
[0047] FIG. 8 demonstrates a reduction in the production of
antigen-specific IgE antibodies.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particularly
exemplified materials or process parameters as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments of the
invention only, and is not intended to be limiting of the use of
alternative terminology to describe the present invention.
[0049] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety for all purposes.
[0050] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the content clearly dictates otherwise. For example, reference to
"a polymer" includes a mixture of two or more such molecules or a
mixture of differing molecular weights of a single polymer species,
reference to "a synthetic nanocarrier" includes a mixture of two or
more such synthetic nanocarriers or a plurality of such synthetic
nanocarriers, reference to "a DNA molecule" includes a mixture of
two or more such DNA molecules or a plurality of such DNA
molecules, reference to "an immunosuppressant" includes a mixture
of two or more such materials or a plurality of immunosuppressant
molecules, and the like.
[0051] As used herein, the term "comprise" or variations thereof
such as "comprises" or "comprising" are to be read to indicate the
inclusion of any recited integer (e.g. a feature, element,
characteristic, property, method/process step or limitation) or
group of integers (e.g. features, element, characteristics,
properties, method/process steps or limitations) but not the
exclusion of any other integer or group of integers. Thus, as used
herein, the term "comprising" is inclusive and does not exclude
additional, unrecited integers or method/process steps.
[0052] In embodiments of any of the compositions and methods
provided herein, "comprising" may be replaced with "consisting
essentially of" or "consisting of". The phrase "consisting
essentially of" is used herein to require the specified integer(s)
or steps as well as those which do not materially affect the
character or function of the claimed invention. As used herein, the
term "consisting" is used to indicate the presence of the recited
integer (e.g. a feature, element, characteristic, property,
method/process step or limitation) or group of integers (e.g.
features, element, characteristics, properties, method/process
steps or limitations) alone.
A. INTRODUCTION
[0053] As previously mentioned, current conventional
immunosuppressants are broad acting and generally result in an
overall systemic down regulation of the immune system. The
compositions and methods provided herein allow for more targeted
immune effects by, for example, allowing for the targeted delivery
to immune cells of interest. Thus, the compositions and methods can
achieve immune suppression in a more directed manner. It has been
found that delivering immunosuppressants and MHC Class
II-restricted epitopes of an allergen more directly to cells of
interest, in particular APCs, can result in beneficial tolerogenic
immune responses, such as the reduction in antibody production,
CD4+ T cell proliferation and/or activity etc., specific to the
allergen. Such immune responses can be beneficial in subjects who
suffer from allergies. This invention is useful, for example, to
promote tolerogenic immune responses in subjects who are
experiencing or are at risk of experiencing undesired immune
responses to allergens. Such subjects include those who have or are
at risk of having an allergy.
[0054] The inventors have unexpectedly and surprisingly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. In particular, the
inventors have unexpectedly discovered that it is possible to
provide synthetic nanocarrier compositions, and related methods,
that induce a tolerogenic immune response to allergens that
comprise MHC Class II-restricted epitopes of an allergen. Such
compositions can reduce an undesired immune response to the
allergen. The compositions described herein include compositions
that comprise (i) a first population of synthetic nanocarriers that
are coupled to immunosuppressants, and (ii) a second population of
synthetic nanocarriers that are coupled to MHC Class II-restricted
epitopes of an allergen. In embodiments, MHC Class I-restricted
epitopes may also be coupled to the synthetic nanocarriers. In some
embodiments, substantially no B cell epitopes of the allergen are
coupled and such epitopes may be specifically excluded from the
compositions and methods provided herein.
[0055] In another aspect, dosage forms of any of the compositions
herein are provided. Such dosage forms can be administered to a
subject, such as one in need of allergen-specific tolerogenic
immune responses. In one embodiment, the subject is one who has
had, is having or is expected to have an undesired immune response
against an allergen. Such subjects include those that have or are
at risk of having an allergy.
[0056] In another aspect, any of the compositions provided herein
is administered to a subject. The composition may be administered
in an amount effective to reduce the generation of an undesired
immune response to an allergen. In one embodiment, a composition is
administered to a subject according to a protocol that was
previously shown to reduce the generation of an undesired immune
response to an allergen in one or more subjects.
[0057] The compositions may be administered to a subject prior to,
concomitantly with or after the exposure of a subject to an
allergen. In embodiments, the compositions provided may also be
administered as one or more maintenance doses to a subject that has
or is at risk of having an allergy. In such embodiments, the
compositions provided are administered such that the generation of
an undesired immune response is reduced for a certain length of
time. Examples of such lengths of time are provided elsewhere
herein.
[0058] In yet another aspect, a method of (i) producing a first
population of synthetic nanocarriers that are coupled to
immunosuppressants, and (ii) producing a second population of
synthetic nanocarriers that are coupled to MHC Class II-restricted
epitopes of an allergen is provided. In embodiments, MHC Class
I-restricted epitopes of the allergen may also be coupled to the
synthetic nanocarriers. In another embodiment, substantially no B
cell epitopes of the allergen are coupled to the synthetic
nanocarriers.
[0059] The invention will now be described in more detail
below.
B. DEFINITIONS
[0060] "Administering" or "administration" means providing a
material to a subject in a manner that is pharmacologically
useful.
[0061] "Allergen-specific" refers to any immune response that
results from the presence of the allergen, or portion thereof, or
that generates molecules that specifically recognize or bind the
allergen. For example, where the immune response is
allergen-specific antibody production, antibodies are produced that
specifically bind the allergen. As another example, where the
immune response is allergen-specific B cell or CD4+ T cell
proliferation and/or activity, the proliferation and/or activity
results from recognition of the allergen, or portion thereof, alone
or in complex with MHC molecules, B cells, etc.
[0062] "Allergens" are any substances that can cause an undesired
(e.g., a Type 1 hypersensitive) immune response (i.e., an allergic
response or reaction) in a subject. Allergens include, but are not
limited to, plant allergens (e.g., pollen, ragweed allergen),
insect allergens, insect sting allergens (e.g., bee sting
allergens), animal allergens (e.g., pet allergens, such as animal
dander or cat Fel d 1 antigen), latex allergens, mold allergens,
fungal allergens, cosmetic allergens, drug allergens, food
allergens, dust, insect venom, viruses, bacteria, etc. Food
allergens include, but are not limited to milk allergens, egg
allergens, nut allergens (e.g., peanut or tree nut allergens, etc.
(e.g., walnuts, cashews, etc.)), fish allergens, shellfish
allergens, soy allergens, legume allergens, seed allergens and
wheat allergens. Insect sting allergens include allergens that are
or are associated with bee stings, wasp stings, hornet stings,
yellow jacket stings, etc. Insect allergens also include house dust
mite allergens (e.g., Der P1 antigen) and cockroach allergens. Drug
allergens include allergens that are or are associated with
antibiotics, NSAIDs, anaesthetics, etc. Pollen allergens include
grass allergens, tree allergens, weed allergens, flower allergens,
etc. Subjects that develop or are at risk of developing an
undesired immune response to any of the allergens provided herein
may be treated with any of the compositions and methods provided
herein. Subjects that may be treated with any of the compositions
and methods provided also include those who have or are at risk of
having an allergy to any of the allergens provided. "Allergens
associated with an allergy" are allergens that generate an
undesired immune response that results in, or would be expected by
a clinician to result in, alone or in combination with other
allergens, an allergic response or reaction or a symptom of an
allergic response or reaction in a subject. "Type(s) of allergens"
means molecules that share the same, or substantially the same,
antigenic characteristics in the context of an undesired immune
response. In some embodiments, the allergens may be proteins,
polypeptides, peptides, lipoproteins or are contained or expressed
in cells.
[0063] It is intended that MHC Class II-restricted epitopes are
preferably coupled to the synthetic nanocarriers as provided
herein. The epitopes themselves may be coupled or proteins,
polypeptides, peptides, etc. that comprise these epitopes may be
coupled to the synthetic nanocarriers. Thus an allergen itself or a
portion thereof that comprises MHC Class II-restricted epitopes may
be coupled to the synthetic nanocarriers in the compositions
provided herein. In some embodiments, MHC Class I-restricted
epitopes may also be coupled. Therefore, in some embodiments, the
allergen itself or portion thereof comprises both MHC Class
II-restricted and MHC Class I-restricted epitopes. The epitopes for
use in the compositions and methods provided herein can be
presented for recognition by cells of the immune system, such as
presented by antigen presenting cells, which include but are not
limited to dendritic cells, B cells or macrophages. The epitopes
can be presented for recognition by, for example, T cells. Such
epitopes may normally be recognized by and trigger an immune
response in a T cell via presentation major histocompatability
complex molecule (MHC), but in the compositions provided herein the
presence of such epitopes in combination with an immunosuppressant
can result in tolerogenic immune responses instead. In some
embodiments, substantially no B cell epitopes are coupled to the
synthetic nanocarriers, such as when the inclusion of the B cell
epitopes would exacerbate an undesired immune response and thus,
the allergens or portions thereof do not comprise B cell epitopes
or do comprise B cell epitopes but such epitopes do not
significantly negatively impact the desired immune responses.
[0064] An allergen can be coupled to the synthetic nanocarriers in
the same form as what a subject is exposed to that causes an
undesired immune response but may also be a fragment or derivative
thereof. When a fragment or derivative, however, a desired immune
response to the form encountered by such a subject is the
preferable result with the compositions and methods provided.
[0065] An "allergy" also referred to herein as an "allergic
condition," is any condition where there is an undesired (e.g., a
Type 1 hypersensitive) immune response (i.e., allergic response or
reaction) to a substance. Such substances are referred to herein as
allergens. Allergies or allergic conditions include, but are not
limited to, allergic asthma, hay fever, hives, eczema, plant
allergies, bee sting allergies, pet allergies, latex allergies,
mold allergies, cosmetic allergies, food allergies, allergic
rhinitis or coryza, topic allergic reactions, anaphylaxis, atopic
dermatitis, hypersensitivity reactions and other allergic
conditions. The allergic reaction may be the result of an immune
reaction to any allergen. In some embodiments, the allergy is a
food allergy. Food allergies include, but are not limited to, milk
allergies, egg allergies, nut allergies, fish allergies, shellfish
allergies, soy allergies or wheat allergies.
[0066] "Amount effective" in the context of a composition or dosage
form for administration to a subject refers to an amount of the
composition or dosage form that produces one or more desired immune
responses in the subject, for example, the generation of a
tolerogenic immune response (e.g, a reduction in the proliferation,
activation, induction, recruitment of allergen-specific CD4+ T
cells or allergen-specific B cells or a reduction in the production
of allergen-specific antibodies). Therefore, in some embodiments,
an amount effective is any amount of a composition provided herein
that produces one or more of these desired immune responses. This
amount can be for in vitro or in vivo purposes. For in vivo
purposes, the amount can be one that a clinician would believe may
have a clinical benefit for a subject in need of allergen-specific
tolerization. Such subjects include those that have or are at risk
of having an allergy or an allergic response against an
allergen.
[0067] Amounts effective can involve only reducing the level of an
undesired immune response, although in some embodiments, it
involves preventing an undesired immune response altogether.
Amounts effective can also involve delaying the occurrence of an
undesired immune response. An amount that is effective can also be
an amount of a composition provided herein that produces a desired
therapeutic endpoint or a desired therapeutic result. Amounts
effective, preferably, result in a tolerogenic immune response in a
subject to an allergen. The achievement of any of the foregoing can
be monitored by routine methods.
[0068] In some embodiments of any of the compositions and methods
provided, the amount effective is one in which the desired immune
response persists in the subject for at least 1 week, at least 2
weeks, at least 1 month, at least 2 months, at least 3 months, at
least 4 months, at least 5 months, at least 6 months, at least 9
months, at least 1 year, at least 2 years, at least 5 years, or
longer. In other embodiments of any of the compositions and methods
provided, the amount effective is one which produces a measurable
desired immune response, for example, a measurable decrease in an
immune response (e.g., to a specific allergen), for at least 1
week, at least 2 weeks, at least 1 month, at least 2 months, at
least 3 months, at least 4 months, at least 5 months, at least 6
months, at least 9 months, at least 1 year, at least 2 years, at
least 5 years, or longer.
[0069] Amounts effective will depend, of course, on the particular
subject being treated; the severity of a condition, disease or
disorder; the individual patient parameters including age, physical
condition, size and weight; the duration of the treatment; the
nature of concurrent therapy (if any); the specific route of
administration and like factors within the knowledge and expertise
of the health practitioner. These factors are well known to those
of ordinary skill in the art and can be addressed with no more than
routine experimentation. It is generally preferred that a maximum
dose be used, that is, the highest safe dose according to sound
medical judgment. It will be understood by those of ordinary skill
in the art, however, that a patient may insist upon a lower dose or
tolerable dose for medical reasons, psychological reasons or for
virtually any other reason.
[0070] In general, doses of the immunosuppressants and/or antigens
in the compositions of the invention can range from about 10
.mu.g/kg to about 100,000 .mu.g/kg. In some embodiments, the doses
can range from about 0.1 mg/kg to about 100 mg/kg. In still other
embodiments, the doses can range from about 0.1 mg/kg to about 25
mg/kg, about 25 mg/kg to about 50 mg/kg, about 50 mg/kg to about 75
mg/kg or about 75 mg/kg to about 100 mg/kg. Alternatively, the dose
can be administered based on the number of synthetic nanocarriers
that provide the desired amount of immunosuppressants and/or
antigens. For example, useful doses include greater than 10.sup.6,
10.sup.7, 10.sup.8, 10.sup.9 or 10.sup.10 synthetic nanocarriers
per dose. Other examples of useful doses include from about
1.times.10.sup.6 to about 1.times.10.sup.10, about 1.times.10.sup.7
to about 1.times.10.sup.9 or about 1.times.10.sup.8 to about
1.times.10.sup.9 synthetic nanocarriers per dose.
[0071] "Antigen" means a B cell antigen or T cell antigen. Antigens
include allergens or fragments or derivatives of allergens that can
generate an immune response alone or in conjunction with another
agent, carrier, etc.
[0072] "Assessing an immune response" refers to any measurement or
determination of the level, presence or absence, reduction,
increase in, etc. of an immune response in vitro or in vivo. Such
measurements or determinations may be performed on one or more
samples obtained from a subject. Such assessing can be performed
with any of the methods provided herein or otherwise known in the
art.
[0073] An "at risk" subject is one in which a health practitioner
believes has a chance of having a disease, disorder or condition as
provided herein or is one a health practitioner believes has a
chance of experiencing an undesired immune response as provided
herein.
[0074] "Average", as used herein, refers to the arithmetic mean
unless otherwise noted.
[0075] "B cell antigen" means any antigen that triggers an immune
response in a B cell (e.g., an antigen that is specifically
recognized by a B cell or a receptor thereon). In some embodiments,
an antigen that is a T cell antigen is also a B cell antigen. In
other embodiments, the T cell antigen is not also a B cell antigen.
B cell antigens include, but are not limited to proteins, peptides,
small molecules, and carbohydrates. In some embodiments, the B cell
antigen comprises a non-protein antigen (i.e., not a protein or
peptide antigen). In some embodiments, the B cell antigen is
obtained or derived from an allergen.
[0076] "Concomitantly" means administering two or more substances
to a subject in a manner that is correlated in time, preferably
sufficiently correlated in time so as to provide a modulation in an
immune response. In embodiments, concomitant administration may
occur through administration of two or more substances in the same
dosage form. In other embodiments, concomitant administration may
encompass administration of two or more substances in different
dosage forms, but within a specified period of time, preferably
within 1 month, more preferably within 1 week, still more
preferably within 1 day, and even more preferably within 1
hour.
[0077] "Couple" or "Coupled" or "Couples" (and the like) means to
chemically associate one entity (for example a moiety) with
another. In some embodiments, the coupling is covalent, meaning
that the coupling occurs in the context of the presence of a
covalent bond between the two entities. In non-covalent
embodiments, the non-covalent coupling is mediated by non-covalent
interactions including but not limited to charge interactions,
affinity interactions, metal coordination, physical adsorption,
host-guest interactions, hydrophobic interactions, TT stacking
interactions, hydrogen bonding interactions, van der Waals
interactions, magnetic interactions, electrostatic interactions,
dipole-dipole interactions, and/or combinations thereof. In
embodiments, encapsulation is a form of coupling.
[0078] "Derived" means prepared from a material or information
related to a material but is not "obtained" from the material. Such
materials may be substantially modified or processed forms of
materials taken directly from a biological material. Such materials
also include materials produced from information related to a
biological material.
[0079] "Dosage form" means a pharmacologically and/or
immunologically active material in a medium, carrier, vehicle, or
device suitable for administration to a subject.
[0080] "Encapsulate" means to enclose at least a portion of a
substance within a synthetic nanocarrier. In some embodiments, a
substance is enclosed completely within a synthetic nanocarrier. In
other embodiments, most or all of a substance that is encapsulated
is not exposed to the local environment external to the synthetic
nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%,
10% or 5% (weight/weight) is exposed to the local environment.
Encapsulation is distinct from absorption, which places most or all
of a substance on a surface of a synthetic nanocarrier, and leaves
the substance exposed to the local environment external to the
synthetic nanocarrier.
[0081] "Epitope", also known as an antigenic determinant, is the
part of an antigen that is recognized by the immune system,
specifically by, for example, antibodies, B cells, or T cells. As
used herein, "MHC Class I-restricted epitopes" are epitopes that
are presented to immune cells by MHC class I molecules found on
nucleated cells. "MHC Class II-restricted epitopes" are epitopes
that are presented to immune cells by MHC class II molecules found
on antigen-presenting cells (APCs), for example, on professional
antigen-presenting immune cells, such as on macrophages, B cells,
and dendritic cells, or on non-hematopoietic cells, such as
hepatocytes. "B cell epitopes" are molecular structures that are
recognized by antibodies or B cells. In some embodiments, the
epitope itself is an antigen.
[0082] A number of epitopes are known to those of skill in the art,
and exemplary epitopes suitable according to some aspects of this
invention include, but are not limited to those listed in the
Immune Epitope Database (www.immuneepitope.org, Vita R, Zarebski L,
Greenbaum J A, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters
B. The immune epitope database 2.0. Nucleic Acids Res. 2010
January; 38 (Database issue):D854-62; the entire contents of which
as well as all database entries of IEDB version 2.4, August 2011,
and particularly all epitopes disclosed therein, are incorporated
herein by reference). Epitopes can also be identified with publicly
available algorithms, for example, the algorithms described in Wang
P, Sidney J, Kim Y, Sette A, Lund 0, Nielsen M, Peters B. 2010.
peptide binding predictions for HLA DR, DP and DQ molecules. BMC
Bioinformatics 2010, 11:568; Wang P, Sidney J, Dow C, Mothe B,
Sette A, Peters B. 2008. A systematic assessment of MHC class II
peptide binding predictions and evaluation of a consensus approach.
PLoS Comput Biol. 4 (4):e1000048; Nielsen M, Lund O. 2009.
NN-align. An artificial neural network-based alignment algorithm
for MHC class II peptide binding prediction. BMC Bioinformatics.
10:296; Nielsen M, Lundegaard C, Lund O. 2007. Prediction of MHC
class II binding affinity using SMM-align, a novel stabilization
matrix alignment method. BMC Bioinformatics. 8:238; Bui H H, Sidney
J, Peters B, Sathiamurthy M, Sinichi A, Purton K A, Mothe B R,
Chisari F V, Watkins D I, Sette A. 2005. Immunogenetics.
57:304-314; Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O,
Sahin U, Braxenthaler M, Gallazzi F, Protti M P, Sinigaglia F,
Hammer J. 1999. Generation of tissue-specific and promiscuous HLA
ligand databases using DNA microarrays and virtual HLA class II
matrices. Nat Biotechnol. 17(6):555-561; Nielsen M, Lundegaard C,
Worning P, Lauemoller S L, Lamberth K, Buus S, Brunak S, Lund O.
2003. Reliable prediction of T-cell epitopes using neural networks
with novel sequence representations. Protein Sci 12:1007-1017; Bui
H H, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton K A,
Mothe B R, Chisari F V, Watkins D I, Sette A. 2005. Automated
generation and evaluation of specific MHC binding predictive tools:
ARB matrix applications. Immunogenetics 57:304-314; Peters B, Sette
A. 2005. Generating quantitative models describing the sequence
specificity of biological processes with the stabilized matrix
method. BMC Bioinformatics 6:132; Chou P Y, Fasman G D. 1978.
Prediction of the secondary structure of proteins from their amino
acid sequence. Adv Enzymol Relat Areas Mol Biol 47:45-148; Emini E
A, Hughes J V, Perlow D S, Boger J. 1985. Induction of hepatitis A
virus-neutralizing antibody by a virus-specific synthetic peptide.
J Virol 55:836-839; Karplus P A, Schulz G E. 1985. Prediction of
chain flexibility in proteins. Naturwissenschaften 72:212-213;
Kolaskar A S, Tongaonkar P C. 1990. A semi-empirical method for
prediction of antigenic determinants on protein antigens. FEBS Lett
276:172-174; Parker J M, Guo D, Hodges R S. 1986. New
hydrophilicity scale derived from high-performance liquid
chromatography peptide retention data: correlation of predicted
surface residues with antigenicity and X-ray-derived accessible
sites. Biochemistry 25:5425-5432; Larsen J E, Lund O, Nielsen M.
2006. Improved method for predicting linear B-cell epitopes.
Immunome Res 2:2; Ponomarenko J V, Bourne P E. 2007.
Antibody-protein interactions: benchmark datasets and prediction
tools evaluation. BMC Struct Biol 7:64; Haste Andersen P, Nielsen
M, Lund O. 2006. Prediction of residues in discontinuous B-cell
epitopes using protein 3D structures. Protein Sci 15:2558-2567;
Ponomarenko J V, Bui H, Li W, Fusseder N, Bourne P E, Sette A,
Peters B. 2008. ElliPro: a new structure-based tool for the
prediction of antibody epitopes. BMC Bioinformatics 9:514; Nielsen
M, Lundegaard C, Blicher T, Peters B, Sette A, Justesen S, Buus S,
and Lund O. 2008. PLoS Comput Biol. 4 (7)e1000107. Quantitative
predictions of peptide binding to any HLA-DR molecule of known
sequence: NetMHCIIpan; the entire contents of each of which are
incorporated herein by reference for disclosure of methods and
algorithms for the identification of epitopes.
[0083] Other examples of epitopes as provided herein include any of
the allergen-associated MHC Class II-restricted and B cell epitopes
as provided as SEQ ID NOs: 1-516. Without wishing to being bound by
any particular theory, MHC Class II-restricted epitopes include
those set forth in SEQ ID NOs: 1-338 and B cell epitopes include
those set forth in SEQ ID NOs: 339-516.
[0084] "Generating" means causing an action, such as an immune
response (e.g., a tolerogenic immune response) to occur, either
directly oneself or indirectly, such as, but not limited to, an
unrelated third party that takes an action through reliance on
one's words or deeds.
[0085] "Identifying" is any action or set of actions that allows a
clinician to recognize a subject as one who may benefit from the
methods and compositions provided herein. Preferably, the
identified subject is one who is in need of a tolerogenic immune
response as provided herein. The action or set of actions may be
either directly oneself or indirectly, such as, but not limited to,
an unrelated third party that takes an action through reliance on
one's words or deeds.
[0086] "Immunosuppressant" means a compound that causes an APC to
have an immunosuppressive (e.g., tolerogenic effect). An
immunosuppressive effect generally refers to the production or
expression of cytokines or other factors by the APC that reduces,
inhibits or prevents an undesired immune response or that promotes
a desired immune response. When the APC results in an
immunosuppressive effect on immune cells that recognize an antigen
presented by the APC, the immunosuppressive effect is said to be
specific to the presented antigen. Such effect is also referred to
herein as a tolerogenic effect. Without being bound by any
particular theory, it is thought that the immunosuppressive or
tolerogenic effect is a result of the immunosuppressant being
delivered to the APC, preferably in the presence of an antigen
(e.g., an administered antigen or one that is already present in
vivo). Accordingly, the immunosuppressant includes compounds that
provide a tolerogenic immune response to an antigen that may or may
not be provided in the same composition or a different composition.
In one embodiment, the immunosuppressant is one that causes an APC
to promote a regulatory phenotype in one or more immune effector
cells. For example, the regulatory phenotype may be characterized
by the inhibition of the production, induction, stimulation or
recruitment of allergen-specific CD4+ T cells or B cells, the
inhibition of the production of allergen-specific antibodies, the
production, induction, stimulation or recruitment of Treg cells
(e.g., CD4+CD25highFoxP3+ Treg cells), etc. This may be the result
of the conversion of CD4+ T cells or B cells to a regulatory
phenotype. This may also be the result of induction of FoxP3 in
other immune cells, such as CD8+ T cells, macrophages and iNKT
cells. In one embodiment, the immunosuppressant is one that affects
the response of the APC after it processes an antigen. In another
embodiment, the immunosuppressant is not one that interferes with
the processing of the antigen. In a further embodiment, the
immunosuppressant is not an apoptotic-signaling molecule. In
another embodiment, the immunosuppressant is not a
phospholipid.
[0087] Immunosuppressants include, but are not limited to, statins;
mTOR inhibitors, such as rapamycin or a rapamycin analog;
TGF-.beta. signaling agents; TGF-.beta. receptor agonists; histone
deacetylase inhibitors, such as Trichostatin A; corticosteroids;
inhibitors of mitochondrial function, such as rotenone; P38
inhibitors; NF-.kappa..beta. inhibitors, such as 6Bio,
Dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists;
prostaglandin E2 agonists (PGE2), such as Misoprostol;
phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitor
(PDE4), such as Rolipram; proteasome inhibitors; kinase inhibitors;
G-protein coupled receptor agonists; G-protein coupled receptor
antagonists; glucocorticoids; retinoids; cytokine inhibitors;
cytokine receptor inhibitors; cytokine receptor activators;
peroxisome proliferator-activated receptor antagonists; peroxisome
proliferator-activated receptor agonists; histone deacetylase
inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3 KB
inhibitors, such as TGX-221; autophagy inhibitors, such as
3-Methyladenine; aryl hydrocarbon receptor inhibitors; proteasome
inhibitor I (PSI); and oxidized ATPs, such as P2X receptor
blockers. Immunosuppressants also include IDO, vitamin D3,
cyclosporins, such as cyclosporine A, aryl hydrocarbon receptor
inhibitors, resveratrol, azathiopurine (Aza), 6-mercaptopurine
(6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol,
mycophenolate mofetil (MMF), aspirin and other COX inhibitors,
niflumic acid, estriol and triptolide. In embodiments, the
immunosuppressant may comprise any of the agents provided
herein.
[0088] The immunosuppressant can be a compound that directly
provides the immunosuppressive (e.g., tolerogenic) effect on APCs
or it can be a compound that provides the immunosuppressive (e.g.,
tolerogenic) effect indirectly (i.e., after being processed in some
way after administration). Immunosuppressants, therefore, include
prodrug forms of any of the compounds provided herein.
[0089] Immunosuppressants also include nucleic acids that encode
the peptides, polypeptides or proteins provided herein that result
in an immunosuppressive (e.g., tolerogenic) immune response. In
embodiments, therefore, the immunosuppressant is a nucleic acid
that encodes a peptide, polypeptide or protein that results in an
immunosuppressive (e.g., tolerogenic) immune response, and it is
the nucleic acid that is coupled to the synthetic nanocarrier.
[0090] The nucleic acid may be DNA or RNA, such as mRNA. In
embodiments, the inventive compositions comprise a complement, such
as a full-length complement, or a degenerate (due to degeneracy of
the genetic code) of any of the nucleic acids provided herein. In
embodiments, the nucleic acid is an expression vector that can be
transcribed when transfected into a cell line. In embodiments, the
expression vector may comprise a plasmid, retrovirus, or an
adenovirus amongst others. Nucleic acids can be isolated or
synthesized using standard molecular biology approaches, for
example by using a polymerase chain reaction to produce a nucleic
acid fragment, which is then purified and cloned into an expression
vector. Additional techniques useful in the practice of this
invention may be found in Current Protocols in Molecular Biology
2007 by John Wiley and Sons, Inc.; Molecular Cloning: A Laboratory
Manual (Third Edition) Joseph Sambrook, Peter MacCallum Cancer
Institute, Melbourne, Australia; David Russell, University of Texas
Southwestern Medical Center, Dallas, Cold Spring Harbor.
[0091] In embodiments, the immunosuppressants provided herein are
coupled to synthetic nanocarriers. In preferable embodiments, the
immunosuppressant is an element that is in addition to the material
that makes up the structure of the synthetic nanocarrier. For
example, in one embodiment, where the synthetic nanocarrier is made
up of one or more polymers, the immunosuppressant is a compound
that is in addition and coupled to the one or more polymers. As
another example, in one embodiment, where the synthetic nanocarrier
is made up of one or more lipids, the immunosuppressant is again in
addition and coupled to the one or more lipids. In embodiments,
such as where the material of the synthetic nanocarrier also
results in an immunosuppressive (e.g., tolerogenic) effect, the
immunosuppressant is an element present in addition to the material
of the synthetic nanocarrier that results in an immunosuppressive
(e.g., tolerogenic) effect.
[0092] Other exemplary immunosuppressants include, but are not
limited, small molecule drugs, natural products, antibodies (e.g.,
antibodies against CD20, CD3, CD4), biologics-based drugs,
carbohydrate-based drugs, nanoparticles, liposomes, RNAi, antisense
nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod;
natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506), etc.
Further immunosuppressants, are known to those of skill in the art,
and the invention is not limited in this respect.
[0093] "Load" of the immunosuppressant or antigen is the amount of
the immunosuppressant or antigen coupled to a synthetic nanocarrier
based on the total weight of materials in an entire synthetic
nanocarrier (weight/weight). Generally, the load is calculated as
an average across a population of synthetic nanocarriers. In one
embodiment, the load of the immunosuppressant on average across the
first population of synthetic nanocarriers is between 0.0001% and
50%. In another embodiment, the load of the antigen on average
across the first and/or second population of synthetic nanocarriers
is between 0.0001% and 50%. In yet another embodiment, the load of
the immunosuppressant and/or antigen is between 0.01% and 20%. In a
further embodiment, the load of the immunosuppressant and/or
antigen is between 0.1% and 10%. In still a further embodiment, the
load of the immunosuppressant and/or antigen is between 1% and 10%.
In yet another embodiment, the load of the immunosuppressant and/or
the antigen is at least 0.1%, at least 0.2%, at least 0.3%, at
least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least
0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at
least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at
least 9%, at least 10%, at least 11%, at least 12%, at least 13%,
at least 14%, at least 15%, at least 16%, at least 17%, at least
18%, at least 19% or at least 20% on average across a population of
synthetic nanocarriers. In yet a further embodiment, the load of
the immunosuppressant and/or the antigen is 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% on average
across a population of synthetic nanocarriers. In some embodiments
of the above embodiments, the load of the immunosuppressant and/or
the antigen is no more than 25% on average across a population of
synthetic nanocarriers. In embodiments, the load is calculated as
described in the Examples.
[0094] In embodiments of any of the compositions and methods
provided, the load may be calculated as follows: Approximately 3 mg
of synthetic nanocarriers are collected and centrifuged to separate
supernatant from synthetic nanocarrier pellet. Acetonitrile is
added to the pellet, and the sample is sonicated and centrifuged to
remove any insoluble material. The supernatant and pellet are
injected on RP-HPLC and absorbance is read at 278 nm. The .mu.g
found in the pellet is used to calculate % entrapped (load), .mu.g
in supernatant and pellet are used to calculate total .mu.g
recovered.
[0095] "Maintenance dose" refers to a dose that is administered to
a subject, after an initial dose has resulted in an
immunosuppressive (e.g., tolerogenic) response in a subject, to
sustain a desired immunosuppressive (e.g., tolerogenic) response. A
maintenance dose, for example, can be one that maintains the
tolerogenic effect achieved after the initial dose, prevents an
undesired immune response in the subject, or prevents the subject
becoming a subject at risk of experiencing an undesired immune
response, including an undesired level of an immune response. In
some embodiments, the maintenance dose is one that is sufficient to
sustain an appropriate level of a desired immune response.
[0096] "Maximum dimension of a synthetic nanocarrier" means the
largest dimension of a nanocarrier measured along any axis of the
synthetic nanocarrier. "Minimum dimension of a synthetic
nanocarrier" means the smallest dimension of a synthetic
nanocarrier measured along any axis of the synthetic nanocarrier.
For example, for a spheroidal synthetic nanocarrier, the maximum
and minimum dimension of a synthetic nanocarrier would be
substantially identical, and would be the size of its diameter.
Similarly, for a cuboidal synthetic nanocarrier, the minimum
dimension of a synthetic nanocarrier would be the smallest of its
height, width or length, while the maximum dimension of a synthetic
nanocarrier would be the largest of its height, width or length. In
an embodiment, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or greater than 100 nm. In
an embodiment, a maximum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is equal to or less than 5 .mu.m.
Preferably, a minimum dimension of at least 75%, preferably at
least 80%, more preferably at least 90%, of the synthetic
nanocarriers in a sample, based on the total number of synthetic
nanocarriers in the sample, is greater than 110 nm, more preferably
greater than 120 nm, more preferably greater than 130 nm, and more
preferably still greater than 150 nm. Aspects ratios of the maximum
and minimum dimensions of inventive synthetic nanocarriers may vary
depending on the embodiment. For instance, aspect ratios of the
maximum to minimum dimensions of the synthetic nanocarriers may
vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1,
more preferably from 1:1 to 10,000:1, more preferably from 1:1 to
1000:1, still more preferably from 1:1 to 100:1, and yet more
preferably from 1:1 to 10:1. Preferably, a maximum dimension of at
least 75%, preferably at least 80%, more preferably at least 90%,
of the synthetic nanocarriers in a sample, based on the total
number of synthetic nanocarriers in the sample is equal to or less
than 3 .mu.m, more preferably equal to or less than 2 .mu.m, more
preferably equal to or less than 1 .mu.m, more preferably equal to
or less than 800 nm, more preferably equal to or less than 600 nm,
and more preferably still equal to or less than 500 nm. In
preferred embodiments, a minimum dimension of at least 75%,
preferably at least 80%, more preferably at least 90%, of the
synthetic nanocarriers in a sample, based on the total number of
synthetic nanocarriers in the sample, is equal to or greater than
100 nm, more preferably equal to or greater than 120 nm, more
preferably equal to or greater than 130 nm, more preferably equal
to or greater than 140 nm, and more preferably still equal to or
greater than 150 nm. Measurement of synthetic nanocarrier
dimensions (e.g., diameter) is obtained by suspending the synthetic
nanocarriers in a liquid (usually aqueous) media and using dynamic
light scattering (DLS) (e.g. using a Brookhaven ZetaPALS
instrument). For example, a suspension of synthetic nanocarriers
can be diluted from an aqueous buffer into purified water to
achieve a final synthetic nanocarrier suspension concentration of
approximately 0.01 to 0.1 mg/mL. The diluted suspension may be
prepared directly inside, or transferred to, a suitable cuvette for
DLS analysis. The cuvette may then be placed in the DLS, allowed to
equilibrate to the controlled temperature, and then scanned for
sufficient time to acquire a stable and reproducible distribution
based on appropriate inputs for viscosity of the medium and
refractive indicies of the sample. The effective diameter, or mean
of the distribution, is then reported. "Dimension" or "size" or
"diameter" of synthetic nanocarriers means the mean of a particle
size distribution obtained using dynamic light scattering.
[0097] "MHC" refers to major histocompatibility complex, a large
genomic region or gene family found in most vertebrates that
encodes MHC molecules that display fragments or epitopes of
processed proteins on the cell surface. The presentation of
MHC:peptide on cell surfaces allows for surveillance by immune
cells, usually a T cell. There are two general classes of MHC
molecules: Class I and Class II. Generally, Class I MHC molecules
are found on nucleated cells and present peptides to cytotoxic T
cells. Class II MHC molecules are found on certain immune cells,
chiefly macrophages, B cells and dendritic cells, collectively
known as professional APCs. The best-known genes in the MHC region
are the subset that encodes antigen-presenting proteins on the cell
surface. In humans, these genes are referred to as human leukocyte
antigen (HLA) genes.
[0098] "Non-methoxy-terminated polymer" means a polymer that has at
least one terminus that ends with a moiety other than methoxy. In
some embodiments, the polymer has at least two termini that ends
with a moiety other than methoxy. In other embodiments, the polymer
has no termini that ends with methoxy. "Non-methoxy-terminated,
pluronic polymer" means a polymer other than a linear pluronic
polymer with methoxy at both termini. Polymeric nanoparticles as
provided herein can comprise non-methoxy-terminated polymers or
non-methoxy-terminated, pluronic polymers.
[0099] "Obtained" means taken directly from a material and used
with substantially no modification and/or processing.
[0100] "Pharmaceutically acceptable excipient" means a
pharmacologically inactive material used together with the recited
synthetic nanocarriers to formulate the inventive compositions.
Pharmaceutically acceptable excipients comprise a variety of
materials known in the art, including but not limited to
saccharides (such as glucose, lactose, and the like), preservatives
such as antimicrobial agents, reconstitution aids, colorants,
saline (such as phosphate buffered saline), and buffers.
[0101] "Protocol" refers to any dosing regimen of one or more
substances to a subject. A dosing regimen may include the amount,
frequency and/or mode of administration. In some embodiments, such
a protocol may be used to administer one or more compositions of
the invention to one or more test subjects. Immune responses in
these test subject can then be assessed to determine whether or not
the protocol was effective in reducing an undesired immune response
or generating a desired immune response (e.g., the promotion of a
tolerogenic effect). Any other therapeutic and/or prophylactic
effect may also be assessed instead of or in addition to the
aforementioned immune responses. Whether or not a protocol had a
desired effect can be determined using any of the methods provided
herein or otherwise known in the art. For example, a population of
cells may be obtained from a subject to which a composition
provided herein has been administered according to a specific
protocol in order to determine whether or not specific immune
cells, cytokines, antibodies, etc. were reduced, generated,
activated, etc. Useful methods for detecting the presence and/or
number of immune cells include, but are not limited to, flow
cytometric methods (e.g., FACS) and immunohistochemistry methods.
Antibodies and other binding agents for specific staining of immune
cell markers, are commercially available. Such kits typically
include staining reagents for multiple antigens that allow for
FACS-based detection, separation and/or quantitation of a desired
cell population from a heterogeneous population of cells.
[0102] "Providing a subject" is any action or set of actions that
causes a clinician to come in contact with a subject and administer
a composition provided herein thereto or to perform a method
provided herein thereupon. Preferably, the subject is one who is in
need of a tolerogenic immune response as provided herein. The
action or set of actions may be either directly oneself or
indirectly, such as, but not limited to, an unrelated third party
that takes an action through reliance on one's words or deeds.
[0103] "Subject" means animals, including warm blooded mammals such
as humans and primates; avians; domestic household or farm animals
such as cats, dogs, sheep, goats, cattle, horses and pigs;
laboratory animals such as mice, rats and guinea pigs; fish;
reptiles; zoo and wild animals; and the like.
[0104] "Substantially no B cell epitopes" refers to the absence of
B cell epitopes in an amount (by itself, within the context of the
allergen, in conjunction with a carrier or in conjunction with an
inventive composition) that stimulates substantial activation of a
B cell response. In embodiments, a composition with substantially
no B cell epitopes does not contain a measurable amount of B cell
epitopes of an allergen. In other embodiments, such a composition
may comprise a measurable amount of B cell epitopes of an allergen
but said amount is not effective to generate a measurable B cell
immune response (by itself, within the context of the antigen, in
conjunction with a carrier, or in conjunction with an inventive
composition), such as allergen-specific antibody production or
allergen-specific B cell proliferation and/or activity, or is not
effective to generate a significant measurable B cell immune
response (by itself, within the context of the antigen, in
conjunction with a carrier or in conjunction with an inventive
composition). In some embodiments, a significant measurable B cell
immune response is one that produces or would be expected to
produce an adverse clinical result in a subject. In other
embodiments, a significant measurable B cell immune response is one
that is greater than the level of the same type of immune response
(e.g., allergen-specific antibody production or allergen-specific B
cell proliferation and/or activity) produced by a control antigen
(e.g., one known not to comprise B cell epitopes of the allergen or
to stimulate B cell immune responses). In some embodiments, a
significant measurable B cell immune response, such as a
measurement of antibody titers (e.g., by ELISA) is 2-fold, 3-fold,
4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold,
20-fold or more greater than the same type of response produced by
a control (e.g., a control antigen). In other embodiments, a
composition with substantially no B cell epitopes is one that
produces little to no allergen-specific antibody titers (by itself,
within the context of the antigen, in conjunction with a carrier or
in conjunction with an inventive composition). Such compositions
include those that produce an antibody titer (as an EC50 value) of
less than 500, 400, 300, 200, 100, 50, 40, 30, 20 or 10. In other
embodiments, a significant measurable B cell immune response, is a
measurement of the number or proliferation of B cells that is 10%,
25%, 50%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, 10-fold, 15-fold, 20-fold or more greater that the
same type of response produced by a control. Other methods for
measuring B cell responses are known to those of ordinary skill in
the art. In embodiments, to ensure that a composition comprises
substantially no B cell epitopes, antigens are selected such that
they do not comprise B cell epitopes for coupling to the synthetic
nanocarriers as provided herein. In other embodiments, to ensure
that a composition comprises substantially no B cell epitopes of an
allergen, the synthetic nanocarriers coupled to the epitopes are
produced and tested for B cell immune responses (e.g., B cell
proliferation and/or activity, allergen-specific antibody
production). Compositions that exhibit the desired properties may
then be selected.
[0105] "Synthetic nanocarrier(s)" means a discrete object that is
not found in nature, and that possesses at least one dimension that
is less than or equal to 5 microns in size. Albumin nanoparticles
are generally included as synthetic nanocarriers, however in
certain embodiments the synthetic nanocarriers do not comprise
albumin nanoparticles. In embodiments, inventive synthetic
nanocarriers do not comprise chitosan. In other embodiments,
inventive synthetic nanocarriers are not lipid-based nanoparticles.
In further embodiments, inventive synthetic nanocarriers do not
comprise a phospholipid.
[0106] A synthetic nanocarrier can be, but is not limited to, one
or a plurality of lipid-based nanoparticles (also referred to
herein as lipid nanoparticles, i.e., nanoparticles where the
majority of the material that makes up their structure are lipids),
polymeric nanoparticles, metallic nanoparticles, surfactant-based
emulsions, dendrimers, buckyballs, nanowires, virus-like particles
(i.e., particles that are primarily made up of viral structural
proteins but that are not infectious or have low infectivity),
peptide or protein-based particles (also referred to herein as
protein particles, i.e., particles where the majority of the
material that makes up their structure are peptides or proteins)
(such as albumin nanoparticles) and/or nanoparticles that are
developed using a combination of nanomaterials such as
lipid-polymer nanoparticles. Synthetic nanocarriers may be a
variety of different shapes, including but not limited to
spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and
the like. Synthetic nanocarriers according to the invention
comprise one or more surfaces. Exemplary synthetic nanocarriers
that can be adapted for use in the practice of the present
invention comprise: (1) the biodegradable nanoparticles disclosed
in U.S. Pat. No. 5,543,158 to Gref et al., (2) the polymeric
nanoparticles of Published US Patent Application 20060002852 to
Saltzman et al., (3) the lithographically constructed nanoparticles
of Published US Patent Application 20090028910 to DeSimone et al.,
(4) the disclosure of WO 2009/051837 to von Andrian et al., (5) the
nanoparticles disclosed in Published US Patent Application
2008/0145441 to Penades et al., (6) the protein nanoparticles
disclosed in Published US Patent Application 20090226525 to de los
Rios et al., (7) the virus-like particles disclosed in published US
Patent Application 20060222652 to Sebbel et al., (8) the nucleic
acid coupled virus-like particles disclosed in published US Patent
Application 20060251677 to Bachmann et al., (9) the virus-like
particles disclosed in WO2010047839A1 or WO2009106999A2, (10) the
nanoprecipitated nanoparticles disclosed in P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010), or (11) apoptotic cells, apoptotic bodies or
the synthetic or semisynthetic mimics disclosed in U.S. Publication
2002/0086049. In embodiments, synthetic nanocarriers may possess an
aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0107] Synthetic nanocarriers according to the invention that have
a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
with hydroxyl groups that activate complement or alternatively
comprise a surface that consists essentially of moieties that are
not hydroxyl groups that activate complement. In a preferred
embodiment, synthetic nanocarriers according to the invention that
have a minimum dimension of equal to or less than about 100 nm,
preferably equal to or less than 100 nm, do not comprise a surface
that substantially activates complement or alternatively comprise a
surface that consists essentially of moieties that do not
substantially activate complement. In a more preferred embodiment,
synthetic nanocarriers according to the invention that have a
minimum dimension of equal to or less than about 100 nm, preferably
equal to or less than 100 nm, do not comprise a surface that
activates complement or alternatively comprise a surface that
consists essentially of moieties that do not activate complement.
In embodiments, synthetic nanocarriers exclude virus-like
particles. In embodiments, synthetic nanocarriers may possess an
aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or
greater than 1:10.
[0108] "T cell antigen" means a CD4+ T-cell antigen or CD8+ cell
antigen. "CD4+ T-cell antigen" means any antigen that is recognized
by and triggers an immune response in a CD4+ T-cell e.g., an
antigen that is specifically recognized by a T-cell receptor on a
CD4+ T cell via presentation of the antigen or portion thereof
bound to a Class II major histocompatability complex molecule
(MHC). "CD8+ T cell antigen" means any antigen that is recognized
by and triggers an immune response in a CD8+ T-cell e.g., an
antigen that is specifically recognized by a T-cell receptor on a
CD8+ T cell via presentation of the antigen or portion thereof
bound to a Class I major histocompatability complex molecule (MHC).
In some embodiments, an antigen that is a T cell antigen is also a
B cell antigen. In other embodiments, the T cell antigen is not
also a B cell antigen. T cell antigens generally are proteins or
peptides.
[0109] "Tolerogenic immune response" means any immune response that
can lead to immune suppression specific to an antigen or a cell,
tissue, organ, etc. that expresses such an antigen. Such immune
responses include any reduction, delay or inhibition in an
undesired immune response specific to the antigen or cell, tissue,
organ, etc. that expresses such antigen. Such immune responses also
include any stimulation, production, induction, promotion or
recruitment in a desired immune response specific to the antigen or
cell, tissue, organ, etc. that expresses such antigen. Tolerogenic
immune responses, therefore, include the absence of or reduction in
an undesired immune response to an antigen that can be mediated by
antigen reactive cells as well as the presence or promotion of
suppressive cells. Tolerogenic immune responses as provided herein
include immunological tolerance. To "generate a tolerogenic immune
response" refers to the generation of any of the foregoing immune
responses specific to an antigen or cell, tissue, organ, etc. that
expresses such antigen. The tolerogenic immune response can be the
result of MHC Class I-restricted presentation and/or MHC Class
II-restricted presentation and/or B cell presentation and/or
presentation by CD1d, etc.
[0110] Tolerogenic immune responses include any reduction, delay or
inhibition in CD4+ T cell, CD8+ T cell or B cell proliferation
and/or activity. Tolerogenic immune responses also include a
reduction in antigen-specific antibody production. Tolerogenic
immune responses can also include any response that leads to the
stimulation, induction, production or recruitment of regulatory
cells, such as CD4+ Treg cells, CD8+ Treg cells, Breg cells, etc.
In some embodiments, the tolerogenic immune response, is one that
results in the conversion to a regulatory phenotype characterized
by the production, induction, stimulation or recruitment of
regulatory cells.
[0111] Tolerogenic immune responses also include any response that
leads to the stimulation, production or recruitment of CD4+ Treg
cells and/or CD8+ Treg cells. CD4+ Treg cells can express the
transcription factor FoxP3 and inhibit inflammatory responses and
auto-immune inflammatory diseases (Human regulatory T cells in
autoimmune diseases. Cvetanovich G L, Hafler D A. Curr Opin
Immunol. 2010 December; 22(6):753-60. Regulatory T cells and
autoimmunity. Vila J, Isaacs J D, Anderson A E. Curr Opin Hematol.
2009 July; 16(4):274-9). Such cells also suppress T-cell help to
B-cells and induce tolerance to both self and foreign antigens
(Therapeutic approaches to allergy and autoimmunity based on FoxP3+
regulatory T-cell activation and expansion. Miyara M, Wing K,
Sakaguchi S. J Allergy Clin Immunol. 2009 April; 123(4):749-55).
CD4+ Treg cells recognize antigen when presented by Class II
proteins on APCs. CD8+ Treg cells, which recognize antigen
presented by Class I (and Qa-1), can also suppress T-cell help to
B-cells and result in activation of antigen-specific suppression
inducing tolerance to both self and foreign antigens. Disruption of
the interaction of Qa-1 with CD8+ Treg cells has been shown to
dysregulate immune responses and results in the development of
auto-antibody formation and an auto-immune lethal
systemic-lupus-erythematosus (Kim et al., Nature. 2010 Sep. 16, 467
(7313): 328-32). CD8+Treg cells have also been shown to inhibit
models of autoimmune inflammatory diseases including rheumatoid
arthritis and colitis (CD4+CD25+ regulatory T cells in autoimmune
arthritis. Oh S, Rankin A L, Caton A J. Immunol Rev. 2010 January;
233(1):97-111. Regulatory T cells in inflammatory bowel disease.
Boden E K, Snapper S B. Curr Opin Gastroenterol. 2008 November;
24(6):733-41). In some embodiments, the compositions provided can
effectively result in both types of responses (CD4+ Treg and CD8+
Treg). In other embodiments, FoxP3 can be induced in other immune
cells, such as macrophages, iNKT cells, etc., and the compositions
provided herein can result in one or more of these responses as
well.
[0112] Tolerogenic immune responses also include, but are not
limited to, the induction of regulatory cytokines, such as Treg
cytokines; induction of inhibitory cytokines; the inhibition of
inflammatory cytokines (e.g., IL-4, IL-1b, IL-5, TNF-.alpha., IL-6,
GM-CSF, IFN-.gamma., IL-2, IL-9, IL-12, IL-17, IL-18, IL-21, IL-22,
IL-23, M-CSF, C reactive protein, acute phase protein, chemokines
(e.g., MCP-1, RANTES, MIP-1.alpha., MIP-1.beta., MIG, ITAC or
IP-10), the production of anti-inflammatory cytokines (e.g., IL-4,
IL-13, IL-10, etc.), chemokines (e.g., CCL-2, CXCL8), proteases
(e.g., MMP-3, MMP-9), leukotrienes (e.g., CysLT-1, CysLT-2),
prostaglandins (e.g., PGE2) or histamines; the inhibition of
polarization to a Th17, Th1 or Th2 immune response; the inhibition
of effector cell-specific cytokines: Th17 (e.g., IL-17, IL-25), Th1
(IFN-.gamma.), Th2 (e.g., IL-4, IL-13); the inhibition of Th1-,
Th2- or TH17-specific transcription factors; the inhibition of
proliferation of effector T cells; the induction of apoptosis of
effector T cells; the induction of tolerogenic dendritic
cell-specific genes, the induction of FoxP3 expression, the
inhibition of IgE induction or IgE-mediated immune responses; the
inhibition of antibody responses (e.g., antigen-specific antibody
production); the inhibition of T helper cell response; the
production of TGF-.beta. and/or IL-10; the inhibition of effector
function of autoantibodies (e.g., inhibition in the depletion of
cells, cell or tissue damage or complement activation); etc.
[0113] Any of the foregoing may be measured in vivo in one or more
animal models or may be measured in vitro. One of ordinary skill in
the art is familiar with such in vivo or in vitro measurements.
Undesired immune responses or tolerogenic immune responses can be
monitored using, for example, methods of assessing immune cell
number and/or function, tetramer analysis, ELISPOT, flow
cytometry-based analysis of cytokine expression, cytokine
secretion, cytokine expression profiling, gene expression
profiling, protein expression profiling, analysis of cell surface
markers, PCR-based detection of immune cell receptor gene usage
(see T. Clay et al., "Assays for Monitoring Cellular Immune
Response to Active Immunotherapy of Cancer" Clinical Cancer
Research 7:1127-1135 (2001)), etc. Undesired immune responses or
tolerogenic immune responses may also be monitored using, for
example, methods of assessing protein levels in plasma or serum,
immune cell proliferation and/or functional assays, etc. In some
embodiments, tolerogenic immune responses can be monitored by
assessing the induction of FoxP3. In addition, specific methods are
described in more detail in the Examples.
[0114] Preferably, tolerogenic immune responses lead to the
inhibition of the development, progression or pathology of the
diseases, disorders or conditions described herein. Whether or not
the inventive compositions can lead to the inhibition of the
development, progression or pathology of the diseases, disorders or
conditions described herein can be measured with animal models of
such diseases, disorders or conditions. In some embodiments, the
reduction of an undesired immune response or generation of a
tolerogenic immune response may be assessed by determining clinical
endpoints, clinical efficacy, clinical symptoms, disease biomarkers
and/or clinical scores. Undesired immune responses or tolerogenic
immune responses can also be assessed with diagnostic tests to
assess the presence or absence of a disease, disorder or condition
as provided herein. In embodiments, methods for monitoring or
assessing undesired immune (e.g., allergic) responses include
assessing an immune response in a subject by skin reactivity and/or
allergen-specific antibody production.
[0115] In some embodiments, monitoring or assessing the generation
of an undesired immune response or a tolerogenic immune response in
a subject can be prior to the administration of a composition of
synthetic nanocarriers provided herein and/or prior to exposure to
an allergen. In other embodiments, assessing the generation of an
undesired immune response or tolerogenic immune response can be
after administration of a composition of synthetic nanocarriers
provided herein and/or and after exposure to an allergen. In some
embodiments, the assessment is done after administration of the
composition of synthetic nanocarriers, but prior to exposure to an
allergen. In other embodiments, the assessment is done after
exposure to an allergen, but prior to administration of the
composition. In still other embodiments, the assessment is
performed prior to both the administration of the synthetic
nanocarriers and exposure to an allergen, while in yet other
embodiments the assessment is performed after both the
administration of synthetic nanocarriers and the exposure to an
allergen. In further embodiments, the assessment is performed both
prior to and after the administration of the synthetic nanocarriers
and/or exposure to the allergen. In still other embodiments, the
assessment is performed more than once on the subject to determine
that a desirable immune state is maintained in the subject, such as
a subject that has or is at risk of having an allergy.
[0116] An antibody response can be assessed by determining one or
more antibody titers. "Antibody titer" means a measurable level of
antibody production. Methods for measuring antibody titers are
known in the art and include Enzyme-linked Immunosorbent Assay
(ELISA). In embodiments, the antibody response can be quantitated,
for example, as the number of antibodies, concentration of
antibodies or titer. The values can be absolute or they can be
relative. Assays for quantifying an antibody response include
antibody capture assays, enzyme-linked immunosorbent assays
(ELISAs), inhibition liquid phase absorption assays (ILPAAs),
rocket immunoelectrophoresis (RIE) assays and line
immunoelectrophoresis (LIE) assays. When an antibody response is
compared to another antibody response the same type of quantitative
value (e.g., titer) and method of measurement (e.g., ELISA) is
preferably used to make the comparison.
[0117] An ELISA method for measuring an antibody titer, for
example, a typical sandwich ELISA, may consist of the following
steps (i) preparing an ELISA-plate coating material such that the
antibody target of interest is coupled to a substrate polymer or
other suitable material (ii) preparing the coating material in an
aqueous solution (such as PBS) and delivering the coating material
solution to the wells of a multiwell plate for overnight deposition
of the coating onto the multiwell plate (iii) thoroughly washing
the multiwell plate with wash buffer (such as 0.05% Tween-20 in
PBS) to remove excess coating material (iv) blocking the plate for
nonspecific binding by applying a diluent solution (such as 10%
fetal bovine serum in PBS), (v) washing the blocking/diluent
solution from the plate with wash buffer (vi) diluting the serum
sample(s) containing antibodies and appropriate standards (positive
controls) with diluent as required to obtain a concentration that
suitably saturates the ELISA response (vii) serially diluting the
plasma samples on the multiwell plate such to cover a range of
concentrations suitable for generating an ELISA response curve
(viii) incubating the plate to provide for antibody-target binding
(ix) washing the plate with wash buffer to remove antibodies not
bound to antigen (x) adding an appropriate concentration of a
secondary detection antibody in same diluent such as a
biotin-coupled detection antibody capable of binding the primary
antibody (xi) incubating the plate with the applied detection
antibody, followed by washing with wash buffer (xii) adding an
enzyme such as streptavidin-HRP (horse radish peroxidase) that will
bind to biotin found on biotinylated antibodies and incubating
(xiii) washing the multiwell plate (xiv) adding substrate(s) (such
as TMB solution) to the plate (xv) applying a stop solution (such
as 2N sulfuric acid) when color development is complete (xvi)
reading optical density of the plate wells at a specific wavelength
for the substrate (450 nm with subtraction of readings at 570 nm)
(xvi) applying a suitable multiparameter curve fit to the data and
defining half-maximal effective concentration (EC50) as the
concentration on the curve at which half the maximum OD value for
the plate standards is achieved.
[0118] "Undesired immune response" refers to any undesired immune
response that results from exposure to an antigen, promotes or
exacerbates a disease, disorder or condition provided herein (or a
symptom thereof), or is symptomatic of a disease, disorder or
condition provided herein. Such immune responses generally have a
negative impact on a subject's health or is symptomatic of a
negative impact on a subject's health. Undesired immune responses
include antigen-specific antibody production, antigen-specific B
cell proliferation and/or activity or antigen-specific CD4+ T cell
proliferation and/or activity.
C. INVENTIVE COMPOSITIONS
[0119] Provided herein are tolerogenic synthetic nanocarrier
compositions comprising immunosuppressants and MHC Class
II-restricted epitopes of an allergen and related methods. Such
compositions and methods are useful for reducing the generation of
undesired immune responses (e.g., undesired B cell or CD4+ T cell
proliferation and/or activity, undesired antibody production, etc.)
and promoting the generation of tolerogenic immune responses that
are specific to the allergen. The compositions can be administered
to subjects in which a tolerogenic immune response to an allergen
is desired. Such subjects include those that have been, are being
or will be exposed to an allergen. Such subjects include those that
have experienced, are experiencing or are expected to experience an
undesired immune (e.g., allergic) response to any of the allergens
described herein. Such subjects also include those that have or are
at risk of having an allergy to any of the allergens described
herein.
[0120] As mentioned above, the synthetic nanocarriers are designed
to comprise immunosuppressants and, in some embodiments, MHC Class
II-restricted epitopes of an allergen against which a tolerogenic
effect is desired. A wide variety of synthetic nanocarriers can be
used according to the invention. In some embodiments, synthetic
nanocarriers are spheres or spheroids. In some embodiments,
synthetic nanocarriers are flat or plate-shaped. In some
embodiments, synthetic nanocarriers are cubes or cubic. In some
embodiments, synthetic nanocarriers are ovals or ellipses. In some
embodiments, synthetic nanocarriers are cylinders, cones, or
pyramids.
[0121] In some embodiments, it is desirable to use a population of
synthetic nanocarriers that is relatively uniform in terms of size,
shape, and/or composition so that each synthetic nanocarrier has
similar properties. For example, at least 80%, at least 90%, or at
least 95% of the synthetic nanocarriers, based on the total number
of synthetic nanocarriers, may have a minimum dimension or maximum
dimension that falls within 5%, 10%, or 20% of the average diameter
or average dimension of the synthetic nanocarriers. In some
embodiments, a population of synthetic nanocarriers may be
heterogeneous with respect to size, shape, and/or composition.
[0122] Synthetic nanocarriers can be solid or hollow and can
comprise one or more layers. In some embodiments, each layer has a
unique composition and unique properties relative to the other
layer(s). To give but one example, synthetic nanocarriers may have
a core/shell structure, wherein the core is one layer (e.g. a
polymeric core) and the shell is a second layer (e.g. a lipid
bilayer or monolayer). Synthetic nanocarriers may comprise a
plurality of different layers.
[0123] In some embodiments, synthetic nanocarriers may optionally
comprise one or more lipids. In some embodiments, a synthetic
nanocarrier may comprise a liposome. In some embodiments, a
synthetic nanocarrier may comprise a lipid bilayer. In some
embodiments, a synthetic nanocarrier may comprise a lipid
monolayer. In some embodiments, a synthetic nanocarrier may
comprise a micelle. In some embodiments, a synthetic nanocarrier
may comprise a core comprising a polymeric matrix surrounded by a
lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In some
embodiments, a synthetic nanocarrier may comprise a non-polymeric
core (e.g., metal particle, quantum dot, ceramic particle, bone
particle, viral particle, proteins, nucleic acids, carbohydrates,
etc.) surrounded by a lipid layer (e.g., lipid bilayer, lipid
monolayer, etc.).
[0124] In other embodiments, synthetic nanocarriers may comprise
metal particles, quantum dots, ceramic particles, etc. In some
embodiments, a non-polymeric synthetic nanocarrier is an aggregate
of non-polymeric components, such as an aggregate of metal atoms
(e.g., gold atoms).
[0125] In some embodiments, synthetic nanocarriers may optionally
comprise one or more amphiphilic entities. In some embodiments, an
amphiphilic entity can promote the production of synthetic
nanocarriers with increased stability, improved uniformity, or
increased viscosity. In some embodiments, amphiphilic entities can
be associated with the interior surface of a lipid membrane (e.g.,
lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities
known in the art are suitable for use in making synthetic
nanocarriers in accordance with the present invention. Such
amphiphilic entities include, but are not limited to,
phosphoglycerides; phosphatidylcholines; dipalmitoyl
phosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine
(DOPE); dioleyloxypropyltriethylammonium (DOTMA);
dioleoylphosphatidylcholine; cholesterol; cholesterol ester;
diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol
(DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol
(PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid,
such as palmitic acid or oleic acid; fatty acids; fatty acid
monoglycerides; fatty acid diglycerides; fatty acid amides;
sorbitan trioleate (Span.RTM.85) glycocholate; sorbitan monolaurate
(Span.RTM.20); polysorbate 20 (Tween.RTM.20); polysorbate 60
(Tween.RTM.60); polysorbate 65 (Tween.RTM.65); polysorbate 80
(Tween.RTM.80); polysorbate 85 (Tween.RTM.85); polyoxyethylene
monostearate; surfactin; a poloxomer; a sorbitan fatty acid ester
such as sorbitan trioleate; lecithin; lysolecithin;
phosphatidylserine; phosphatidylinositol; sphingomyelin;
phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic
acid; cerebrosides; dicetylphosphate;
dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;
hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl
sterate; isopropyl myristate; tyloxapol; poly(ethylene
glycol)5000-phosphatidylethanolamine; poly(ethylene
glycol)400-monostearate; phospholipids; synthetic and/or natural
detergents having high surfactant properties; deoxycholates;
cyclodextrins; chaotropic salts; ion pairing agents; and
combinations thereof. An amphiphilic entity component may be a
mixture of different amphiphilic entities. Those skilled in the art
will recognize that this is an exemplary, not comprehensive, list
of substances with surfactant activity. Any amphiphilic entity may
be used in the production of synthetic nanocarriers to be used in
accordance with the present invention.
[0126] In some embodiments, synthetic nanocarriers may optionally
comprise one or more carbohydrates. Carbohydrates may be natural or
synthetic. A carbohydrate may be a derivatized natural
carbohydrate. In certain embodiments, a carbohydrate comprises
monosaccharide or disaccharide, including but not limited to
glucose, fructose, galactose, ribose, lactose, sucrose, maltose,
trehalose, cellbiose, mannose, xylose, arabinose, glucoronic acid,
galactoronic acid, mannuronic acid, glucosamine, galatosamine, and
neuramic acid. In certain embodiments, a carbohydrate is a
polysaccharide, including but not limited to pullulan, cellulose,
microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC),
hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran,
glycogen, hydroxyethylstarch, carageenan, glycon, amylose,
chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,
starch, chitin, inulin, konjac, glucommannan, pustulan, heparin,
hyaluronic acid, curdlan, and xanthan. In embodiments, the
inventive synthetic nanocarriers do not comprise (or specifically
exclude) carbohydrates, such as a polysaccharide. In certain
embodiments, the carbohydrate may comprise a carbohydrate
derivative such as a sugar alcohol, including but not limited to
mannitol, sorbitol, xylitol, erythritol, maltitol, and
lactitol.
[0127] In some embodiments, synthetic nanocarriers can comprise one
or more polymers. In some embodiments, the synthetic nanocarriers
comprise one or more polymers that is a nonmethoxy-terminated,
pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the
polymers that make up the synthetic nanocarriers are
non-methoxy-terminated, pluronic polymers. In some embodiments, all
of the polymers that make up the synthetic nanocarriers are
non-methoxy-terminated, pluronic polymers. In some embodiments, the
synthetic nanocarriers comprise one or more polymers that is a
non-methoxy-terminated polymer. In some embodiments, at least 1%,
2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight)
of the polymers that make up the synthetic nanocarriers are
nonmethoxy-terminated polymers. In some embodiments, all of the
polymers that make up the synthetic nanocarriers are
non-methoxy-terminated polymers. In some embodiments, the synthetic
nanocarriers comprise one or more polymers that does not comprise
of pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight/weight) of the
polymers that make up the synthetic nanocarriers do not comprise
pluronic polymer. In some embodiments, all of the polymers that
make up the synthetic nanocarriers do not comprise pluronic
polymer. In some embodiments, such a polymer can be surrounded by a
coating layer (e.g., liposome, lipid monolayer, micelle, etc.). In
some embodiments, various elements of the synthetic nanocarriers
can be coupled with the polymer.
[0128] The immunosuppressants and/or antigens can be coupled to the
synthetic nanocarriers by any of a number of methods. Generally,
the coupling can be a result of bonding between the
immunosuppressants and/or antigens and the synthetic nanocarriers.
This bonding can result in the immunosuppressants and/or antigens
being attached to the surface of the synthetic nanocarrierss and/or
contained within (encapsulated) the synthetic nanocarriers. In some
embodiments, however, the immunosuppressants and/or antigens are
encapsulated by the synthetic nanocarriers as a result of the
structure of the synthetic nanocarriers rather than bonding to the
synthetic nanocarriers. In preferable embodiments, the synthetic
nanocarriers comprise a polymer as provided herein, and the
immunosuppressants and/or antigens are coupled to the polymer.
[0129] When coupling occurs as a result of bonding between the
immunosuppressants and/or antigens and synthetic nanocarriers, the
coupling may occur via a coupling moiety. A coupling moiety can be
any moiety through which an immunosuppressant and/or antigen is
bonded to a synthetic nanocarrier. Such moieties include covalent
bonds, such as an amide bond or ester bond, as well as separate
molecules that bond (covalently or non-covalently) the
immunosuppressant and/or antigen to the synthetic nanocarrier. Such
molecules include linkers or polymers or a unit thereof. For
example, the coupling moiety can comprise a charged polymer to
which an immunosuppressant and/or antigen electrostatically binds.
As another example, the coupling moiety can comprise a polymer or
unit thereof to which it is covalently bonded.
[0130] In preferred embodiments, the synthetic nanocarriers
comprise a polymer as provided herein. These synthetic nanocarriers
can be completely polymeric or they can be a mix of polymers and
other materials.
[0131] In some embodiments, the polymers of a synthetic nanocarrier
associate to form a polymeric matrix. In some of these embodiments,
a component, such as an immunosuppressant or antigen can be
covalently associated with one or more polymers of the polymeric
matrix. In some embodiments, covalent association is mediated by a
linker. In some embodiments, a component can be noncovalently
associated with one or more polymers of the polymeric matrix. For
example, in some embodiments a component can be encapsulated
within, surrounded by, and/or dispersed throughout the polymeric
matrix. Alternatively or additionally, a component can be
associated with one or more polymers of a polymeric matrix by
hydrophobic interactions, charge interactions, van der Waals
forces, etc. A wide variety of polymers and methods for forming
polymeric matrices therefrom are known conventionally.
[0132] Polymers may be natural or unnatural (synthetic) polymers.
Polymers may be homopolymers or copolymers comprising two or more
monomers. In terms of sequence, copolymers may be random, block, or
comprise a combination of random and block sequences. Typically,
polymers in accordance with the present invention are organic
polymers.
[0133] In some embodiments, the polymer comprises a polyester,
polycarbonate, polyamide, or polyether, or unit thereof. In other
embodiments, the polymer comprises poly(ethylene glycol) (PEG),
polypropylene glycol, poly(lactic acid), poly(glycolic acid),
poly(lactic-co-glycolic acid), or a polycaprolactone, or unit
thereof. In some embodiments, it is preferred that the polymer is
biodegradable. Therefore, in these embodiments, it is preferred
that if the polymer comprises a polyether, such as poly(ethylene
glycol) or polypropylene glycol or unit thereof, the polymer
comprises a block-co-polymer of a polyether and a biodegradable
polymer such that the polymer is biodegradable. In other
embodiments, the polymer does not solely comprise a polyether or
unit thereof, such as poly(ethylene glycol) or polypropylene glycol
or unit thereof.
[0134] Other examples of polymers suitable for use in the present
invention include, but are not limited to polyethylenes,
polycarbonates (e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g.
poly(sebacic anhydride)), polypropylfumerates, polyamides (e.g.
polycaprolactam), polyacetals, polyethers, polyesters (e.g.,
polylactide, polyglycolide, polylactide-co-glycolide,
polycaprolactone, polyhydroxy acid (e.g.
poly(.beta.-hydroxyalkanoate))), poly(orthoesters),
polycyanoacrylates, polyvinyl alcohols, polyurethanes,
polyphosphazenes, polyacrylates, polymethacrylates, polyureas,
polystyrenes, and polyamines, polylysine, polylysine-PEG
copolymers, and poly(ethyleneimine), poly(ethylene imine)-PEG
copolymers.
[0135] In some embodiments, polymers in accordance with the present
invention include polymers which have been approved for use in
humans by the U.S. Food and Drug Administration (FDA) under 21
C.F.R. .sctn.177.2600, including but not limited to polyesters
(e.g., polylactic acid, poly(lactic-co-glycolic acid),
polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2one));
polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g.,
polyethylene glycol); polyurethanes; polymethacrylates;
polyacrylates; and polycyanoacrylates.
[0136] In some embodiments, polymers can be hydrophilic. For
example, polymers may comprise anionic groups (e.g., phosphate
group, sulphate group, carboxylate group); cationic groups (e.g.,
quaternary amine group); or polar groups (e.g., hydroxyl group,
thiol group, amine group). In some embodiments, a synthetic
nanocarrier comprising a hydrophilic polymeric matrix generates a
hydrophilic environment within the synthetic nanocarrier. In some
embodiments, polymers can be hydrophobic. In some embodiments, a
synthetic nanocarrier comprising a hydrophobic polymeric matrix
generates a hydrophobic environment within the synthetic
nanocarrier. Selection of the hydrophilicity or hydrophobicity of
the polymer may have an impact on the nature of materials that are
incorporated (e.g., coupled) within the synthetic nanocarrier.
[0137] In some embodiments, polymers may be modified with one or
more moieties and/or functional groups. A variety of moieties or
functional groups can be used in accordance with the present
invention. In some embodiments, polymers may be modified with
polyethylene glycol (PEG), with a carbohydrate, and/or with acyclic
polyacetals derived from polysaccharides (Papisov, 2001, ACS
Symposium Series, 786:301). Certain embodiments may be made using
the general teachings of U.S. Pat. No. 5,543,158 to Gref et al., or
WO publication WO2009/051837 by Von Andrian et al.
[0138] In some embodiments, polymers may be modified with a lipid
or fatty acid group. In some embodiments, a fatty acid group may be
one or more of butyric, caproic, caprylic, capric, lauric,
myristic, palmitic, stearic, arachidic, behenic, or lignoceric
acid. In some embodiments, a fatty acid group may be one or more of
palmitoleic, oleic, vaccenic, linoleic, alpha-linoleic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0139] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEG copolymers and copolymers of lactide and
glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG
copolymers, and derivatives thereof. In some embodiments,
polyesters include, for example, poly(caprolactone),
poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),
poly(serine ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0140] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA are characterized by the
ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid:glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention is characterized by a lactic acid:glycolic acid
ratio of approximately 85:15, approximately 75:25, approximately
60:40, approximately 50:50, approximately 40:60, approximately
25:75, or approximately 15:85.
[0141] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
anhydride), methyl methacrylate, polymethacrylate, poly(methyl
methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate
copolymer, glycidyl methacrylate copolymers, polycyanoacrylates,
and combinations comprising one or more of the foregoing polymers.
The acrylic polymer may comprise fully-polymerized copolymers of
acrylic and methacrylic acid esters with a low content of
quaternary ammonium groups.
[0142] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids (e.g. DNA, or
derivatives thereof). Amine-containing polymers such as
poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and
Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethylene
imine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA,
1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo
et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al.,
1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,
Bioconjugate Chem., 4:372) are positively-charged at physiological
pH, form ion pairs with nucleic acids, and mediate transfection in
a variety of cell lines. In embodiments, the inventive synthetic
nanocarriers may not comprise (or may exclude) cationic
polymers.
[0143] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains (Putnam et al., 1999, Macromolecules,
32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon
et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am.
Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules,
23:3399). Examples of these polyesters include
poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem.
Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,
Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam
et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am.
Chem. Soc., 121:5633).
[0144] The properties of these and other polymers and methods for
preparing them are well known in the art (see, for example, U.S.
Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404;
6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600;
5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and
4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et
al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem.
Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et
al., 1999, Chem. Rev., 99:3181). More generally, a variety of
methods for synthesizing certain suitable polymers are described in
Concise Encyclopedia of Polymer Science and Polymeric Amines and
Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles
of Polymerization by Odian, John Wiley & Sons, Fourth Edition,
2004; Contemporary Polymer Chemistry by Allcock et al.,
Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in
U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.
[0145] In some embodiments, polymers can be linear or branched
polymers. In some embodiments, polymers can be dendrimers. In some
embodiments, polymers can be substantially cross-linked to one
another. In some embodiments, polymers can be substantially free of
cross-links. In some embodiments, polymers can be used in
accordance with the present invention without undergoing a
cross-linking step. It is further to be understood that inventive
synthetic nanocarriers may comprise block copolymers, graft
copolymers, blends, mixtures, and/or adducts of any of the
foregoing and other polymers. Those skilled in the art will
recognize that the polymers listed herein represent an exemplary,
not comprehensive, list of polymers that can be of use in
accordance with the present invention.
[0146] In other embodiments, synthetic nanocarriers may comprise
metal particles, quantum dots, ceramic particles, etc. In some
embodiments, a non-polymeric synthetic nanocarrier is an aggregate
of non-polymeric components, such as an aggregate of metal atoms
(e.g., gold atoms).
[0147] Compositions according to the invention comprise synthetic
nanocarriers in combination with pharmaceutically acceptable
excipients, such as preservatives, buffers, saline, or phosphate
buffered saline. The compositions may be made using conventional
pharmaceutical manufacturing and compounding techniques to arrive
at useful dosage forms. In an embodiment, inventive synthetic
nanocarriers are suspended in sterile saline solution for injection
together with a preservative.
[0148] In embodiments, when preparing synthetic nanocarriers as
carriers, methods for coupling components to the synthetic
nanocarriers may be useful. If the component is a small molecule it
may be of advantage to attach the component to a polymer prior to
the assembly of the synthetic nanocarriers. In embodiments, it may
also be an advantage to prepare the synthetic nanocarriers with
surface groups that are used to couple the components to the
synthetic nanocarriers through the use of these surface groups
rather than attaching the components to a polymer and then using
this polymer conjugate in the construction of synthetic
nanocarriers.
[0149] In certain embodiments, the coupling can be a covalent
linker. In embodiments, peptides according to the invention can be
covalently coupled to the external surface via a 1,2,3-triazole
linker formed by the 1,3-dipolar cycloaddition reaction of azido
groups on the surface of the nanocarrier with antigen or
immunosuppressant containing an alkyne group or by the 1,3-dipolar
cycloaddition reaction of alkynes on the surface of the nanocarrier
with antigens or immunosuppressants containing an azido group. Such
cycloaddition reactions are preferably performed in the presence of
a Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing
agent to reduce Cu(II) compound to catalytic active Cu(I) compound.
This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be
referred as the click reaction.
[0150] Additionally, the covalent coupling may comprise a covalent
linker that comprises an amide linker, a disulfide linker, a
thioether linker, a hydrazone linker, a hydrazide linker, an imine
or oxime linker, an urea or thiourea linker, an amidine linker, an
amine linker, and a sulfonamide linker.
[0151] An amide linker is formed via an amide bond between an amine
on one component with the carboxylic acid group of a second
component. The amide bond in the linker can be made using any of
the conventional amide bond forming reactions with suitably
protected amino acids and activated carboxylic acid such
N-hydroxysuccinimide-activated ester.
[0152] A disulfide linker is made via the formation of a disulfide
(S--S) bond between two sulfur atoms of the form, for instance, of
R1-S--S--R2. A disulfide bond can be formed by thiol exchange of a
component containing thiol/mercaptan group (--SH) with another
activated thiol group and another containing thiol/mercaptan groups
with a component containing activated thiol group.
[0153] A triazole linker, specifically a 1,2,3-triazole of the
form
##STR00001##
wherein R1 and R2 may be any chemical entities, is made by the
1,3-dipolar cycloaddition reaction of an azide attached to a first
component such as the nanocarrier with a terminal alkyne attached
to a second component such as the immunosuppressant antigen. The
1,3-dipolar cycloaddition reaction is performed with or without a
catalyst, preferably with Cu(I)-catalyst, which links the two
components through a 1,2,3-triazole function. This chemistry is
described in detail by Sharpless et al., Angew. Chem. Int. Ed.
41(14), 2596, (2002) and Meldal, et al, Chem. Rev., 2008, 108(8),
2952-3015 and is often referred to as a "click" reaction or
CuAAC.
[0154] In embodiments, a polymer containing an azide or alkyne
group, terminal to the polymer chain is prepared. This polymer is
then used to prepare a synthetic nanocarrier in such a manner that
a plurality of the alkyne or azide groups are positioned on the
surface of that nanocarrier. Alternatively, the synthetic
nanocarrier can be prepared by another route, and subsequently
functionalized with alkyne or azide groups. The component is
prepared with the presence of either an alkyne (if the polymer
contains an azide) or an azide (if the polymer contains an alkyne)
group. The component is then allowed to react with the nanocarrier
via the 1,3-dipolar cycloaddition reaction with or without a
catalyst which covalently couples the component to the particle
through the 1,4-disubstituted 1,2,3-triazole linker.
[0155] A thioether linker is made by the formation of a
sulfur-carbon (thioether) bond in the form, for instance, of
R1-S--R2. Thioether can be made by either alkylation of a
thiol/mercaptan (--SH) group on one component with an alkylating
group such as halide or epoxide on a second component. Thioether
linkers can also be formed by Michael addition of a thiol/mercaptan
group on one component to an electron-deficient alkene group on a
second component containing a maleimide group or vinyl sulfone
group as the Michael acceptor. In another way, thioether linkers
can be prepared by the radical thiol-ene reaction of a
thiol/mercaptan group on one component with an alkene group on a
second component.
[0156] A hydrazone linker is made by the reaction of a hydrazide
group on one component with an aldehyde/ketone group on the second
component.
[0157] A hydrazide linker is formed by the reaction of a hydrazine
group on one component with a carboxylic acid group on the second
component. Such reaction is generally performed using chemistry
similar to the formation of amide bond where the carboxylic acid is
activated with an activating reagent.
[0158] An imine or oxime linker is formed by the reaction of an
amine or N-alkoxyamine (or aminooxy) group on one component with an
aldehyde or ketone group on the second component.
[0159] An urea or thiourea linker is prepared by the reaction of an
amine group on one component with an isocyanate or thioisocyanate
group on the second component.
[0160] An amidine linker is prepared by the reaction of an amine
group on one component with an imidoester group on the second
component.
[0161] An amine linker is made by the alkylation reaction of an
amine group on one component with an alkylating group such as
halide, epoxide, or sulfonate ester group on the second component.
Alternatively, an amine linker can also be made by reductive
amination of an amine group on one component with an aldehyde or
ketone group on the second component with a suitable reducing
reagent such as sodium cyanoborohydride or sodium
triacetoxyborohydride.
[0162] A sulfonamide linker is made by the reaction of an amine
group on one component with a sulfonyl halide (such as sulfonyl
chloride) group on the second component.
[0163] A sulfone linker is made by Michael addition of a
nucleophile to a vinyl sulfone. Either the vinyl sulfone or the
nucleophile may be on the surface of the nanocarrier or attached to
a component.
[0164] The component can also be conjugated to the nanocarrier via
non-covalent conjugation methods. For example, a negative charged
antigen or immunosuppressant can be conjugated to a positive
charged nanocarrier through electrostatic adsorption. A component
containing a metal ligand can also be conjugated to a nanocarrier
containing a metal complex via a metal-ligand complex.
[0165] In embodiments, the component can be attached to a polymer,
for example polylactic acid-block-polyethylene glycol, prior to the
assembly of the synthetic nanocarrier or the synthetic nanocarrier
can be formed with reactive or activatible groups on its surface.
In the latter case, the component may be prepared with a group
which is compatible with the attachment chemistry that is presented
by the synthetic nanocarriers' surface. In other embodiments, a
peptide component can be attached to VLPs or liposomes using a
suitable linker. A linker is a compound or reagent that capable of
coupling two molecules together. In an embodiment, the linker can
be a homobifuntional or heterobifunctional reagent as described in
Hermanson 2008. For example, an VLP or liposome synthetic
nanocarrier containing a carboxylic group on the surface can be
treated with a homobifunctional linker, adipic dihydrazide (ADH),
in the presence of EDC to form the corresponding synthetic
nanocarrier with the ADH linker. The resulting ADH linked synthetic
nanocarrier is then conjugated with a peptide component containing
an acid group via the other end of the ADH linker on NC to produce
the corresponding VLP or liposome peptide conjugate.
[0166] For detailed descriptions of available conjugation methods,
see Hermanson G T "Bioconjugate Techniques", 2nd Edition Published
by Academic Press, Inc., 2008. In addition to covalent attachment
the component can be coupled by adsorption to a pre-formed
synthetic nanocarrier or it can be coupled by encapsulation during
the formation of the synthetic nanocarrier.
[0167] Any immunosuppressant as provided herein can be coupled to
the synthetic nanocarrier. Immunosuppressants include, but are not
limited to, statins; mTOR inhibitors, such as rapamycin or a
rapamycin analog; TGF-.beta. signaling agents; TGF-.beta. receptor
agonists; histone deacetylase (HDAC) inhibitors; corticosteroids;
inhibitors of mitochondrial function, such as rotenone; P38
inhibitors; NF-.kappa..beta. inhibitors; adenosine receptor
agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors,
such as phosphodiesterase 4 inhibitor; proteasome inhibitors;
kinase inhibitors; G-protein coupled receptor agonists; G-protein
coupled receptor antagonists; glucocorticoids; retinoids; cytokine
inhibitors; cytokine receptor inhibitors; cytokine receptor
activators; peroxisome proliferator-activated receptor antagonists;
peroxisome proliferator-activated receptor agonists; histone
deacetylase inhibitors; calcineurin inhibitors; phosphatase
inhibitors and oxidized ATPs. Immunosuppressants also include IDO,
vitamin D3, cyclosporine A, aryl hydrocarbon receptor inhibitors,
resveratrol, azathiopurine, 6-mercaptopurine, aspirin, niflumic
acid, estriol, tripolide, interleukins (e.g., IL-1, IL-10),
cyclosporine A, siRNAs targeting cytokines or cytokine receptors
and the like.
[0168] Examples of statins include atorvastatin (LIPITOR.RTM.,
TORVAST.RTM.), cerivastatin, fluvastatin (LESCOL.RTM., LESCOL.RTM.
XL), lovastatin (MEVACOR.RTM., ALTOCOR.RTM., ALTOPREV.RTM.),
mevastatin (COMPACTIN.RTM.), pitavastatin (LIVALO.RTM.,
PIAVA.RTM.), rosuvastatin (PRAVACHOL.RTM., SELEKTINE.RTM.,
LIPOSTAT.RTM.), rosuvastatin (CRESTOR.RTM.), and simvastatin
(ZOCOR.RTM., LIPEX.RTM.).
[0169] Examples of mTOR inhibitors include rapamycin and analogs
thereof (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin
(C20-Marap), C16-(S)-butylsulfonamidorapamycin (C16-BSrap),
C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry
& Biology 2006, 13:99-107)), AZD8055, BEZ235 (NVP-BEZ235),
chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus
(RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354
(available from Selleck, Houston, Tex., USA).
[0170] Examples of TGF-.beta. signaling agents include TGF-.beta.
ligands (e.g., activin A, GDF1, GDF11, bone morphogenic proteins,
nodal, TGF-.beta.s) and their receptors (e.g., ACVR1B, ACVR1C,
ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGF.beta.RI, TGF.beta.RII),
R-SMADS/co-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMADS, SMAD8),
and ligand inhibitors (e.g, follistatin, noggin, chordin, DAN,
lefty, LTBP1, THBS1, Decorin).
[0171] Examples of inhibitors of mitochondrial function include
atractyloside (dipotassium salt), bongkrekic acid (triammonium
salt), carbonyl cyanide m-chlorophenylhydrazone,
carboxyatractyloside (e.g., from Atractylis gummifera), CGP-37157,
(-)-Deguelin (e.g., from Mundulea sericea), F16, hexokinase II VDAC
binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and
valinomycin (e.g., from Streptomyces fulvissimus) (EMD4
Biosciences, USA).
[0172] Examples of P38 inhibitors include SB-203580
(4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole)-
, SB-239063
(trans-1-(4hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-y-
l) imidazole), SB-220025
(5-(2amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole))-
, and ARRY-797.
[0173] Examples of NF (e.g., NK-.kappa..beta.) inhibitors include
IFRD1, 2-(1,8-naphthyridin-2-yl)-Phenol, 5-aminosalicylic acid, BAY
11-7082, BAY 11-7085, CAPE (Caffeic Acid Phenethylester),
diethylmaleate, IKK-2 Inhibitor IV, IMD 0354, lactacystin, MG-132
[Z-Leu-Leu-Leu-CHO], NF.kappa.B Activation Inhibitor III,
NF-.kappa.B Activation Inhibitor II, JSH-23, parthenolide,
Phenylarsine Oxide (PAO), PPM-18, pyrrolidinedithiocarbamic acid
ammonium salt, QNZ, RO 106-9920, rocaglamide, rocaglamide AL,
rocaglamide C, rocaglamide I, rocaglamide J, rocaglaol, (R)-MG-132,
sodium salicylate, triptolide (PG490), wedelolactone.
[0174] Examples of adenosine receptor agonists include CGS-21680
and ATL-146e.
[0175] Examples of prostaglandin E2 agonists include E-Prostanoid 2
and E-Prostanoid 4.
[0176] Examples of phosphodiesterase inhibitors (non-selective and
selective inhibitors) include caffeine, aminophylline, IBMX
(3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline,
theobromine, theophylline, methylated xanthines, vinpocetine, EHNA
(erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone
(PERFAN.TM.), milrinone, levosimendon, mesembrine, ibudilast,
piclamilast, luteolin, drotaverine, roflumilast (DAXAS.TM.,
DALIRESP.TM.), sildenafil (REVATION.RTM., VIAGRA.RTM.), tadalafil
(ADCIRCA.RTM., CIALIS.RTM.), vardenafil (LEVITRA.RTM.,
STAXYN.RTM.), udenafil, avanafil, icariin, 4-methylpiperazine, and
pyrazolo pyrimidin-7-1.
[0177] Examples of proteasome inhibitors include bortezomib,
disulfiram, epigallocatechin-3-gallate, and salinosporamide A.
[0178] Examples of kinase inhibitors include bevacizumab, BIBW
2992, cetuximab (ERBITUX.RTM.), imatinib (GLEEVEC.RTM.),
trastuzumab (HERCEPTIN.RTM.), gefitinib (IRESSA.RTM.), ranibizumab
(LUCENTIS.RTM.), pegaptanib, sorafenib, dasatinib, sunitinib,
erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080,
pazopanib, mubritinib.
[0179] Examples of glucocorticoids include hydrocortisone
(cortisol), cortisone acetate, prednisone, prednisolone,
methylprednisolone, dexamethasone, betamethasone, triamcinolone,
beclometasone, fludrocortisone acetate, deoxycorticosterone acetate
(DOCA), and aldosterone.
[0180] Examples of retinoids include retinol, retinal, tretinoin
(retinoic acid, RETIN-A.RTM.), isotretinoin (ACCUTANE.RTM.,
AMNESTEEM.RTM., CLARAVIS.RTM., SOTRET.RTM.), alitretinoin
(PANRETIN.RTM.), etretinate (TEGISON.TM.) and its metabolite
acitretin (SORIATANE.RTM.), tazarotene (TAZORAC.RTM., AVAGE.RTM.,
ZORAC.RTM.), bexarotene (TARGRETIN.RTM.), and adapalene
(DIFFERIN.RTM.).
[0181] Examples of cytokine inhibitors include IL1ra, IL1 receptor
antagonist, IGFBP, TNF-BF, uromodulin, Alpha-2-Macroglobulin,
Cyclosporin A, Pentamidine, and Pentoxifylline (PENTOPAK.RTM.,
PENTOXIL.RTM., TRENTAL.RTM.).
[0182] Examples of peroxisome proliferator-activated receptor
antagonists include GW9662, PPAR.gamma. antagonist III, G335,
T0070907 (EMD4Biosciences, USA).
[0183] Examples of peroxisome proliferator-activated receptor
agonists include pioglitazone, ciglitazone, clofibrate, GW1929,
GW7647, L-165,041, LY 171883, PPAR.gamma. activator, Fmoc-Leu,
troglitazone, and WY-14643 (EMD4Biosciences, USA).
[0184] Examples of histone deacetylase inhibitors include
hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic
tetrapeptides (such as trapoxin B) and depsipeptides, benzamides,
electrophilic ketones, aliphatic acid compounds such as
phenylbutyrate and valproic acid, hydroxamic acids such as
vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat
(LBH589), benzamides such as entinostat (MS-275), CI994, and
mocetinostat (MGCD0103), nicotinamide, derivatives of NAD,
dihydrocoumarin, naphthopyranone, and 2-hydroxynaphaldehydes.
[0185] Examples of calcineurin inhibitors include cyclosporine,
pimecrolimus, voclosporin, and tacrolimus.
[0186] Examples of phosphatase inhibitors include BN82002
hydrochloride, CP-91149, calyculin A, cantharidic acid,
cantharidin, cypermethrin, ethyl-3,4-dephostatin, fostriecin sodium
salt, MAZ51, methyl-3,4-dephostatin, NSC 95397, norcantharidin,
okadaic acid ammonium salt from prorocentrum concavum, okadaic
acid, okadaic acid potassium salt, okadaic acid sodium salt,
phenylarsine oxide, various phosphatase inhibitor cocktails,
protein phosphatase 1C, protein phosphatase 2A inhibitor protein,
protein phosphatase 2A1, protein phosphatase 2A2, sodium
orthovanadate.
[0187] In some embodiments, antigens as described herein are also
coupled to synthetic nanocarriers. In some embodiments, the
antigens are coupled to the same or different synthetic
nanocarriers as to which the immunosuppressants are coupled. In
other embodiments, the antigens are not coupled to any synthetic
nanocarriers.
[0188] In some embodiments, a component, such as an antigen or
immunosuppressant, may be isolated. Isolated refers to the element
being separated from its native environment and present in
sufficient quantities to permit its identification or use. This
means, for example, the element may be (i) selectively produced by
expression cloning or (ii) purified as by chromatography or
electrophoresis. Isolated elements may be, but need not be,
substantially pure. Because an isolated element may be admixed with
a pharmaceutically acceptable excipient in a pharmaceutical
preparation, the element may comprise only a small percentage by
weight of the preparation. The element is nonetheless isolated in
that it has been separated from the substances with which it may be
associated in living systems, i.e., isolated from other lipids or
proteins. Any of the elements provided herein may be isolated and
can be included in the compositions in isolated form.
D. METHODS OF MAKING AND USING THE INVENTIVE COMPOSITIONS AND
RELATED METHODS
[0189] Synthetic nanocarriers may be prepared using a wide variety
of methods known in the art. For example, synthetic nanocarriers
can be formed by methods as nanoprecipitation, flow focusing using
fluidic channels, spray drying, single and double emulsion solvent
evaporation, solvent extraction, phase separation, milling,
microemulsion procedures, microfabrication, nanofabrication,
sacrificial layers, simple and complex coacervation, and other
methods well known to those of ordinary skill in the art.
Alternatively or additionally, aqueous and organic solvent
syntheses for monodisperse semiconductor, conductive, magnetic,
organic, and other nanomaterials have been described (Pellegrino et
al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci.,
30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional
methods have been described in the literature (see, e.g., Doubrow,
Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy,"
CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control.
Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275;
and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S.
Pat. Nos. 5,578,325 and 6,007,845; P. Paolicelli et al.,
"Surface-modified PLGA-based Nanoparticles that can Efficiently
Associate and Deliver Virus-like Particles" Nanomedicine.
5(6):843-853 (2010)).
[0190] Various materials may be encapsulated into synthetic
nanocarriers as desirable using a variety of methods including but
not limited to C. Astete et al., "Synthesis and characterization of
PLGA nanoparticles" J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3,
pp. 247-289 (2006); K. Avgoustakis "Pegylated Poly(Lactide) and
Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties
and Possible Applications in Drug Delivery" Current Drug Delivery
1:321-333 (2004); C. Reis et al., "Nanoencapsulation I. Methods for
preparation of drug-loaded polymeric nanoparticles" Nanomedicine
2:8-21 (2006); P. Paolicelli et al., "Surface-modified PLGA-based
Nanoparticles that can Efficiently Associate and Deliver Virus-like
Particles" Nanomedicine. 5(6):843-853 (2010). Other methods
suitable for encapsulating materials into synthetic nanocarriers
may be used, including without limitation methods disclosed in U.S.
Pat. No. 6,632,671 to Unger Oct. 14, 2003.
[0191] In certain embodiments, synthetic nanocarriers are prepared
by a nanoprecipitation process or spray drying. Conditions used in
preparing synthetic nanocarriers may be altered to yield particles
of a desired size or property (e.g., hydrophobicity,
hydrophilicity, external morphology, "stickiness," shape, etc.).
The method of preparing the synthetic nanocarriers and the
conditions (e.g., solvent, temperature, concentration, air flow
rate, etc.) used may depend on the materials to be coupled to the
synthetic nanocarriers and/or the composition of the polymer
matrix.
[0192] If particles prepared by any of the above methods have a
size range outside of the desired range, particles can be sized,
for example, using a sieve.
[0193] Elements (i.e., components) of the inventive synthetic
nanocarriers (such as moieties of which an immunofeature surface is
comprised, targeting moieties, polymeric matrices, antigens,
immunosuppressants and the like) may be coupled to the overall
synthetic nanocarrier, e.g., by one or more covalent bonds, or may
be coupled by means of one or more linkers. Additional methods of
functionalizing synthetic nanocarriers may be adapted from
Published US Patent Application 2006/0002852 to Saltzman et al.,
Published US Patent Application 2009/0028910 to DeSimone et al., or
Published International Patent Application WO/2008/127532 A1 to
Murthy et al.
[0194] Alternatively or additionally, synthetic nanocarriers can be
coupled to components directly or indirectly via non-covalent
interactions. In non-covalent embodiments, the non-covalent
coupling is mediated by non-covalent interactions including but not
limited to charge interactions, affinity interactions, metal
coordination, physical adsorption, host-guest interactions,
hydrophobic interactions, TT stacking interactions, hydrogen
bonding interactions, van der Waals interactions, magnetic
interactions, electrostatic interactions, dipole-dipole
interactions, and/or combinations thereof. Such couplings may be
arranged to be on an external surface or an internal surface of an
inventive synthetic nanocarrier. In embodiments, encapsulation
and/or absorption is a form of coupling. In embodiments, the
inventive synthetic nanocarriers can be combined with an antigen by
admixing in the same vehicle or delivery system.
[0195] Populations of synthetic nanocarriers may be combined to
form pharmaceutical dosage forms according to the present invention
using traditional pharmaceutical mixing methods. These include
liquid-liquid mixing in which two or more suspensions, each
containing one or more subsets of nanocarriers, are directly
combined or are brought together via one or more vessels containing
diluent. As synthetic nanocarriers may also be produced or stored
in a powder form, dry powder-powder mixing could be performed as
could the re-suspension of two or more powders in a common media.
Depending on the properties of the nanocarriers and their
interaction potentials, there may be advantages conferred to one or
another route of mixing.
[0196] Typical inventive compositions that comprise synthetic
nanocarriers may comprise inorganic or organic buffers (e.g.,
sodium or potassium salts of phosphate, carbonate, acetate, or
citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium
or potassium hydroxide, salts of citrate or acetate, amino acids
and their salts) antioxidants (e.g., ascorbic acid,
alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate
80, polyoxyethylene 9-10 nonyl phenol, sodium desoxycholate),
solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose,
mannitol, trehalose), osmotic adjustment agents (e.g., salts or
sugars), antibacterial agents (e.g., benzoic acid, phenol,
gentamicin), antifoaming agents (e.g., polydimethylsilozone),
preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric
stabilizers and viscosity-adjustment agents (e.g.,
polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and
co-solvents (e.g., glycerol, polyethylene glycol, ethanol).
[0197] Compositions according to the invention comprise inventive
synthetic nanocarriers in combination with pharmaceutically
acceptable excipients. The compositions may be made using
conventional pharmaceutical manufacturing and compounding
techniques to arrive at useful dosage forms. Techniques suitable
for use in practicing the present invention may be found in
Handbook of Industrial Mixing: Science and Practice, Edited by
Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004
John Wiley & Sons, Inc.; and Pharmaceutics: The Science of
Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill
Livingstone. In an embodiment, inventive synthetic nanocarriers are
suspended in sterile saline solution for injection together with a
preservative.
[0198] It is to be understood that the compositions of the
invention can be made in any suitable manner, and the invention is
in no way limited to compositions that can be produced using the
methods described herein. Selection of an appropriate method may
require attention to the properties of the particular moieties
being associated.
[0199] In some embodiments, inventive synthetic nanocarriers are
manufactured under sterile conditions or are terminally sterilized.
This can ensure that resulting compositions are sterile and
non-infectious, thus improving safety when compared to non-sterile
compositions. This provides a valuable safety measure, especially
when subjects receiving synthetic nanocarriers have immune defects,
are suffering from infection, and/or are susceptible to infection.
In some embodiments, inventive synthetic nanocarriers may be
lyophilized and stored in suspension or as lyophilized powder
depending on the formulation strategy for extended periods without
losing activity.
[0200] The compositions of the invention can be administered by a
variety of routes, including but not limited to subcutaneous,
intranasal, oral, intravenous, intraperitoneal, intramuscular,
transmucosal, transmucosal, sublingual, rectal, ophthalmic,
pulmonary, intradermal, transdermal, transcutaneous or intradermal
or by a combination of these routes. Routes of administration also
include administration by inhalation or pulmonary aerosol.
Techniques for preparing aerosol delivery systems are well known to
those of skill in the art (see, for example, Sciarra and Cutie,
"Aerosols," in Remington's Pharmaceutical Sciences, 18th edition,
1990, pp. 1694-1712; incorporated by reference).
[0201] The compositions of the invention can be administered in
effective amounts, such as the effective amounts described
elsewhere herein. Doses of dosage forms contain varying amounts of
populations of synthetic nanocarriers and/or varying amounts of
antigens and/or immunosuppressants, according to the invention. The
amount of synthetic nanocarriers and/or antigens and/or
immunosuppressants present in the inventive dosage forms can be
varied according to the nature of the antigens and/or
immunosuppressants, the therapeutic benefit to be accomplished, and
other such parameters. In embodiments, dose ranging studies can be
conducted to establish optimal therapeutic amount of the population
of synthetic nanocarriers and the amount of antigens and/or
immunosuppressants to be present in the dosage form. In
embodiments, the synthetic nanocarriers and/or the antigens and/or
immunosuppressants are present in the dosage form in an amount
effective to generate a tolerogenic immune response to the antigens
upon administration to a subject. It may be possible to determine
amounts of the antigens and/or immunosuppressants effective to
generate a tolerogenic immune response using conventional dose
ranging studies and techniques in subjects. Inventive dosage forms
may be administered at a variety of frequencies. In a preferred
embodiment, at least one administration of the dosage form is
sufficient to generate a pharmacologically relevant response. In
more preferred embodiments, at least two administrations, at least
three administrations, or at least four administrations, of the
dosage form are utilized to ensure a pharmacologically relevant
response.
[0202] Prophylactic administration of the inventive compositions
can be initiated prior to the onset of disease, disorder or
condition or therapeutic administration can be initiated after a
disorder, disorder or condition is established.
[0203] In some embodiments, administration of synthetic
nanocarriers is undertaken e.g., prior to exposure to an allergen.
In exemplary embodiments, synthetic nanocarriers are administered
at one or more times including, but not limited to, 30, 25, 20, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 days prior to
exposure to an allergen. In addition or alternatively, synthetic
nanocarriers can be administered to a subject following exposure to
an allergen. In exemplary embodiments, synthetic nanocarriers are
administered at one or more times including, but not limited to, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, etc.
days following exposure to an allergen.
[0204] In some embodiments, a maintenance dose (e.g., of a
synthetic nanocarrier composition provided herein) is administered
to a subject after an initial administration has resulted in a
tolerogenic response in the subject, for example to maintain the
tolerogenic effect achieved after the initial dose, to prevent an
undesired immune reaction in the subject, or to prevent the subject
becoming a subject at risk of experiencing an undesired immune
response or an undesired level of an immune response. In some
embodiments, the maintenance dose is the same dose as the initial
dose the subject received. In some embodiments, the maintenance
dose is a lower dose than the initial dose. For example, in some
embodiments, the maintenance dose is about 3/4, about 2/3, about
1/2, about 1/3, about 1/4, about 1/8, about 1/10, about 1/20, about
1/25, about 1/50, about 1/100, about 1/1,000, about 1/10,000, about
1/100,000, or about 1/1,000,000 (weight/weight) of the initial
dose.
[0205] The compositions and methods described herein can be used to
induce or enhance a tolerogenic immune response and/or to suppress,
modulate, direct or redirect an undesired immune response for the
purpose of immune suppression. The compositions and methods
described herein can be used for the generation of a tolerogenic
immune response in a subject that has been, is being or will be
exposed to an allergen.
EXAMPLES
Example 1
Immune Response of Synthetic Nanocarriers with Coupled Rapamycin
with and without Ovalbumin Peptide (323-339)
Materials
[0206] Ovalbumin peptide 323-339, a 17 amino acid peptide known to
be a T and B cell epitope of Ovalbumin protein, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505;
Part #4065609). Rapamycin was purchased from TSZ CHEM (185 Wilson
Street, Framingham, Mass. 01702; Product Catalogue # R1017). PLGA
with a lactide:glycolide ratio of 3:1 and an inherent viscosity of
0.75 dL/g was purchased from SurModics Pharmaceuticals (756 Tom
Martin Drive, Birmingham, Ala. 35211; Product Code 7525 DLG 7A).
Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD
Chemicals (Product Number 1.41350.1001).
[0207] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature.
[0208] Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The
solution was prepared by dissolving rapamycin in pure methylene
chloride.
[0209] Solution 3: PLGA @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLGA in pure methylene
chloride.
[0210] Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
Method for Preparing Synthetic Nanocarrier Containing Rapamycin and
Ovalbumin (323-339)
[0211] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining solution 1 (0.2 mL), solution 2 (0.2 mL),
and solution 3 (1.0 mL) in a small pressure tube and sonicating at
50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
A secondary emulsion (W1/O1/W2) was then prepared by combining
solution 4 (3.0 mL) with the primary W1/O1 emulsion, vortexing for
10 s, and sonicating at 30% amplitude for 60 seconds using the
Branson Digital Sonifier 250.
[0212] The W1/O1/W2 emulsion was added to a beaker containing 70 mM
pH 8 phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the methylene chloride to
evaporate and for the synthetic nanocarriers to form. A portion of
the synthetic nanocarriers were washed by transferring the
synthetic nanocarrier suspension to a centrifuge tube and
centrifuging at 21,000.times.g and 4.degree. C. for one hour,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure was repeated, and the pellet
was re-suspended in phosphate buffered saline for a final synthetic
nanocarrier dispersion of about 10 mg/mL.
[0213] The amounts of peptide and rapamycin in the synthetic
nanocarriers were determined by HPLC analysis. The total
dry-synthetic nanocarrier mass per mL of suspension was determined
by a gravimetric method.
Method for Synthetic Nanocarrier Containing Rapamycin
[0214] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining 0.13 M hydrochloric acid solution (0.2
mL), solution 2 (0.2 mL), and solution 3 (1.0 mL) in a small
pressure tube and sonicating at 50% amplitude for 40 seconds using
a Branson Digital Sonifier 250. A secondary emulsion (W1/O1/W2) was
then prepared by combining solution 4 (3.0 mL) with the primary
W1/O1 emulsion, vortexing for 10 s, and sonicating at 30% amplitude
for 60 seconds using the Branson Digital Sonifier 250.
[0215] The W1/O1/W2 emulsion was added to a beaker containing 70 mM
pH 8 phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the methylene chloride to
evaporate and for the synthetic nanocarriers to form. A portion of
the synthetic nanocarriers were washed by transferring the
synthetic nanocarrier suspension to a centrifuge tube and
centrifuging at 21,000.times.g and 4.degree. C. for one hour,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure was repeated, and the pellet
was re-suspended in phosphate buffered saline for a final synthetic
nanocarrier dispersion of about 10 mg/mL.
[0216] The amount of rapamycin in the synthetic nanocarrier was
determined by HPLC analysis. The total dry-synthetic nanocarrier
mass per mL of suspension was determined by a gravimetric
method.
Method for Measuring Rapamycin Load
[0217] Approximately 3 mg of synthetic nanocarriers were collected
and centrifuged to separate supernatant from synthetic nanocarrier
pellet. Acetonitrile was added to the pellet, and the sample was
sonicated and centrifuged to remove any insoluble material. The
supernatant and pellet were injected on RP-HPLC and absorbance was
read at 278 nm. The .mu.g found in the pellet were used to
calculate % entrapped (load), .mu.g in supernatant and pellet were
used to calculate total .mu.g recovered.
Method for Measuring Ovalbumin (323-339) Load
[0218] Approximately 3 mg of synthetic nanocarriers were collected
and centrifuged to separate supernatant from synthetic nanocarrier
pellet. Trifluoroethanol was added to the pellet and the sample was
sonicated to dissolve the polymer, 0.2% trifluoroacetic acid was
added and sample was sonicated and then centrifuged to remove any
insoluble material. The supernatant and pellet were injected on
RP-HPLC and absorbance was read at 215 nm. The .mu.g found in the
pellet were used to calculate % entrapped (load), .mu.g in
supernatant and pellet were used to calculate total .mu.g
recovered.
Antigen-Specific Tolerogenic Dendritic Cells (Tdc) Activity on Treg
Cell Development
[0219] The assay included the use of OTII mice which have a
transgenic T-cell receptor specific for an immune-dominant
ovalbumin (323-339). In order to create antigen-specific tDCs,
CD11c+ splenocytes were isolated, and the ovalbumin (323-339)
peptide added in vitro at 1 .mu.g/ml or no antigen. Soluble or
nanocarrier-encapsulated rapamycin was then added to the DCs for 2
hours which were then washed extensively to remove free rapamycin
from the culture. Purified responder CD4+CD25- cells were isolated
from OTII mice and added to tDC at a 10:1 T to DC ratio. The
mixture of tDC and OTII T-cells were then cultured for 4-5 days,
and the frequency of Treg cells (CD4+CD25highFoxP3+) were analyzed
by flow cytometry as shown in FIG. 1. Regions were selected based
on isotype controls.
Example 2
Mesoporous Silica Nanoparticles with Coupled Ibuprofen
(Prophetic)
[0220] Mesoporous SiO2 nanoparticle cores are created through a
sol-gel process. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5 g)
is dissolved in deionized water (500 mL), and then 2 M aqueous NaOH
solution (3.5 mL) is added to the CTAB solution. The solution is
stirred for 30 min, and then Tetraethoxysilane (TEOS) (2.5 mL) is
added to the solution. The resulting gel is stirred for 3 h at a
temperature of 80.degree. C. The white precipitate which forms is
captured by filtration, followed by washing with deionized water
and drying at room temperature. The remaining surfactant is then
extracted from the particles by suspension in an ethanolic solution
of HCl overnight. The particles are washed with ethanol,
centrifuged, and redispersed under ultrasonication. This wash
procedure is repeated two additional times.
[0221] The SiO2 nanoparticles are then functionalized with amino
groups using (3-aminopropyl)-triethoxysilane (APTMS). To do this,
the particles are suspended in ethanol (30 mL), and APTMS (50
.mu.L) is added to the suspension. The suspension is allowed to
stand at room temperature for 2 h and then is boiled for 4 h,
keeping the volume constant by periodically adding ethanol.
Remaining reactants are removed by five cycles of washing by
centrifugation and redispersing in pure ethanol.
[0222] In a separate reaction, 1-4 nm diameter gold seeds are
created. All water used in this reaction is first deionized and
then distilled from glass. Water (45.5 mL) is added to a 100 mL
round-bottom flask. While stirring, 0.2 M aqueous NaOH (1.5 mL) is
added, followed by a 1% aqueous solution of
tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL). Two
minutes after the addition of THPC solution, a 10 mg/mL aqueous
solution of chloroauric acid (2 mL), which has been aged at least
15 min, is added. The gold seeds are purified through dialysis
against water.
[0223] To form the core-shell nanocarriers, the
amino-functionalized SiO2 nanoparticles formed above are first
mixed with the gold seeds for 2 h at room temperature. The
gold-decorated SiO2 particles are collected through centrifugation
and mixed with an aqueous solution of chloroauric acid and
potassium bicarbonate to form the gold shell. The particles are
then washed by centrifugation and redispersed in water. Ibuprofen
is loaded by suspending the particles in a solution of sodium
ibuprofen (1 mg/L) for 72 h. Free ibuprofen is then washed from the
particles by centrifugation and redispersing in water.
Example 3
Liposomes Containing Cyclosporine A (Prophetic)
[0224] The liposomes are formed using thin film hydration.
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 .mu.mol),
cholesterol (32 .mu.mol), and cyclosporin A (6.4 .mu.mol) are
dissolved in pure chloroform (3 mL). This lipid solution is added
to a 50 mL round-bottom flask, and the solvent is evaporated on a
rotary evaporator at a temperature of 60.degree. C. The flask is
then flushed with nitrogen gas to remove remaining solvent.
Phosphate buffered saline (2 mL) and five glass beads are added to
the flask, and the lipid film is hydrated by shaking at 60.degree.
C. for 1 h to form a suspension. The suspension is transferred to a
small pressure tube and sonicated at 60.degree. C. for four cycles
of 30s pulses with a 30 s delay between each pulse. The suspension
is then left undisturbed at room temperature for 2 h to allow for
complete hydration. The liposomes are washed by centrifugation
followed by resuspension in fresh phosphate buffered saline.
Example 4
Polymeric Nanocarrier Containing Polymer-Rapamycin Conjugate
(Prophetic)
[0225] Preparation of PLGA-Rapamycin Conjugate:
[0226] PLGA polymer with acid end group (7525 DLG1A, acid number
0.46 mmol/g, Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 eq) is
dissolved in 30 mL of dichloromethane (DCM).
N,N-Dicyclohexylcarbodimide (1.2 eq, 2.8 mmol, 0.57 g) is added
followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and
4-dimethylaminopyridine (DMAP) (2.0 eq, 4.6 mmol, 0.56 g). The
mixture is stirred at rt for 2 days. The mixture is then filtered
to remove insoluble dicyclohexylurea. The filtrate is concentrated
to ca. 10 mL in volume and added to 100 mL of isopropyl alcohol
(IPA) to precipitate out the PLGA-rapamycin conjugate. The IPA
layer is removed and the polymer is then washed with 50 mL of IPA
and 50 mL of methyl t-butyl ether (MTBE). The polymer is then dried
under vacuum at 35 C for 2 days to give PLGA-rapamycin as a white
solid (ca. 6.5 g).
[0227] Preparation of nanocarrier containing PLGA-rapamycin
conjugate and ovalbumin peptide (323-339):
[0228] Nanocarrier containing PLGA-rapamycin is prepared according
to the procedure described in Example 1 as follows:
[0229] Solutions for nanocarrier formation are prepared as
follows:
[0230] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution is prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature. Solution 2: PLGA-rapamycin @ 100 mg/mL in
methylene chloride. The solution is prepared by dissolving
PLGA-rapamycin in pure methylene chloride. Solution 3: PLA-PEG @
100 mg/mL in methylene chloride. The solution is prepared by
dissolving PLA-PEG in pure methylene chloride. Solution 4:
Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8 phosphate buffer.
[0231] A primary water-in-oil emulsion is prepared first. W1/O1 is
prepared by combining solution 1 (0.2 mL), solution 2 (0.75 mL),
and solution 3 (0.25 mL) in a small pressure tube and sonicating at
50% amplitude for 40 seconds using a Branson Digital Sonifier 250.
A secondary emulsion (W1/O1/W2) is then prepared by combining
solution 4 (3.0 mL) with the primary W1/O1 emulsion, vortexing for
10 s, and sonicating at 30% amplitude for 60 seconds using the
Branson Digital Sonifier 250. The W1/O1/W2 emulsion is added to a
beaker containing 70 mM pH 8 phosphate buffer solution (30 mL) and
stirred at room temperature for 2 hours to allow the methylene
chloride to evaporate and for the nanocarriers to form. A portion
of the nanocarriers is washed by transferring the nanocarrier
suspension to a centrifuge tube and centrifuging at 75,600.times.g
and 4.degree. C. for 35 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure is repeated, and the pellet is re-suspended in phosphate
buffered saline for a final nanocarrier dispersion of about 10
mg/mL.
Example 5
Preparation of Gold Nanocarriers (AuNCs) Containing Rapamycin
(Prophetic)
[0232] Preparation of HS-PEG-Rapamycin:
[0233] A solution of PEG acid disulfide (1.0 eq), rapamycin
(2.0-2.5 eq), DCC (2.5 eq) and DMAP (3.0 eq) in dry DMF is stirred
at rt overnight. The insoluble dicyclohexylurea is removed by
filtration and the filtrate is added to isopropyl alcohol (IPA) to
precipitate out the PEG-disulfide-di-rapamycin ester and washed
with IPA and dried. The polymer is then treated with
tris(2-carboxyethyl)phosphine hydrochloride in DMF to reduce the
PEG disulfide to thiol PEG rapamycin ester (HS-PEG-rapamycin). The
resulting polymer is recovered by precipitation from IPA and dried
as previously described and analyzed by H NMR and GPC.
[0234] Formation of Gold NCs (AuNCs):
[0235] An aq. solution of 500 mL of 1 mM HAuCl4 is heated to reflux
for 10 min with vigorous stirring in a 1 L round-bottom flask
equipped with a condenser. A solution of 50 mL of 40 mM of
trisodium citrate is then rapidly added to the stirring solution.
The resulting deep wine red solution is kept at reflux for 25-30
min and the heat is withdrawn and the solution is cooled to room
temperature. The solution is then filtered through a 0.8 .mu.m
membrane filter to give the AuNCs solution. The AuNCs are
characterized using visible spectroscopy and transmission electron
microscopy. The AuNCs are ca. 20 nm diameter capped by citrate with
peak absorption at 520 nm.
[0236] AuNCs Conjugate with HS-PEG-Rapamycin:
[0237] A solution of 150 .mu.l of HS-PEG-rapamycin (10 .mu.M in 10
mM pH 9.0 carbonate buffer) is added to 1 mL of 20 nm diameter
citrate-capped gold nanocarriers (1.16 nM) to produce a molar ratio
of thiol to gold of 2500:1. The mixture is stirred at room
temperature under argon for 1 hour to allow complete exchange of
thiol with citrate on the gold nanocarriers. The AuNCs with
PEG-rapamycin on the surface is then purified by centrifuge at
12,000 g for 30 minutes. The supernatant is decanted and the pellet
containing AuNC--S-PEG-rapamycin is then pellet washed with
1.times.PBS buffer. The purified Gold-PEG-rapamycin nanocarriers
are then resuspend in suitable buffer for further analysis and
bioassays.
Example 6
Mesoporous Silica-Gold Core-Shell Nanocarriers Containing Ovalbumin
(Prophetic)
[0238] Mesoporous SiO.sub.2 nanoparticle cores are created through
a sol-gel process. Hexadecyltrimethyl-ammonium bromide (CTAB) (0.5
g) is dissolved in deionized water (500 mL), and then 2 M aqueous
NaOH solution (3.5 mL) is added to the CTAB solution. The solution
is stirred for 30 min, and then Tetraethoxysilane (TEOS) (2.5 mL)
is added to the solution. The resulting gel is stirred for 3 h at a
temperature of 80.degree. C. The white precipitate which forms is
captured by filtration, followed by washing with deionized water
and drying at room temperature. The remaining surfactant is then
extracted from the particles by suspension in an ethanolic solution
of HCl overnight. The particles are washed with ethanol,
centrifuged, and redispersed under ultrasonication. This wash
procedure is repeated two additional times.
[0239] The SiO.sub.2 nanoparticles are then functionalized with
amino groups using (3-aminopropyl)-triethoxysilane (APTMS). To do
this, the particles are suspended in ethanol (30 mL), and APTMS (50
.mu.L) is added to the suspension. The suspension is allowed to
stand at room temperature for 2 h and then is boiled for 4 h,
keeping the volume constant by periodically adding ethanol.
Remaining reactants are removed by five cycles of washing by
centrifugation and redispersing in pure ethanol.
[0240] In a separate reaction, 1-4 nm diameter gold seeds are
created. All water used in this reaction is first deionized and
then distilled from glass. Water (45.5 mL) is added to a 100 mL
round-bottom flask. While stirring, 0.2 M aqueous NaOH (1.5 mL) is
added, followed by a 1% aqueous solution of
tetrakis(hydroxymethyl)phosphonium chloride (THPC) (1.0 mL). Two
minutes after the addition of THPC solution, a 10 mg/mL aqueous
solution of chloroauric acid (2 mL), which has been aged at least
15 min, is added. The gold seeds are purified through dialysis
against water.
[0241] To form the core-shell nanocarriers, the
amino-functionalized SiO.sub.2 nanoparticles formed above are first
mixed with the gold seeds for 2 h at room temperature. The
gold-decorated SiO.sub.2 particles are collected through
centrifugation and mixed with an aqueous solution of chloroauric
acid and potassium bicarbonate to form the gold shell. The
particles are then washed by centrifugation and redispersed in
water. Thiolated Ovalbumin (made by treating Ovalbumin with
2-iminothiolane hydrochloride) is loaded by suspending the
particles in a solution of thiolated Ovalbumin (1 mg/L) for 72 h.
The particles is then pellet washed with 1.times.PBS (pH 7.4) to
remove free protein. The resulting silica-gold core-shell
nanocarriers containing Ovalbumin are then re-suspended in
1.times.PBS for further analysis and assays.
Example 7
Liposomes Containing Rapamycin and Ovalbumin (Prophetic)
[0242] The liposomes are formed by thin film hydration.
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 .mu.mol),
cholesterol (32 .mu.mol), and rapamycin (6.4 .mu.mol) are dissolved
in pure chloroform (3 mL). This lipid solution is added to a 10 mL
glass tube and the solvent is removed under nitrogen gas stream and
desiccated for 6 hr. under vacuum. Multilamellar vesicles are
obtained by hydration of the film with 2.0 ml of 25 mM MOPS buffer
pH 8.5, containing excess amount of Ovalbumin. The tube is vortexed
until the lipid film is peeled of from the tube surface. To break
the multilamellar vesicles into monolamellar, ten cycles of
freezing (liquid nitrogen) and thawing (30.degree. C. water bath)
are applied. The sample is then diluted to 1 ml in 25 mM MOPS
buffer pH 8.5. Size of the resulting liposome is homogenized by
extrusion by passing the sample 10 fold through a 200 nm pore
polycarbonate filters. The resulting liposomes are then used for
further analysis and bioassays.
Example 8
Polymeric Nanocarriers Composed of Modified Polyamino Acid with
Surface Conjugated Ovalbumin (Prophetic)
[0243] Step-1. Preparation of Poly(.gamma.-glutamic acid)
(.gamma.-PGA) modified with L-phenylalanine ethyl ester (L-PAE):
4.7 unit mmol of .gamma.-PGA (Mn=300 kD) is dissolved in 0.3
N--NaHCO3 aqueous solution (50 mL). L-PAE (4.7 mmol) and EDC.HCl
(4.7 mmol) are added to the solution and stirred for 30 min at 4 C.
The solution is then maintained at room temperature with stirring
for 24 h. Low-molecular-weight chemicals are removed by dialysis
using dialysis membrane with MWCO 50 kD. The resulting
.gamma.-PGA-graft-L-PAE is obtained by freeze-drying.
[0244] Step-2. Preparation of nanoparticles from
.gamma.-PGA-graft-L-PAE polymer: Nanoparticles composed of
.gamma.-PGA-graft-L-PAE are prepared by a precipitation and
dialysis method. .gamma.-PGA-graft-L-PAE (20 mg) was dissolved in 2
ml of DMSO followed by addition of 2 mL of water to form a
translucent solution. The solution is then dialyzed against
distilled water using cellulose membrane tubing (50,000 MWCO) to
form the nanoparticles and to remove the organic solvents for 72 h
at room temperature. The distilled water is exchanged at intervals
of 12 h. The resulting nanoparticle solution (10 mg/mL in water) is
then used for antigen conjugation.
[0245] Step-3. Ovalbumin conjugation to .gamma.-PGA nanoparticles:
Surface carboxylic acid groups of the .gamma.-PGA nanoparticles (10
mg/ml) are first activated by EDC and NHS (10 mg/mL each in
phosphate buffer, pH 5.8) for 2 h at ambient temperature. After
pellet washing to remove excess EDC/NHS, the activated
nanoparticles are mixed with 1 mL of Ovalbumin (10 mg/ml) in
phosphate-buffered saline (PBS, pH 7.4) and the mixture is
incubated at 4-8 C for 24 h. The resulting Ovalbumin conjugated
.gamma.-PGA nanoparticles are washed twice with PBS and resuspended
at 5 mg/mL in PBS for further analysis and bioassays.
Example 9
Erythropoietin (EPO)-Encapsulated .gamma.-PGA Nanoparticles
(Prophetic)
[0246] To prepare the EPO-encapsulated .gamma.-PGA nanoparticles,
0.25-4 mg of EPO is dissolved in 1 mL of PBS (pH 7.4) and 1 mL of
the .gamma.-PGA-graft-L-PAE (10 mg/mL in DMSO) is added to the EPO
solution. The resulting solution is centrifuged at 14,000.times.g
for 15 min and repeatedly rinsed with PBS. The resulting
EPO-encapsulated .gamma.-PGA nanoparticles are then resuspended in
PBS (5 mg/mL) for further analysis and bioassay.
Example 10
Preparation of Gold Nanocarriers (AuNCs) Containing Ovalbumin
(Prophetic)
[0247] Step-1. Formation of Gold NCs (AuNCs): An aq. solution of
500 mL of 1 mM HAuCl4 is heated to reflux for 10 min with vigorous
stirring in a 1 L round-bottom flask equipped with a condenser. A
solution of 50 mL of 40 mM of trisodium citrate is then rapidly
added to the stirring solution. The resulting deep wine red
solution is kept at reflux for 25-30 min and the heat is withdrawn
and the solution is cooled to room temperature. The solution is
then filtered through a 0.8 .mu.m membrane filter to give the AuNCs
solution. The AuNCs are characterized using visible spectroscopy
and transmission electron microscopy. The AuNCs are ca. 20 nm
diameter capped by citrate with peak absorption at 520 nm.
[0248] Step-2. Conjugation of Ovalbumin to AuNCs: A solution of 150
.mu.l of thiolated Ovalbumin (10 .mu.M in 10 mM pH 9.0 carbonate
buffer) is added to 1 mL of 20 nm diameter citrate-capped gold
nanocarriers (1.16 nM) to produce a molar ratio of thiol to gold of
2500:1. The mixture is stirred at room temperature under argon for
1 hour to allow complete exchange of thiol with citrate on the gold
nanocarriers. The AuNCs with Ovalbumin on the surface is then
purified by centrifuge at 12,000 g for 30 minutes. The supernatant
is decanted and the pellet containing AuNC-Ovalbumin is then pellet
washed with 1.times.PBS buffer. The purified Gold-Ovalbumin
nanocarriers are then resuspend in suitable buffer for further
analysis and bioassays.
Example 11
Evaluating Tolerogenic Immune Response to Der P1 Antigen In Vivo
(Prophetic)
[0249] Balb/c mice are immunized with a Der P1 antigen in
incomplete Freunds adjuvant to induce CD4+ T-cell proliferation,
the level of which is assessed. Subsequently, a composition of the
invention comprising MHC Class II-restricted epitopes of Der P1
antigen and an immunosuppressant is administered subcutaneously in
a dose-dependent manner. The same mice are then again exposed to
the Der P1 antigen, and the level of CD4+ T cell proliferation is
again assessed. Changes in the CD4+ T cell population are then
monitored with a reduction in CD4+ T cell proliferation upon
subsequent challenge with the Der P1 antigen indicating a
tolerogenic immune response.
Example 12
Evaluating Tolerogenic Immune Responses with Synthetic Nanocarriers
Comprising Immunosuppressant and APC Presentable Antigen In
Vivo
Materials and Methods of Synthetic Nanocarrier Production
Nanocarrier 1
[0250] Rapamycin was purchased from TSZ CHEM (185 Wilson Street,
Framingham, Mass. 01702; Product Catalogue # R1017). PLGA with a
lactide:glycolide ratio of 3:1 and an inherent viscosity of 0.75
dL/g was purchased from SurModics Pharmaceuticals (756 Tom Martin
Drive, Birmingham, Ala. 35211; Product Code 7525 DLG 7A). PLA-PEG
block co-polymer with a PEG block of approximately 5,000 Da and PLA
block of approximately 20,000 Da was synthesized. Polyvinyl alcohol
(85-89% hydrolyzed) was purchased from EMD Chemicals (Product
Number 1.41350.1001).
[0251] Solutions were Prepared as Follows:
[0252] Solution 1: Rapamycin @ 50 mg/mL in methylene chloride. The
solution was prepared by dissolving rapamycin in pure methylene
chloride.
[0253] Solution 2: PLGA @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLGA in pure methylene
chloride.
[0254] Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLA-PEG in pure methylene
chloride.
[0255] Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0256] An oil-in-water emulsion was used to prepare the
nanocarriers. The O/W emulsion was prepared by combining solution 1
(0.2 mL), solution 2 (0.75 mL), solution 3 (0.25 mL), and solution
4 (3 mL) in a small pressure tube and sonicating at 30% amplitude
for 60 seconds using a Branson Digital Sonifier 250. The O/W
emulsion was added to a beaker containing 70 mM pH 8 phosphate
buffer solution (30 mL) and stirred at room temperature for 2 hours
to allow the methylene chloride to evaporate and for the
nanocarriers to form. A portion of the nanocarriers was washed by
transferring the nanocarrier suspension to a centrifuge tube and
centrifuging at 21,000.times.g and 4.degree. C. for 45 min,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure was repeated, and the pellet
was re-suspended in phosphate buffered saline for a final
nanocarrier dispersion of about 10 mg/mL.
[0257] Nanocarrier size was determined by dynamic light scattering.
The amount of rapamycin in the nanocarrier was determined by HPLC
analysis. The total dry-nanocarrier mass per mL of suspension was
determined by a gravimetric method.
TABLE-US-00001 Nanocarrier Effective Rapamycin ID Diameter (nm)
Content (% w/w) Nanocarrier 1 215 9.5
Nanocarrier 2
[0258] Ovalbumin peptide 323-339, a 17 amino acid peptide known to
be a T and B cell epitope of Ovalbumin protein, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505;
Part #4065609). PLGA with a lactide:glycolide ratio of 3:1 and an
inherent viscosity of 0.75 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;
Product Code 7525 DLG 7A). PLA-PEG block co-polymer with a PEG
block of approximately 5,000 Da and PLA block of approximately
20,000 Da was synthesized. Polyvinyl alcohol (85-89% hydrolyzed)
was purchased from EMD Chemicals (Product Number 1.41350.1001).
[0259] Solutions were Prepared as Follows:
[0260] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature.
[0261] Solution 2: PLGA @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLGA in pure methylene
chloride.
[0262] Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLA-PEG in pure methylene
chloride.
[0263] Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0264] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining solution 1 (0.2 mL), solution 2 (0.75
mL), and solution 3 (0.25 mL) in a small pressure tube and
sonicating at 50% amplitude for 40 seconds using a Branson Digital
Sonifier 250. A secondary emulsion (W1/O1/W2) was then prepared by
combining solution 4 (3.0 mL) with the primary W1/O1 emulsion,
vortexing for 10 s, and sonicating at 30% amplitude for 60 seconds
using the Branson Digital Sonifier 250.
[0265] The W1/O1/W2 emulsion was added to a beaker containing 70 mM
pH 8 phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the methylene chloride to
evaporate and for the nanocarriers to form. A portion of the
nanocarriers were washed by transferring the nanocarrier suspension
to a centrifuge tube and centrifuging at 75,600.times.g and
4.degree. C. for 35 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure was repeated, and the pellet was re-suspended in
phosphate buffered saline for a final nanocarrier dispersion of
about 10 mg/mL.
[0266] Nanocarrier size was determined by dynamic light scattering.
The amount of peptide in the nanocarrier was determined by HPLC
analysis. The total dry-nanocarrier mass per mL of suspension was
determined by a gravimetric method.
TABLE-US-00002 Nanocarrier Effective Peptide ID Diameter (nm)
Content (% w/w) Nanocarrier 2 234 2.1
Nanocarrier 3
[0267] Simvastatin was purchased from LKT Laboratories, Inc. (2233
University Avenue West, St. Paul, Minn. 55114; Product Catalogue #
S3449). PLGA with a lactide:glycolide ratio of 3:1 and an inherent
viscosity of 0.75 dL/g was purchased from SurModics Pharmaceuticals
(756 Tom Martin Drive, Birmingham, Ala. 35211; Product Code 7525
DLG 7A). PLA-PEG block co-polymer with a PEG block of approximately
5,000 Da and PLA block of approximately 20,000 Da was synthesized.
Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD
Chemicals (Product Number 1.41350.1001).
[0268] Solutions were Prepared as Follows:
[0269] Solution 1: Simvastatin @ 50 mg/mL in methylene chloride.
The solution was prepared by dissolving simvastatin in pure
methylene chloride.
[0270] Solution 2: PLGA @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLGA in pure methylene
chloride.
[0271] Solution 3: PLA-PEG @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLA-PEG in pure methylene
chloride.
[0272] Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0273] An oil-in-water emulsion was used to prepare the
nanocarriers. The O/W emulsion was prepared by combining solution 1
(0.15 mL), solution 2 (0.75 mL), solution 3 (0.25 mL), and solution
4 (3 mL) in a small pressure tube and sonicating at 30% amplitude
for 60 seconds using a Branson Digital Sonifier 250. The O/W
emulsion was added to a beaker containing 70 mM pH 8 phosphate
buffer solution (30 mL) and stirred at room temperature for 2 hours
to allow the methylene chloride to evaporate and for the
nanocarriers to form. A portion of the nanocarriers was washed by
transferring the nanocarrier suspension to a centrifuge tube and
centrifuging at 75,600.times.g and 4.degree. C. for 35 min,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure was repeated, and the pellet
was re-suspended in phosphate buffered saline for a final
nanocarrier dispersion of about 10 mg/mL.
[0274] Nanocarrier size was determined by dynamic light scattering.
The amount of simvastatin in the nanocarrier was determined by HPLC
analysis. The total dry-nanocarrier mass per mL of suspension was
determined by a gravimetric method.
TABLE-US-00003 Nanocarrier Effective Simvastatin ID Diameter (nm)
Content (% w/w) Nanocarrier 3 196 8.0
Nanocarrier 4
[0275] Ovalbumin peptide 323-339, a 17 amino acid peptide known to
be a T and B cell epitope of Ovalbumin protein, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505;
Part #4065609). Rapamycin was purchased from TSZ CHEM (185 Wilson
Street, Framingham, Mass. 01702; Product Catalogue # R1017). PLGA
with a lactide:glycolide ratio of 3:1 and an inherent viscosity of
0.75 dL/g was purchased from SurModics Pharmaceuticals (756 Tom
Martin Drive, Birmingham, Ala. 35211; Product Code 7525 DLG 7A).
PLA-PEG block co-polymer with a PEG block of approximately 5,000 Da
and PLA block of approximately 20,000 Da was synthesized. Polyvinyl
alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals
(Product Number 1.41350.1001).
[0276] Solutions were Prepared as Follows:
[0277] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature.
[0278] Solution 2: Rapamycin @ 50 mg/mL in methylene chloride. The
solution was prepared by dissolving rapamycin in pure methylene
chloride.
[0279] Solution 3: PLGA @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLGA in pure methylene
chloride.
[0280] Solution 4: PLA-PEG @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLA-PEG in pure methylene
chloride.
[0281] Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0282] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining solution 1 (0.2 mL), solution 2 (0.2 mL),
solution 3 (0.75 mL), and solution 4 (0.25 mL) in a small pressure
tube and sonicating at 50% amplitude for 40 seconds using a Branson
Digital Sonifier 250. A secondary emulsion (W1/O1/W2) was then
prepared by combining solution 5 (3.0 mL) with the primary W1/O1
emulsion, vortexing for 10 s, and sonicating at 30% amplitude for
60 seconds using the Branson Digital Sonifier 250.
[0283] The W1/O1/W2 emulsion was added to a beaker containing 70 mM
pH 8 phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the methylene chloride to
evaporate and for the nanocarriers to form. A portion of the
nanocarriers were washed by transferring the nanocarrier suspension
to a centrifuge tube and centrifuging at 21,000.times.g and
4.degree. C. for 45 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure was repeated, and the pellet was re-suspended in
phosphate buffered saline for a final nanocarrier dispersion of
about 10 mg/mL.
[0284] Nanocarrier size was determined by dynamic light scattering.
The amounts of peptide and rapamycin in the nanocarrier were
determined by HPLC analysis. The total dry-nanocarrier mass per mL
of suspension was determined by a gravimetric method.
TABLE-US-00004 Nanocarrier Effective Rapamycin Peptide ID Diameter
(nm) Content (% w/w) Content (% w/w) 4 227 9.0 2.5
Nanocarrier 5
[0285] Ovalbumin peptide 323-339, a 17 amino acid peptide known to
be a T and B cell epitope of Ovalbumin protein, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505;
Part #4065609). Simvastatin was purchased from LKT Laboratories,
Inc. (2233 University Avenue West, St. Paul, Minn. 55114; Product
Catalogue # S3449). PLGA with a lactide:glycolide ratio of 3:1 and
an inherent viscosity of 0.75 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;
Product Code 7525 DLG 7A). PLA-PEG block co-polymer with a PEG
block of approximately 5,000 Da and PLA block of approximately
20,000 Da was synthesized. Polyvinyl alcohol (85-89% hydrolyzed)
was purchased from EMD Chemicals (Product Number 1.41350.1001).
[0286] Solutions were Prepared as Follows:
[0287] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature.
[0288] Solution 2: Simvastatin @ 50 mg/mL in methylene chloride.
The solution was prepared by dissolving simvastatin in pure
methylene chloride.
[0289] Solution 3: PLGA @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLGA in pure methylene
chloride.
[0290] Solution 4: PLA-PEG @ 100 mg/mL in methylene chloride. The
solution was prepared by dissolving PLA-PEG in pure methylene
chloride.
[0291] Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100 mM pH 8
phosphate buffer.
[0292] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining solution 1 (0.2 mL), solution 2 (0.15
mL), solution 3 (0.75 mL), and solution 4 (0.25 mL) in a small
pressure tube and sonicating at 50% amplitude for 40 seconds using
a Branson Digital Sonifier 250. A secondary emulsion (W1/O1/W2) was
then prepared by combining solution 5 (3.0 mL) with the primary
W1/O1 emulsion, vortexing for 10 s, and sonicating at 30% amplitude
for 60 seconds using the Branson Digital Sonifier 250.
[0293] The W1/O1/W2 emulsion was added to a beaker containing 70 mM
pH 8 phosphate buffer solution (30 mL) and stirred at room
temperature for 2 hours to allow the methylene chloride to
evaporate and for the nanocarriers to form. A portion of the
nanocarriers were washed by transferring the nanocarrier suspension
to a centrifuge tube and centrifuging at 75,600.times.g and
4.degree. C. for 35 min, removing the supernatant, and
re-suspending the pellet in phosphate buffered saline. The washing
procedure was repeated, and the pellet was re-suspended in
phosphate buffered saline for a final nanocarrier dispersion of
about 10 mg/mL.
[0294] Nanocarrier size was determined by dynamic light scattering.
The amounts of peptide and simvastatin in the nanocarrier were
determined by HPLC analysis. The total dry-nanocarrier mass per mL
of suspension was determined by a gravimetric method.
TABLE-US-00005 Nanocarrier Effective Simvastatin Peptide ID
Diameter (nm) Content (% w/w) Content (% w/w) Nanocarrier 5 226 2.7
1.9
In Vivo Administration 1
[0295] Spleens from B6.Cg-Tg(TcraTcrb)425Cbn/J (OTII) and C57BL/6
(B6) mice were harvested, mechanically dissociated and filtered
separately through a 70 .mu.M sieve to yield a single-cell
suspension. Purified CD4.sup.+CD25- cells were then extracted in a
2-step process. Using a Miltenyi Biotec AutoMACS magnetic cell
sorter spleen cells were first labeled with CD4.sup.+ T-cell
isolation kit II and the unlabeled fraction was depleted of
CD25.sup.+ cells with CD25 depletion kit. The purified B6 cells
were stained with an intracellular dye, Carboxyfluorescein
Succinimidyl Ester (CFSE), before being admixed at equal
concentrations with the purified OTII cells. They were then
injected intravenously (i.v.) into B6.SJL-Ptprc.sup.a/BoyAi
(CD45.1) recipient mice.
[0296] The next day the recipient CD45.1 mice were treated with
targeted tolerogenic synthetic vaccine particles (t.sup.2SVP). They
were loaded with combinations of ovalbumin peptide (323-339)
(OVA.sup.323-339), Rapamycin (Rapa) and/or Simvastatin (Simva) and
were administered subcutaneously (s.c.).
[0297] The injection constitutes a tolerogenic treatment and was
followed by 4 more injections each spaced 2 weeks apart. After the
treatment schedule was completed the recipient CD45.1 animals were
killed and their spleens and popliteal lymph nodes were harvested,
mechanically dissociated and filtered separately through a 70 .mu.M
sieve to yield a single-cell suspension. The spleen cells were
depleted of red blood cells (RBCs) by incubation with RBC lysis
buffer (Stem Cell Technologies) and cell counts were performed on
both the spleens and lymph nodes.
[0298] Spleen or lymph node cells were cultured in CM (complete
media) supplemented with 10 U/ml IL-2, restimulated with OPII at
0.3.times.10.sup.6 cells/well in 96-well round bottom (RB) plates
and incubated at 37.degree. C., 5% CO.sub.2. Cells were split at
Day 2 and harvested on Day 5. Supernatants were collected and
frozen while cells were stained for phenotypic analysis by flow
cytometry. The cells were analyzed on a Becton Dickinson FacsCanto
flow cytometer.
In Vivo Administration 2
[0299] Spleens from B6.Cg-Tg(TcraTcrb)425Cbn/J (OTII) and C57BL/6
(B6) mice were harvested, mechanically dissociated and filtered
separately through a 70 .mu.M sieve to yield a single-cell
suspension. Purified CD4.sup.+CD25- cells were then extracted in a
2-step process using a Miltenyi Biotec AutoMACS magnetic cell
sorter. Spleen cells were labeled using Miltenyi's CD4.sup.+ T-cell
isolation kit II. The unlabeled CD4+ T-cell fraction was then
depleted of CD25.sup.+ cells with CD25 depletion kit. The purified
CD4 cells from B6 mice were then stained with an intracellular dye,
Carboxyfluorescein Succinimidyl Ester (CFSE), before being admixed
at equal concentrations with the purified OTII cells. They were
then injected intravenously (i.v.) into B6.SJL-Ptprc.sup.a/BoyAi
(CD45.1) recipient mice.
[0300] The next day the recipient CD45.1 mice were treated with
targeted tolerogenic synthetic vaccine particles. They comprised
combinations of ovalbumin peptide (323-339) (OVA.sup.323-339),
Rapamycin (Rapa) and Simvastatin (Simva) and were administered
subcutaneously (s.c.) or intravenously (i.v.).
[0301] After the treatment schedule was completed the recipient
CD45.1 animals were killed and their spleens and popliteal lymph
nodes were harvested, mechanically dissociated and filtered
separately through a 70 .mu.M sieve to yield a single-cell
suspension. The spleen cells were depleted of red blood cells
(RBCs) by incorporation with RBC lysis buffer (Stem Cell
Technologies) and cell counts were performed on both the spleens
and lymph nodes.
[0302] Spleen or lymph node cells were cultured in CM supplemented
with 10 U/ml IL-2, restimulated with 1 .mu.M OPII at
0.3.times.10.sup.6 cells/well in 96-well round bottom (RB) plates
and incubated at 37.degree. C., 5% CO.sub.2. Cells were split at
Day 2 and harvested on Day 5. Supernatants were collected and
frozen while cells were stained for phenotypic analysis by flow
cytometry. The cells were analyzed on a Becton Dickinson FacsCanto
flow cytometer.
Results
[0303] The results are shown in FIGS. 2 and 3 (Immunomodulator 1:
rapamycin; immunomodulator 2: simvastatin). The figures shows in
vivo effects and demonstrates that antigen-specific expansion of
effector immune cells is reduced with synthetic nanocarriers
comprising antigen and immunosuppressants as compared to antigen
alone or synthetic nanocarriers comprising antigen with and without
an immunostimulatory molecule.
Example 13
Assessing the Effects of Nanocarriers with Antigens and
Immunosuppressants on Immune Responses
Materials and Methods
Nanocarrier 1
[0304] Ovalbumin peptide 323-339, a 17 amino acid peptide known to
be a T and B cell epitope of Ovalbumin protein, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505;
Part #4065609). PLGA with a lactide:glycolide ratio of 3:1 and an
inherent viscosity of 0.75 dL/g was purchased from SurModics
Pharmaceuticals (756 Tom Martin Drive, Birmingham, Ala. 35211;
Product Code 7525 DLG 7A). PLA-PEG block co-polymer with a PEG
block of approximately 5,000 Da and PLA block of approximately
20,000 Da was synthesized. Polyvinyl alcohol (85-89% hydrolyzed)
was purchased from EMD Chemicals (Product Number 1.41350.1001).
[0305] Solutions were Prepared as Follows:
[0306] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature. Solution 2: PLGA @ 100 mg/mL in methylene
chloride. The solution was prepared by dissolving PLGA in pure
methylene chloride. Solution 3: PLA-PEG @ 100 mg/mL in methylene
chloride. The solution was prepared by dissolving PLA-PEG in pure
methylene chloride. Solution 4: Polyvinyl alcohol @ 50 mg/mL in 100
mM pH 8 phosphate buffer.
[0307] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining solution 1 (0.2 mL), solution 2 (0.75
mL), and solution 3 (0.25 mL) in a small pressure tube and
sonicating at 50% amplitude for 40 seconds using a Branson Digital
Sonifier 250. A secondary emulsion (W1/O1/W2) was then prepared by
combining solution 4 (3.0 mL) with the primary W1/O1 emulsion,
vortexing for 10 s, and sonicating at 30% amplitude for 60 seconds
using the Branson Digital Sonifier 250. The W1/O1/W2 emulsion was
added to a beaker containing 70 mM pH 8 phosphate buffer solution
(30 mL) and stirred at room temperature for 2 hours to allow the
methylene chloride to evaporate and for the nanocarriers to form. A
portion of the nanocarriers were washed by transferring the
nanocarrier suspension to a centrifuge tube and centrifuging at
75,600.times.g and 4.degree. C. for 35 min, removing the
supernatant, and re-suspending the pellet in phosphate buffered
saline. The washing procedure was repeated, and the pellet was
re-suspended in phosphate buffered saline for a final nanocarrier
dispersion of about 10 mg/mL.
[0308] Nanocarrier size was determined by dynamic light scattering.
The amount of peptide in the nanocarrier was determined by HPLC
analysis. The total dry-nanocarrier mass per mL of suspension was
determined by a gravimetric method.
TABLE-US-00006 Nanocarrier Effective Peptide ID Diameter (nm)
Content (% w/w) 1 234 2.1
Nanocarrier 2
[0309] Ovalbumin peptide 323-339, a 17 amino acid peptide known to
be a T and B cell epitope of Ovalbumin protein, was purchased from
Bachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505;
Part #4065609). Rapamycin was purchased from TSZ CHEM (185 Wilson
Street, Framingham, Mass. 01702; Product Catalogue # R1017). PLGA
with a lactide:glycolide ratio of 3:1 and an inherent viscosity of
0.75 dL/g was purchased from SurModics Pharmaceuticals (756 Tom
Martin Drive, Birmingham, Ala. 35211; Product Code 7525 DLG 7A).
PLA-PEG block co-polymer with a PEG block of approximately 5,000 Da
and PLA block of approximately 20,000 Da was synthesized. Polyvinyl
alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals
(Product Number 1.41350.1001).
[0310] Solutions were Prepared as Follows:
[0311] Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL in dilute
hydrochloric acid aqueous solution. The solution was prepared by
dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution
at room temperature. Solution 2: Rapamycin @ 50 mg/mL in methylene
chloride. The solution was prepared by dissolving rapamycin in pure
methylene chloride. Solution 3: PLGA @ 100 mg/mL in methylene
chloride. The solution was prepared by dissolving PLGA in pure
methylene chloride. Solution 4: PLA-PEG @ 100 mg/mL in methylene
chloride. The solution was prepared by dissolving PLA-PEG in pure
methylene chloride. Solution 5: Polyvinyl alcohol @ 50 mg/mL in 100
mM pH 8 phosphate buffer.
[0312] A primary water-in-oil emulsion was prepared first. W1/O1
was prepared by combining solution 1 (0.2 mL), solution 2 (0.2 mL),
solution 3 (0.75 mL), and solution 4 (0.25 mL) in a small pressure
tube and sonicating at 50% amplitude for 40 seconds using a Branson
Digital Sonifier 250. A secondary emulsion (W1/O1/W2) was then
prepared by combining solution 5 (3.0 mL) with the primary W1/O1
emulsion, vortexing for 10 s, and sonicating at 30% amplitude for
60 seconds using the Branson Digital Sonifier 250. The W1/O1/W2
emulsion was added to a beaker containing 70 mM pH 8 phosphate
buffer solution (30 mL) and stirred at room temperature for 2 hours
to allow the methylene chloride to evaporate and for the
nanocarriers to form. A portion of the nanocarriers were washed by
transferring the nanocarrier suspension to a centrifuge tube and
centrifuging at 21,000.times.g and 4.degree. C. for 45 min,
removing the supernatant, and re-suspending the pellet in phosphate
buffered saline. The washing procedure was repeated, and the pellet
was re-suspended in phosphate buffered saline for a final
nanocarrier dispersion of about 10 mg/mL.
[0313] Nanocarrier size was determined by dynamic light scattering.
The amounts of peptide and rapamycin in the nanocarrier were
determined by HPLC analysis. The total dry-nanocarrier mass per mL
of suspension was determined by a gravimetric method.
TABLE-US-00007 Nanocarrier Effective Rapamycin Peptide ID Diameter
(nm) Content (% w/w) Content (% w/w) 2 227 9.0 2.5
Immunization
[0314] Animals received immunization every 2 weeks at the same time
they received the treatment. Each of these groups was split into
subgroups to test the capacity of different treatments to modify
the Ig titers induced. A control subgroup did not receive
tolerogenic treatment. Two subgroups received nanocarrier carrying
just OVA.sub.323-339 peptide or in combination with rapamycin.
[0315] Immunization was administered via the following routes
(values are per animal): 20 .mu.l/limb of OVA+CpG (12.5 .mu.g
OVA+10 .mu.g CpG), both hind limbs s.c. Tolerogenic treatments were
administered via the following route (values are per animal): 200
.mu.l nanocarriers were provided at 100 .mu.g/ml of OVA.sub.323-339
content.
Measurement of IgG
[0316] The level of IgG antibodies were measured. This level is
indicative of immunoglobulins in general, including IgEs, which are
of particular relevance in allergy. Blocker Casein in PBS (Thermo
Fisher, Catalog #37528) was used as diluent. 0.05% Tween-20 in PBS
was used as wash buffer, prepared by adding 10 ml of Tween-20
((Sigma, Catalog #P9416-100 mL) to 2 liters of a 10.times.PBS stock
(PBS: OmniPur.RTM. 10.times.PBS Liquid Concentrate, 4 L, EMD
Chemicals, Catalog #6505) and 18 Liters of deionized water. OVA
protein at a stock concentration of 5 mg/ml was used as a coating
material. A 1:1000 dilution to 5 .mu.g/ml was used as a working
concentration. Each well of the assay plates was coated with 100
.mu.l diluted OVA per well, plates were sealed with sealing film
(VWR catalog #60941-120), and incubated overnight at 4.degree. C.
Costar9017 96-well Flat bottom plates were used as assay plates,
Costar9017.
[0317] Low-binding polypropylene 96-well plate or tubes were used
as set-up plates, in which samples were prepared before being
transferred to the assay plate. The setup plates did not contain
any antigen and, therefore, serum antibodies did not bind to the
plate during the setup of the samples. Setup plates were used for
sample preparation to minimize binding that might occur during
preparation or pipetting of samples if an antigen-coated plate was
used to prepare the samples. Before preparing samples in the setup
plate, wells were covered with diluent to block any non-specific
binding and the plate was sealed and incubated at 4.degree. C.
overnight.
[0318] Assay plates were washed three times with wash buffer, and
wash buffer was completely aspirated out of the wells after the
last wash. After washing, 300 .mu.l diluent were added to each well
of assay plate(s) to block non-specific binding and plates were
incubated at least 2 hours at room temperature. Serum samples were
prepared in the setup plate at appropriate starting dilutions.
Starting dilutions were sometimes also prepared in 1.5 ml tubes
using diluent. Appropriate starting dilutions were determined based
on previous data, where available. Where no previous data was
available, the lowest starting dilution was 1:40. Once diluted, 200
.mu.l of the starting dilution of the serum sample was transferred
from to the appropriate well of the setup plate.
[0319] An exemplary setup plate layout is described as follows:
Columns 2 and 11 contained anti-Ovabumin monoclonal IgG2b isotype
(AbCam, ab17291) standard, diluted to 1 .mu.g/mL (1:4000 dilution).
Columns 3-10 contained serum samples (at appropriate dilutions).
Columns 1 and 12 were not used for samples or standards to avoid
any bias of measurements due to edge effect. Instead, columns 1 and
12 contained 200 .mu.l diluent. Normal mouse serum diluted 1:40 was
used as a negative control. Anti-mouse IgG2a diluted 1:500 from 0.5
mg/mL stock (BD Bioscience) was used as an isotype control.
[0320] Once all samples were prepared in the setup plate, the plate
was sealed and stored at 4.degree. C. until blocking of the assay
plates was complete. Assay plates were washed three times with wash
buffer, and wash buffer was completely aspirated after the last
wash. After washing, 100 .mu.L of diluent was added to all wells in
rows B-H of the assay plates. A 12-channel pipet was used to
transfer samples from the setup plate to the assay plate. Samples
were mixed prior to transfer by pipetting 150 .mu.l of diluted
serum up and down 3 times. After mixing, 150 .mu.l of each sample
was transferred from the setup plate and added to row A of the
respective assay plate.
[0321] Once the starting dilutions of each sample were transferred
from the setup plate to row A of the assay plate, serial dilutions
were pipetted on the assay plate as follows: 50 .mu.l of each serum
sample was removed from row A using 12-channel pipet and mixed with
the 100 .mu.l of diluent previously added to each well of row B.
This step was repeated down the entire plate. After pipetting the
dilution of the final row, 50 .mu.l of fluid was removed from the
wells in the final row and discarded, resulting in a final volume
of 100 .mu.l in every well of the assay plate. Once sample
dilutions were prepared in the assay plates, the plates were
incubated at room temperature for at least 2 hours.
[0322] After the incubation, plates were washed three times with
wash buffer. Detection antibody (Goat anti-mouse anti-IgG, HRP
conjugated, AbCam ab98717) was diluted 1:1500 (0.33 .mu.g/mL) in
diluent and 100 .mu.l of the diluted antibody was added to each
well. Plates were incubated for 1 hour at room temperature and then
washed three times with wash buffer, with each washing step
including a soak time of at least 30 seconds.
[0323] After washing, detection substrate was added to the wells.
Equal parts of substrate A and substrate B (BD Biosciences TMB
Substrate Reagent Set, catalog #555214) were combined immediately
before addition to the assay plates, and 100 .mu.l of the mixed
substrate solution were added to each well and incubated for 10
minutes in the dark. The reaction was stopped by adding 50 .mu.l of
stop solution (2NH2SO4) to each well after the 10 minute period.
The optical density (OD) of the wells was assessed immediately
after adding the stop solution on a plate reader at 450 nm with
subtraction at 570 nm. Data analysis was performed using Molecular
Device's software SoftMax Pro v5.4. In some cases, a four-parameter
logistic curve-fit graph was prepared with the dilution on the
x-axis (log scale) and the OD value on the y-axis (linear scale),
and the half maximum value (EC50) for each sample was determined.
The plate template at the top of the layout was adjusted to reflect
the dilution of each sample (1 per column).
Determination of % OVA+Dividing B Cells
[0324] Ovalbumin+ B-cell division was assessed by flow cytometry.
Splenocytes from experimental animals were stained with Cell
Tracker Orange (CTO), a thiol-reactive fluorescent probe suitable
for long-term cell labeling, and cultured in complete media at 37
C, 5% CO.sub.2 with Ovalbumin protein or peptide for 3 days. On day
3 the cells were washed, blocked with anti-CD16/32 antibody and
then stained with conjugated antibodies specific to B220 and CD19.
Alexa 647 conjugated ovalbumin protein was also incubated with the
cells to label Ovalbumin specific BCRs. Those splenocytes that were
CD19+ B220+ OVA- Alexa647+ were assessed for proliferation by
comparing the differential CTO staining. Those that were CTO low
were labeled as proliferating Ovalbumin+ B-cells and were compared
to the CTO high Ovalbumin+ B-cells to quantify the percentages.
Results
[0325] FIG. 4 shows a reduction in antigen-specific IgG levels with
the administration of synthetic nanocarriers comprising ova peptide
and the immunosuppressant rapamycin. The level of IgG antibodies is
reflective of antibody production in general including the
production of IgE antibodies, which are of particular relevance in
allergy and allergic reactions. FIG. 5 also demonstrates a
reduction, but in the number of antigen-specific B cells with the
synthetic nanocarriers. These results demonstrate the reduction in
undesired immune responses relevant to allergy and allergic
responses with synthetic nanocarriers coupled to ova peptide
(comprising an MHC Class II-restricted epitope) and
immunosuppressant.
Example 14
Assessing the Effects of Nanocarriers with Antigens and
Immunosuppressants on Allergic Asthma
Nanocarriers
[0326] Nanocarriers were prepared according to methods provided
above (Example 13).
Immunization
[0327] The nanocarriers were thawed and equilibrated. Initial
dilutions constituted a 10.times. stock solution, and were further
diluted to a concentration of 100 .mu.g/ml in OVA.sub.323-339, or a
1.times. solution. This 1.times. solution was used for injections
at 200 .mu.l per i.v. injection. Animals were immunized with OVA
protein (OVA) and treated with OVA.sub.323-339 peptide to assess
the capacity of nanocarriers to control the allergic response in
absence of B cell antigens. Immunization routes were as follows: 10
.mu.g of OVA+ 4 mg Alum i.p. in 400 .mu.l per each Balb/C
immunologically naive female mouse. Experimental groups consisted
of 5 animals each. Spleen cells were restimulated with antigen
using CFSE or CTO to determine the amount of Ag-specific
proliferation.
Levels of Specific Types of Immune Cells
[0328] FCS files were analyzed using FlowJo software. 7AAD positive
cells (a nuclear dye that label dead cells) positive cells were
excluded and cell morphologies dependent on expression of CD4, CD8,
Gr-1, F4/80, B220, TCRb and CD11b were quantified.
Gating strategy for T-cell subsets.fwdarw.7AAD- F4/80- GR-1- TCRb+
CD4+/- CD8+/- Gating strategy for B-cell subsets.fwdarw.7AAD- B220+
TCRb- Gating strategy for Eosinophils.fwdarw.7AAD- F4/80- Gr-1+
TCRb- CD11b+ Gr-1+
Determination of % Dividing CD4+ T Cells
[0329] The frequency of Ovalbumin reactive CD4.sup.+ T cells was
calculated by way of flow cytometry. Splenocytes from experimental
animals were stained with CFSE, a thiol-reactive Fluorescent Probe
suitable for long-term cell labeling, and cultured in complete
media at 37 C, 5% CO.sub.2 with Ovalbumin protein for 3 days. On
day 3 the cells were washed, blocked with anti-CD16/32 antibody and
then stained with conjugated antibodies specific to TCR CD4 and
CD8a. Splenocytes that were TCR+CD4 or TCR+CD8a+ were assessed for
proliferation by comparing the differential CFSE staining.
Measurement of IgE Antibodies
[0330] IgE antibodies were measured using a Mouse OVA-IgE ELISA kit
provided by MDBioproducts (Cat# M036005) consistent with the
manufacturer's instructions.
Results
[0331] FIGS. 6 and 7 demonstrate the effectiveness of the
nanocarriers in an animal model for allergic asthma. Specifically,
FIG. 6 demonstrates an overall reduction in the number of various
immune cells in lavage samples from asthma model animal subjects
treated with synthetic nanocarriers comprising OVA.sub.323-339 (an
MHC Class II-restricted epitope) and immunosuppressant. FIG. 7
demonstrates a reduction in the percentage of dividing CD4+ T cells
as a result of the same treatment. FIG. 8 demonstrates a reduction
in the production of antigen-specific IgE antibodies.
Sequence CWU 1
1
516120PRTArtificial SequenceArachis hypogaea 2S protein 1 epitope
1Ala His Ala Ser Ala Arg Gln Gln Trp Glu Leu Gln Gly Asp Arg Arg 1
5 10 15 Cys Gln Ser Gln 20 220PRTArtificial SequenceArachis
hypogaea 2S protein 1 epitope 2Ala Lys Leu Thr Ile Leu Val Ala Leu
Ala Leu Phe Leu Leu Ala Ala 1 5 10 15 His Ala Ser Ala 20
319PRTArtificial SequenceArachis hypogaea 2S protein 1 epitope 3Ala
Leu Gln Gln Ile Met Glu Asn Gln Ser Asp Arg Leu Gln Gly Arg 1 5 10
15 Gln Gln Glu 420PRTArtificial SequenceArachis hypogaea 2S protein
1 epitope 4Ala Asn Leu Arg Pro Cys Glu Gln His Leu Met Gln Lys Ile
Gln Arg 1 5 10 15 Asp Glu Asp Ser 20 520PRTArtificial
SequenceArachis hypogaea 2S protein 1 epitope 5Cys Asn Glu Leu Asn
Glu Phe Glu Asn Asn Gln Arg Cys Met Cys Glu 1 5 10 15 Ala Leu Gln
Gln 20 616PRTArtificial SequenceHomo sapiens 5-hydroxytryptamine
receptor 2C (5-HT-2C) (Serotonin receptor 2C) (5-HT2C) (5-HTR2C)
(5HT-1C) epitope 6Pro Arg Gly Thr Met Gln Ala Ile Asn Asn Glu Arg
Lys Ala Ser Lys 1 5 10 15 712PRTArtificial SequenceBos taurus
Allergen Bos d 2 precursor epitope 7Asp Gln Gly Thr Cys Leu Leu Leu
Thr Glu Val Ala 1 5 10 814PRTArtificial SequenceBos taurus Allergen
Bos d 2 precursor epitope 8Glu Leu Glu Lys Tyr Gln Gln Leu Asn Ser
Glu Arg Gly Val 1 5 10 913PRTArtificial SequenceBos taurus Allergen
Bos d 2 precursor epitope 9Gly Glu Arg Ile Thr Lys Met Thr Glu Gly
Leu Ala Lys 1 5 10 1014PRTArtificial SequenceBos taurus Allergen
Bos d 2 precursor epitope 10Pro Gly Glu Trp Arg Ile Ile Tyr Ala Ala
Ala Asp Asn Lys 1 5 10 118PRTArtificial SequenceBos taurus Allergen
Bos d 2 precursor epitope 11Arg Ile Glu Cys Ile Asn Asp Cys 1 5
1212PRTArtificial SequenceBos taurus Allergen Bos d 2 precursor
epitope 12Val Ala Lys Arg Gln Glu Gly Tyr Val Tyr Val Leu 1 5 10
1310PRTArtificial SequenceBos taurus Allergen Bos d 2 precursor
epitope 13Val Ser Glu Asn Met Leu Val Thr Tyr Val 1 5 10
1416PRTArtificial SequenceBos taurus Allergen Bos d 2 precursor
epitope 14Glu Leu Glu Lys Tyr Gln Gln Leu Asn Ser Glu Arg Gly Val
Pro Asn 1 5 10 15 1513PRTArtificial SequenceCryptomeria japonica
Allergen Cry j 2 epitope 15Asp Ile Phe Ala Ser Lys Asn Phe His Leu
Gln Lys Asn 1 5 10 1613PRTArtificial SequenceCryptomeria japonica
Allergen Cry j 2 epitope 16Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala
Ser Trp Lys 1 5 10 1712PRTArtificial SequenceCryptomeria japonica
Allergen Cry j 2 epitope 17Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys
Leu Asn 1 5 10 1812PRTArtificial SequenceCryptomeria japonica
Allergen Cry j 2 epitope 18Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu
Met Gly 1 5 10 198PRTArtificial SequenceAspergillus fumigatus
allergen I/a; Asp f I/a epitope 19Ile Asn Gln Gln Leu Asn Pro Lys 1
5 2015PRTArtificial SequenceAspergillus fumigatus allergen I/a; Asp
f I/a epitope 20Ile Asn Gln Gln Leu Asn Pro Lys Thr Asn Lys Trp Glu
Asp Lys 1 5 10 15 2111PRTArtificial SequenceAspergillus fumigatus
allergen I/a; Asp f I/a epitope 21Leu Asn Pro Lys Thr Asn Lys Trp
Glu Asp Lys 1 5 10 227PRTArtificial SequenceAspergillus fumigatus
allergen I/a; Asp f I/a epitope 22Thr Asn Lys Trp Glu Asp Lys 1 5
2312PRTArtificial SequenceAspergillus fumigatus allergen I/a; Asp f
I/a epitope 23Leu Asn Pro Lys Thr Asn Lys Trp Glu Asp Lys Arg 1 5
10 2415PRTArtificial SequenceDermatophagoides farinae Allergen Mag
epitope 24Pro Arg Leu Ser Trp His Gln Tyr Thr Lys Arg Asp Ser Arg
Glu 1 5 10 15 2515PRTArtificial SequenceDermatophagoides farinae
Allergen Mag epitope 25Thr Val Asp Leu Ile Ser Pro Val Thr Lys Arg
Ala Ser Leu Lys 1 5 10 15 2618PRTArtificial SequenceBos taurus
Alpha-S1-casein precursor epitope 26Ala Trp Tyr Tyr Val Pro Leu Gly
Thr Gln Tyr Thr Asp Ala Pro Ser 1 5 10 15 Phe Ser 2718PRTArtificial
SequenceBos taurus Alpha-S1-casein precursor epitope 27Asp Ala Tyr
Pro Ser Gly Ala Trp Tyr Tyr Val Pro Leu Gly Thr Gln 1 5 10 15 Tyr
Thr 2818PRTArtificial SequenceBos taurus Alpha-S1-casein precursor
epitope 28Asp Ile Gly Ser Glu Ser Thr Glu Asp Gln Ala Met Glu Asp
Ile Lys 1 5 10 15 Gln Met 296PRTArtificial SequenceBos taurus
Alpha-S1-casein precursor epitope 29Glu Asp Ile Lys Gln Met 1 5
3012PRTArtificial SequenceBos taurus Alpha-S1-casein precursor
epitope 30Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr 1 5 10
3118PRTArtificial SequenceBos taurus Alpha-S1-casein precursor
epitope 31Glu Pro Met Ile Gly Val Asn Gln Glu Leu Ala Tyr Phe Tyr
Pro Glu 1 5 10 15 Leu Phe 3217PRTArtificial SequenceArachis
hypogaea Ara h 2.01 allergen epitope 32Glu Leu Asn Glu Phe Glu Asn
Asn Gln Arg Cys Met Cys Glu Ala Leu 1 5 10 15 Gln 3316PRTArtificial
SequenceArachis hypogaea Ara h 2.01 allergen epitope 33Ser Gln Leu
Glu Arg Ala Asn Leu Arg Pro Cys Glu Gln His Leu Met 1 5 10 15
3415PRTArtificial SequenceCryptomeria japonica Cry j 1 precursor
epitope 34Gly Ala Thr Arg Asp Arg Pro Leu Trp Ile Ile Phe Ser Gly
Asn 1 5 10 15 3520PRTArtificial SequenceCryptomeria japonica Cry j
1 precursor epitope 35Ile Phe Ser Gly Asn Met Asn Ile Lys Leu Lys
Met Pro Met Tyr Ile 1 5 10 15 Ala Gly Tyr Lys 20 3620PRTArtificial
SequenceCryptomeria japonica Cry j 1 precursor epitope 36Lys Met
Pro Met Tyr Ile Ala Gly Tyr Lys Thr Phe Asp Gly Arg Gly 1 5 10 15
Ala Gln Val Tyr 20 3720PRTArtificial SequenceCryptomeria japonica
Cry j 1 precursor epitope 37Leu Gly His Asp Asp Ala Tyr Ser Asp Asp
Lys Ser Met Lys Val Thr 1 5 10 15 Val Ala Phe Asn 20
3819PRTArtificial SequenceCryptomeria japonica Cry j 1 precursor
epitope 38Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu
Ala Phe 1 5 10 15 Asn Val Glu 3911PRTArtificial
SequenceCochliobolus lunatus Cytochrome c epitope 39Glu Asn Pro Lys
Lys Tyr Ile Pro Gly Thr Lys 1 5 10 4011PRTArtificial
SequenceCochliobolus lunatus Cytochrome c epitope 40Gly Leu Phe Gly
Arg Lys Thr Gly Ser Val Ala 1 5 10 419PRTArtificial
SequenceCochliobolus lunatus Cytochrome c epitope 41Lys Ile Gly Pro
Glu Leu His Gly Leu 1 5 4212PRTArtificial SequenceCochliobolus
lunatus Cytochrome c epitope 42Leu Lys Ala Gly Glu Gly Asn Lys Ile
Gly Pro Glu 1 5 10 4311PRTArtificial SequenceCochliobolus lunatus
Cytochrome c epitope 43Leu Lys Lys Pro Lys Asp Arg Asn Asp Leu Ile
1 5 10 4418PRTArtificial SequenceDermatophagoides farinae Der f 2
allergen epitope 44Gly Leu Glu Ile Asp Val Pro Gly Ile Asp Thr Asn
Ala Cys His Phe 1 5 10 15 Val Lys 4520PRTArtificial
SequenceDermatophagoides farinae Der f 2 allergen epitope 45Pro Gly
Ile Asp Thr Asn Ala Cys His Phe Val Lys Cys Pro Leu Val 1 5 10 15
Lys Gly Gln Gln 20 4619PRTArtificial SequenceDermatophagoides
pteronyssinus Der p 1 allergen epitope 46Arg Phe Gly Ile Ser Asn
Tyr Cys Gln Ile Tyr Pro Pro Asn Ala Asn 1 5 10 15 Lys Ile Arg
4715PRTArtificial SequenceDermatophagoides pteronyssinus Der p 1
allergen epitope 47Ala Val Asn Ile Val Gly Tyr Ser Asn Ala Gln Gly
Val Asp Tyr 1 5 10 15 4820PRTArtificial SequenceChironomus thummi
globin Ctt 3-1 epitope 48Phe Ala Gly Lys Asp Leu Glu Ser Ile Lys
Gly Thr Ala Pro Phe Glu 1 5 10 15 Thr His Ala Asn 20
4911PRTArtificial SequenceChironomus thummi globin Ctt 3-1 epitope
49Gly Thr Ala Pro Phe Glu Thr His Ala Asn Arg 1 5 10
5021PRTArtificial SequenceChironomus thummi globin Ctt 3-1 epitope
50Lys Gly Thr Ala Pro Phe Glu Thr His Ala Asn Arg Ile Val Gly Phe 1
5 10 15 Phe Ser Lys Ile Ile 20 5121PRTArtificial SequenceChironomus
thummi thummi Globin CTT-III epitope 51Ala His Thr Asp Phe Ala Gly
Ala Glu Ala Ala Trp Gly Ala Thr Leu 1 5 10 15 Asp Thr Phe Phe Gly
20 5220PRTArtificial SequenceChironomus thummi thummi Globin
CTT-III epitope 52Phe Ala Gly Lys Asp Leu Glu Ser Ile Lys Gly Thr
Ala Pro Phe Glu 1 5 10 15 Ile His Ala Asn 20 5321PRTArtificial
SequenceChironomus thummi thummi Globin CTT-III epitope 53Val Asn
Thr Phe Val Ala Ser His Lys Pro Arg Gly Val Thr His Asp 1 5 10 15
Gln Leu Asn Asn Phe 20 548PRTArtificial SequenceChironomus thummi
thummi Globin CTT-III precursor epitope 54Ala Asp Pro Ser Ile Met
Ala Lys 1 5 5521PRTArtificial SequenceChironomus thummi thummi
Globin CTT-III precursor epitope 55Ala Asp Pro Ser Ile Met Ala Lys
Phe Thr Gln Phe Ala Gly Lys Asp 1 5 10 15 Leu Glu Ser Ile Lys 20
565PRTArtificial SequenceChironomus thummi thummi Globin CTT-III
precursor epitope 56Ala Glu Ala Ala Trp 1 5 5720PRTArtificial
SequenceChironomus thummi thummi Globin CTT-III precursor epitope
57Ala Glu Ala Ala Trp Gly Ala Thr Leu Asp Thr Phe Phe Gly Met Ile 1
5 10 15 Phe Ser Lys Met 20 588PRTArtificial SequenceChironomus
thummi thummi Globin CTT-III precursor epitope 58Ala Gly Phe Val
Ser Tyr Met Lys 1 5 5915PRTArtificial SequencePhaseolus vulgaris
Glycine-rich cell wall structural protein 1.8 precursor epitope
59Gly Gly Tyr Gly Asp Gly Gly Ala His Gly Gly Gly Tyr Gly Gly 1 5
10 15 6015PRTArtificial SequencePhleum pratense Group V allergen
Phl p 5 epitope 60Ala Thr Pro Glu Ala Lys Tyr Asp Ala Tyr Val Ala
Thr Leu Ser 1 5 10 15 6115PRTArtificial SequencePhleum pratense
Group V allergen Phl p 5 epitope 61Phe Thr Val Phe Glu Ala Ala Phe
Asn Asn Ala Ile Lys Ala Gly 1 5 10 15 6215PRTArtificial
SequencePhleum pratense Group V allergen Phl p 5 epitope 62Lys Tyr
Asp Ala Tyr Val Ala Thr Leu Ser Glu Ala Leu Arg Ile 1 5 10 15
6315PRTArtificial SequencePhleum pratense Group V allergen Phl p 5
epitope 63Pro Ala Asn Asp Lys Phe Thr Val Phe Glu Ala Ala Phe Asn
Asn 1 5 10 15 6415PRTArtificial SequencePhleum pratense Group V
allergen Phl p 5 epitope 64Pro Lys Gly Gly Ala Glu Ser Ser Ser Lys
Ala Ala Leu Thr Ser 1 5 10 15 6516PRTArtificial SequenceHomo
sapiens KIAA1224 protein epitope 65Asp Leu Glu Ser Tyr Leu Gln Leu
Asn Cys Glu Arg Gly Thr Trp Arg 1 5 10 15 6615PRTArtificial
SequenceLepidoglyphus destructor Lep D 2 precursor epitope 66Lys
Gly Glu Ala Leu Asp Phe Asn Tyr Gly Met Thr Ile Pro Ala 1 5 10 15
6712PRTArtificial SequenceCorylus avellana lipid transfer protein
precursor epitope 67Ala Gly Leu Pro Gly Lys Cys Gly Val Asn Ile Pro
1 5 10 6812PRTArtificial SequenceCorylus avellana lipid transfer
protein precursor epitope 68Ala Lys Gly Ile Ala Gly Leu Asn Pro Asn
Leu Ala 1 5 10 6912PRTArtificial SequenceCorylus avellana lipid
transfer protein precursor epitope 69Cys Gly Val Asn Ile Pro Tyr
Lys Ile Ser Pro Ser 1 5 10 7012PRTArtificial SequenceCorylus
avellana lipid transfer protein precursor epitope 70Cys Lys Gly Val
Arg Ala Val Asn Asp Ala Ser Arg 1 5 10 7112PRTArtificial
SequenceCorylus avellana lipid transfer protein precursor epitope
71Cys Val Leu Tyr Leu Lys Asn Gly Gly Val Leu Pro 1 5 10
7216PRTArtificial SequenceHomo sapiens Lipocalin 1 (tear
prealbumin) epitope 72Lys Pro Val Arg Gly Val Lys Leu Val Gly Arg
Asp Pro Lys Asn Asn 1 5 10 15 7315PRTArtificial
SequenceDermatophagoides farinae Mag3 epitope 73Glu Phe Asn Thr Glu
Phe Thr Ile His Ala Asp Lys Asn Asn Leu 1 5 10 15 7415PRTArtificial
SequenceDermatophagoides farinae Mag3 epitope 74Phe Thr Ile His Ala
Asp Lys Asn Asn Leu Lys Met His Met Asp 1 5 10 15 7515PRTArtificial
SequenceDermatophagoides farinae Mag3 epitope 75Lys Met His Met Asp
Phe Pro Asn Val Phe Gln Ala Asp Leu Thr 1 5 10 15 7613PRTArtificial
SequenceApium graveolens Major allergen Api g 1 epitope 76Ala Leu
Phe Lys Ala Leu Glu Ala Tyr Leu Ile Ala Asn 1 5 10
7712PRTArtificial SequenceApium graveolens Major allergen Api g 1
epitope 77Asp Ala Val Val Pro Glu Glu Asn Ile Lys Tyr Ala 1 5 10
7812PRTArtificial SequenceApium graveolens Major allergen Api g 1
epitope 78Asp Ile Leu Leu Gly Phe Ile Glu Ser Ile Glu Asn 1 5 10
7912PRTArtificial SequenceApium graveolens Major allergen Api g 1
epitope 79Gly Gly Ser Ile Cys Lys Thr Thr Ala Ile Phe His 1 5 10
8012PRTArtificial SequenceApium graveolens Major allergen Api g 1
epitope 80Gly Val Gln Thr His Val Leu Glu Leu Thr Ser Ser 1 5 10
8115PRTArtificial SequenceAspergillus fumigatus Major allergen Asp
f 2 precursor epitope 81Phe Gly Asn Arg Pro Thr Met Glu Ala Val Gly
Ala Tyr Asp Val 1 5 10 15 8215PRTArtificial SequenceAspergillus
fumigatus Major allergen Asp f 2 precursor epitope 82Met Glu Ala
Val Gly Ala Tyr Asp Val Ile Val Asn Gly Asp Lys 1 5 10 15
8316PRTArtificial SequenceCanis lupus familiaris Major allergen Can
f 1 precursor epitope 83Ala Leu Glu Asp Phe Arg Glu Phe Ser Arg Ala
Lys Gly Leu Asn Gln 1 5 10 15 8416PRTArtificial SequenceCanis lupus
familiaris Major allergen Can f 1 precursor epitope 84Asp Gln Glu
Val Pro Glu Lys Pro Asp Ser Val Thr Pro Met Ile Leu 1 5 10 15
8512PRTArtificial SequenceCorylus avellana major allergen Cor a
1.0401 epitope 85Ala Gly Lys Glu Lys Ala Ala Gly Leu Phe Lys Ala 1
5 10 8612PRTArtificial SequenceCorylus avellana major allergen Cor
a 1.0401 epitope 86Ala Gly Leu Phe Lys Ala Val Glu Ala Tyr Leu Leu
1 5 10 8712PRTArtificial SequenceCorylus avellana major allergen
Cor a 1.0401 epitope 87Ala Pro Gln His Phe Thr Ser Ala Glu Asn Leu
Glu 1 5 10 8812PRTArtificial SequenceCorylus avellana major
allergen Cor a 1.0401 epitope 88Ala Arg Leu Phe Lys Ser Phe Val Leu
Asp Ala Asp 1 5 10 8912PRTArtificial SequenceCorylus avellana major
allergen Cor a 1.0401 epitope 89Glu Ile Asp His Ala Asn Phe Lys Tyr
Cys Tyr Ser 1 5 10 9013PRTArtificial SequenceDaucus carota Major
allergen Dau c 1 epitope 90Ala Leu Phe Lys Ala Ile Glu Ala Tyr Leu
Ile Ala Asn 1 5 10 9116PRTArtificial SequenceEquus caballus Major
allergen Equ c 1 precursor epitope 91Asp Gly Tyr Asn Val Phe Arg
Ile Ser Glu Phe Glu Asn Asp Glu His 1 5 10 15 9216PRTArtificial
SequenceEquus caballus Major allergen Equ c 1 precursor epitope
92Asp Lys Asp Arg Pro Phe Gln Leu Phe Glu Phe Tyr Ala Arg Glu Pro 1
5
10 15 9316PRTArtificial SequenceEquus caballus Major allergen Equ c
1 precursor epitope 93Asp Leu Thr Lys Ile Asp Arg Cys Phe Gln Leu
Arg Gly Asn Gly Val 1 5 10 15 9416PRTArtificial SequenceEquus
caballus Major allergen Equ c 1 precursor epitope 94Asp Arg Pro Phe
Gln Leu Phe Glu Phe Tyr Ala Arg Glu Pro Asp Val 1 5 10 15
9516PRTArtificial SequenceEquus caballus Major allergen Equ c 1
precursor epitope 95Asp Val Ser Pro Glu Ile Lys Glu Glu Phe Val Lys
Ile Val Gln Lys 1 5 10 15 9617PRTArtificial SequenceFelis catus
major allergen I epitope 96Glu Asn Ala Arg Ile Leu Lys Asn Cys Val
Asp Ala Lys Met Thr Glu 1 5 10 15 Glu 9717PRTArtificial
SequenceFelis catus major allergen I epitope 97Arg Asp Val Asp Leu
Phe Leu Thr Gly Thr Pro Asp Glu Tyr Val Glu 1 5 10 15 Gln
9817PRTArtificial SequenceFelis catus major allergen I epitope
98Thr Gly Thr Pro Asp Glu Tyr Val Glu Gln Val Ala Gln Tyr Lys Ala 1
5 10 15 Leu 9917PRTArtificial SequenceFelis catus Major allergen I
polypeptide chain 1 precursor epitope 99Asp Val Asp Leu Phe Leu Thr
Gly Thr Pro Asp Glu Tyr Val Glu Gln 1 5 10 15 Val
10017PRTArtificial SequenceFelis catus Major allergen I polypeptide
chain 1 precursor epitope 100Glu Ile Cys Pro Ala Val Lys Arg Asp
Val Asp Leu Phe Leu Thr Gly 1 5 10 15 Thr 10116PRTArtificial
SequenceFelis catus Major allergen I polypeptide chain 1 precursor
epitope 101Glu Gln Val Ala Gln Tyr Lys Ala Leu Pro Val Val Leu Glu
Asn Ala 1 5 10 15 10217PRTArtificial SequenceFelis catus Major
allergen I polypeptide chain 1 precursor epitope 102Lys Ala Leu Pro
Val Val Leu Glu Asn Ala Arg Ile Leu Lys Asn Cys 1 5 10 15 Val
10317PRTArtificial SequenceFelis catus Major allergen I polypeptide
chain 1 precursor epitope 103Leu Phe Leu Thr Gly Thr Pro Asp Glu
Tyr Val Glu Gln Val Ala Gln 1 5 10 15 Tyr 10416PRTArtificial
SequenceFelis catus major allergen I, polypeptide chain 1 epitope
104Lys Glu Asn Ala Leu Ser Leu Leu Asp Lys Ile Tyr Thr Ser Pro Leu
1 5 10 15 10516PRTArtificial SequenceFelis catus major allergen I,
polypeptide chain 1 epitope 105Lys Met Thr Glu Glu Asp Lys Glu Asn
Ala Leu Ser Leu Leu Asp Lys 1 5 10 15 10615PRTArtificial
SequenceMalus x domestica Major allergen Mal d 1 epitope 106Gly Leu
Phe Lys Leu Ile Glu Ser Tyr Leu Lys Asp His Pro Asp 1 5 10 15
10715PRTArtificial SequencePrunus avium Major allergen Pru av 1
epitope 107Asn Leu Phe Lys Leu Ile Glu Thr Tyr Leu Lys Gly His Pro
Asp 1 5 10 15 10820PRTArtificial SequenceHevea brasiliensis Major
latex allergen Hev b 5 epitope 108Ala Ala Pro Ala Glu Gly Glu Lys
Pro Ala Glu Glu Glu Lys Pro Ile 1 5 10 15 Thr Glu Ala Ala 20
10920PRTArtificial SequenceHevea brasiliensis Major latex allergen
Hev b 5 epitope 109Ala Glu Glu Glu Lys Pro Ile Thr Glu Ala Ala Glu
Thr Ala Thr Thr 1 5 10 15 Glu Val Pro Val 20 11020PRTArtificial
SequenceHevea brasiliensis Major latex allergen Hev b 5 epitope
110Ala Pro Ala Glu Pro Glu Ala Pro Ala Pro Glu Thr Glu Lys Ala Glu
1 5 10 15 Glu Val Glu Lys 20 11120PRTArtificial SequenceHevea
brasiliensis Major latex allergen Hev b 5 epitope 111Ala Pro Glu
Ala Asp Gln Thr Thr Pro Glu Glu Lys Pro Ala Glu Pro 1 5 10 15 Glu
Pro Val Ala 20 11220PRTArtificial SequenceHevea brasiliensis Major
latex allergen Hev b 5 epitope 112Ala Ser Glu Gln Glu Thr Ala Asp
Ala Thr Pro Glu Lys Glu Glu Pro 1 5 10 15 Thr Ala Ala Pro 20
11311PRTArtificial SequenceDermatophagoides pteronyssinus Major
mite fecal allergen Der p 1 epitope 113Tyr Ala Tyr Val Ala Arg Glu
Gln Ser Cys Arg 1 5 10 11419PRTArtificial SequenceDermatophagoides
pteronyssinus Major mite fecal allergen Der p 1 epitope 114Ala Leu
Ala Gln Thr His Thr Ala Ile Ala Val Ile Ile Gly Ile Lys 1 5 10 15
Asp Leu Asp 11535PRTArtificial SequenceOlea europaea Major pollen
allergen epitope 115Glu Asp Ile Pro Gln Pro Pro Val Ser Gln Phe His
Ile Gln Gly Gln 1 5 10 15 Val Tyr Cys Asp Thr Cys Arg Ala Gly Phe
Ile Thr Glu Leu Ser Glu 20 25 30 Phe Ile Pro 35 11631PRTArtificial
SequenceOlea europaea Major pollen allergen epitope 116Gly Ala Ser
Leu Arg Leu Gln Cys Lys Asp Lys Glu Asn Gly Asp Val 1 5 10 15 Thr
Phe Thr Glu Val Gly Tyr Thr Arg Ala Glu Gly Leu Tyr Ser 20 25 30
11734PRTArtificial SequenceOlea europaea Major pollen allergen
epitope 117Gly Thr Thr Arg Thr Val Asn Pro Leu Gly Phe Phe Lys Lys
Glu Ala 1 5 10 15 Leu Pro Lys Cys Ala Gln Val Tyr Asn Lys Leu Gly
Met Tyr Pro Pro 20 25 30 Asn Met 11853PRTArtificial SequenceOlea
europaea Major pollen allergen epitope 118Leu Val Glu Arg Asp His
Lys Asn Glu Phe Cys Glu Ile Thr Leu Ile 1 5 10 15 Ser Ser Gly Arg
Lys Asp Cys Asn Glu Ile Pro Thr Glu Gly Trp Ala 20 25 30 Lys Pro
Ser Leu Lys Phe Lys Leu Asn Thr Val Asn Gly Thr Thr Arg 35 40 45
Thr Val Asn Pro Leu 50 11933PRTArtificial SequenceOlea europaea
Major pollen allergen epitope 119Met Leu Val Glu Arg Asp His Lys
Asn Glu Phe Cys Glu Ile Thr Leu 1 5 10 15 Ile Ser Ser Gly Arg Lys
Asp Cys Asn Glu Ile Pro Thr Glu Gly Trp 20 25 30 Ala
12012PRTArtificial SequenceArtemisia vulgaris Major pollen allergen
Art v 1 precursor epitope 120Ala Gly Gly Ser Pro Ser Pro Pro Ala
Asp Gly Gly 1 5 10 12112PRTArtificial SequenceArtemisia vulgaris
Major pollen allergen Art v 1 precursor epitope 121Ala Gly Ser Lys
Leu Cys Glu Lys Thr Ser Lys Thr 1 5 10 12212PRTArtificial
SequenceArtemisia vulgaris Major pollen allergen Art v 1 precursor
epitope 122Cys Asp Lys Lys Cys Ile Glu Trp Glu Lys Ala Gln 1 5 10
12312PRTArtificial SequenceArtemisia vulgaris Major pollen allergen
Art v 1 precursor epitope 123Asp Gly Gly Ser Pro Pro Pro Pro Ala
Asp Gly Gly 1 5 10 12412PRTArtificial SequenceArtemisia vulgaris
Major pollen allergen Art v 1 precursor epitope 124Glu Lys Thr Ser
Lys Thr Tyr Ser Gly Lys Cys Asp 1 5 10 12512PRTArtificial
SequenceBetula pendula Major pollen allergen Bet v 1-A epitope
125Ala Ala Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly 1 5 10
12615PRTArtificial SequenceBetula pendula Major pollen allergen Bet
v 1-A epitope 126Ala Ala Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly
Asp Asn Leu 1 5 10 15 12712PRTArtificial SequenceBetula pendula
Major pollen allergen Bet v 1-A epitope 127Ala Glu Gln Val Lys Ala
Ser Lys Glu Met Gly Glu 1 5 10 12821PRTArtificial SequenceBetula
pendula Major pollen allergen Bet v 1-A epitope 128Ala Phe Ile Leu
Asp Gly Asp Asn Leu Phe Pro Lys Val Ala Pro Gln 1 5 10 15 Ala Ile
Ser Ser Val 20 12912PRTArtificial SequenceBetula pendula Major
pollen allergen Bet v 1-A epitope 129Ala Ile Ser Ser Val Glu Asn
Ile Glu Gly Asn Gly 1 5 10 13015PRTArtificial SequenceBetula
pendula Major pollen allergen Bet v 1-A epitope 130Glu Thr Leu Leu
Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser 1 5 10 15
13116PRTArtificial SequenceBetula pendula Major pollen allergen Bet
v 1-F/I epitope 131Gly Glu Thr Leu Leu Arg Ala Val Glu Ser Tyr Leu
Leu Ala His Ser 1 5 10 15 13220PRTArtificial SequenceChamaecyparis
obtusa Major pollen allergen Cha o 1 precursor epitope 132Ala Asn
Asn Asn Tyr Asp Pro Trp Ser Ile Tyr Ala Ile Gly Gly Ser 1 5 10 15
Ser Asn Pro Thr 20 13320PRTArtificial SequenceChamaecyparis obtusa
Major pollen allergen Cha o 1 precursor epitope 133Ala Ser Thr Gly
Val Thr Ile Ser Asn Asn His Phe Phe Asn His His 1 5 10 15 Lys Val
Met Leu 20 13420PRTArtificial SequenceChamaecyparis obtusa Major
pollen allergen Cha o 1 precursor epitope 134Cys Ala Asn Trp Val
Trp Arg Ser Thr Gln Asp Ser Phe Asn Asn Gly 1 5 10 15 Ala Tyr Phe
Val 20 13520PRTArtificial SequenceChamaecyparis obtusa Major pollen
allergen Cha o 1 precursor epitope 135Asp Ala Ile Thr Met Arg Asn
Val Thr Asp Val Trp Ile Asp His Asn 1 5 10 15 Ser Leu Ser Asp 20
13620PRTArtificial SequenceChamaecyparis obtusa Major pollen
allergen Cha o 1 precursor epitope 136Asp Ala Asn Trp Asp Gln Asn
Arg Met Lys Leu Ala Asp Cys Ala Val 1 5 10 15 Gly Phe Gly Ser 20
13720PRTArtificial SequenceCynodon dactylon Major pollen allergen
Cyn d 1 epitope 137Ala Ile Gly Asp Lys Pro Gly Pro Asn Ile Thr Ala
Thr Tyr Gly Asn 1 5 10 15 Lys Trp Leu Glu 20 13820PRTArtificial
SequenceCynodon dactylon Major pollen allergen Cyn d 1 epitope
138Cys Tyr Glu Ile Lys Cys Lys Glu Pro Val Glu Cys Ser Gly Glu Pro
1 5 10 15 Val Leu Val Lys 20 13920PRTArtificial SequenceCynodon
dactylon Major pollen allergen Cyn d 1 epitope 139Asp His Gly Gly
Ala Cys Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe 1 5 10 15 Asp Gly
Met Thr 20 14020PRTArtificial SequenceCynodon dactylon Major pollen
allergen Cyn d 1 epitope 140Glu Gly Gly Ala His Leu Val Gln Asp Asp
Val Ile Pro Ala Asn Trp 1 5 10 15 Lys Pro Asp Thr 20
14120PRTArtificial SequenceCynodon dactylon Major pollen allergen
Cyn d 1 epitope 141Phe Lys Asp Gly Leu Gly Cys Gly Ala Cys Tyr Glu
Ile Lys Cys Lys 1 5 10 15 Glu Pro Val Glu 20 14215PRTArtificial
SequencePhleum pratense Major pollen allergen Phl p 4 precursor
epitope 142Phe Ala Glu Tyr Lys Ser Asp Tyr Val Tyr Gln Pro Phe Pro
Lys 1 5 10 15 14315PRTArtificial SequencePhleum pratense Major
pollen allergen Phl p 4 precursor epitope 143Met Leu Leu Arg Lys
Tyr Gly Ile Ala Ala Glu Asn Val Ile Asp 1 5 10 15
14415PRTArtificial SequencePhleum pratense Major pollen allergen
Phl p 4 precursor epitope 144Asn Ser Phe Lys Pro Phe Ala Glu Tyr
Lys Ser Asp Tyr Val Tyr 1 5 10 15 14520PRTArtificial SequenceRattus
norvegicus Major urinary protein precursor epitope 145Ala Ser Asn
Lys Arg Glu Lys Ile Glu Glu Asn Gly Ser Met Arg Val 1 5 10 15 Phe
Met Gln His 20 14620PRTArtificial SequenceRattus norvegicus Major
urinary protein precursor epitope 146Asp Ile Lys Glu Lys Phe Ala
Lys Leu Cys Glu Ala His Gly Ile Thr 1 5 10 15 Arg Asp Asn Ile 20
14720PRTArtificial SequenceRattus norvegicus Major urinary protein
precursor epitope 147Glu Glu Ala Ser Ser Thr Arg Gly Asn Leu Asp
Val Ala Lys Leu Asn 1 5 10 15 Gly Asp Trp Phe 20 14820PRTArtificial
SequenceRattus norvegicus Major urinary protein precursor epitope
148Glu Glu Asn Gly Ser Met Arg Val Phe Met Gln His Ile Asp Val Leu
1 5 10 15 Glu Asn Ser Leu 20 14920PRTArtificial SequenceRattus
norvegicus Major urinary protein precursor epitope 149Glu Asn Ser
Leu Gly Phe Lys Phe Arg Ile Lys Glu Asn Gly Glu Cys 1 5 10 15 Arg
Glu Leu Tyr 20 15021PRTArtificial SequenceDermatophagoides farinae
Mite group 2 allergen Der f 2 precursor epitope 150Asp Ile Lys Tyr
Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu 1 5 10 15 Asn Val
Val Val Thr 20 15117PRTArtificial SequenceDermatophagoides farinae
Mite group 2 allergen Der f 2 precursor epitope 151Asp Asn Gly Val
Leu Ala Cys Ala Ile Ala Thr His Gly Lys Ile Arg 1 5 10 15 Asp
15221PRTArtificial SequenceDermatophagoides farinae Mite group 2
allergen Der f 2 precursor epitope 152Glu Ala Leu Phe Asp Ala Asn
Gln Asn Thr Lys Thr Ala Lys Ile Glu 1 5 10 15 Ile Lys Ala Ser Leu
20 15345PRTArtificial SequenceDermatophagoides farinae Mite group 2
allergen Der f 2 precursor epitope 153Gln Tyr Asp Ile Lys Tyr Thr
Trp Asn Val Pro Lys Ile Ala Pro Lys 1 5 10 15 Ser Glu Asn Val Val
Val Thr Val Lys Leu Ile Gly Asp Asn Gly Val 20 25 30 Leu Ala Cys
Ala Ile Ala Thr His Gly Lys Ile Arg Asp 35 40 45 15419PRTArtificial
SequenceDermatophagoides farinae Mite group 2 allergen Der f 2
precursor epitope 154Thr Lys Thr Ala Lys Ile Glu Ile Lys Ala Ser
Leu Asp Gly Leu Glu 1 5 10 15 Ile Asp Val 15514PRTArtificial
SequenceDermatophagoides pteronyssinus Mite group 2 allergen Der p
2 epitope 155Ala Ser Ile Asp Gly Leu Gly Val Asp Val Pro Gly Ile
Asp 1 5 10 15615PRTArtificial SequenceDermatophagoides
pteronyssinus Mite group 2 allergen Der p 2 epitope 156Phe Glu Ala
Val Gln Asn Thr Lys Thr Ala Lys Ile Glu Ile Lys 1 5 10 15
15717PRTArtificial SequenceDermatophagoides pteronyssinus Mite
group 2 allergen Der p 2 epitope 157Arg Gly Lys Pro Pro Gln Leu Glu
Ala Val Phe Glu Ala Val Gln Asn 1 5 10 15 Thr 15815PRTArtificial
SequenceDermatophagoides pteronyssinus Mite group 2 allergen Der p
2 precursor epitope 158Cys His Gly Ser Glu Pro Cys Ile Ile His Arg
Gly Lys Pro Phe 1 5 10 15 15927PRTArtificial
SequenceDermatophagoides pteronyssinus Mite group 2 allergen Der p
2 precursor epitope 159Cys Pro Leu Val Lys Gly Gln Gln Tyr Asp Ile
Lys Tyr Thr Trp Asn 1 5 10 15 Val Pro Lys Ile Ala Pro Lys Ser Glu
Asn Val 20 25 16026PRTArtificial SequenceDermatophagoides
pteronyssinus Mite group 2 allergen Der p 2 precursor epitope
160Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys Ile Ala Pro Lys Ser Glu
1 5 10 15 Asn Val Val Val Thr Val Lys Val Met Gly 20 25
16115PRTArtificial SequenceDermatophagoides pteronyssinus Mite
group 2 allergen Der p 2 precursor epitope 161Asp Gln Val Asp Val
Lys Asp Cys Ala Asn His Glu Ile Lys Lys 1 5 10 15
16220PRTArtificial SequenceDermatophagoides pteronyssinus Mite
group 2 allergen Der p 2 precursor epitope 162Asp Gln Val Asp Val
Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val 1 5 10 15 Leu Val Pro
Gly 20 16315PRTArtificial SequenceLepidoglyphus destructor Mite
group 2 allergen Lep d 2 precursor epitope 163Asp His Gly Val Met
Ala Cys Gly Thr Val His Gly Gln Val Glu 1 5 10 15
16415PRTArtificial SequenceLepidoglyphus destructor Mite group 2
allergen Lep d 2 precursor epitope 164Gly Cys Lys Phe Ile Lys Cys
Pro Val Lys Lys Gly Glu Ala Leu 1 5 10 15 16515PRTArtificial
SequenceLepidoglyphus destructor Mite group 2 allergen Lep d 2
precursor epitope 165Gly Glu Lys Met Thr Leu Glu
Ala Lys Phe Ala Ala Asn Gln Asp 1 5 10 15 16615PRTArtificial
SequenceLepidoglyphus destructor Mite group 2 allergen Lep d 2
precursor epitope 166Gly Glu Val Thr Glu Leu Asp Ile Thr Gly Cys
Ser Gly Asp Thr 1 5 10 15 16715PRTArtificial SequenceLepidoglyphus
destructor Mite group 2 allergen Lep d 2 precursor epitope 167Gly
Lys Met Thr Phe Lys Asp Cys Gly His Gly Glu Val Thr Glu 1 5 10 15
16816PRTArtificial SequenceHomo sapiens Neurofilament heavy
polypeptide (NF-H) (Neurofilament triplet H protein) (200 kDa
neurofilament protein) epitope 168Tyr Gln Glu Ala Ile Gln Gln Leu
Asp Ala Glu Leu Arg Asn Thr Lys 1 5 10 15 16910PRTArtificial
SequencePrunus persica Non-specific lipid-transfer protein 1
epitope 169Ala Ala Ala Leu Pro Gly Lys Cys Gly Val 1 5 10
17010PRTArtificial SequencePrunus persica Non-specific
lipid-transfer protein 1 epitope 170Ala Cys Cys Asn Gly Ile Arg Asn
Val Asn 1 5 10 17110PRTArtificial SequencePrunus persica
Non-specific lipid-transfer protein 1 epitope 171Ala Pro Cys Ile
Pro Tyr Val Arg Gly Gly 1 5 10 17210PRTArtificial SequencePrunus
persica Non-specific lipid-transfer protein 1 epitope 172Ile Arg
Asn Val Asn Asn Leu Ala Arg Thr 1 5 10 17311PRTArtificial
SequencePrunus persica Non-specific lipid-transfer protein 1
epitope 173Ile Ser Ala Ser Thr Asn Cys Ala Thr Val Lys 1 5 10
17410PRTArtificial SequencePrunus persica Non-specific
lipid-transfer protein 1 epitope 174Asn Leu Ala Arg Thr Thr Pro Asp
Arg Gln 1 5 10 17510PRTArtificial SequenceGallus gallus Ovalbumin
epitope 175Cys Phe Asp Val Phe Lys Glu Leu Lys Val 1 5 10
17610PRTArtificial SequenceGallus gallus Ovalbumin epitope 176Gly
Ser Ile Gly Ala Ala Ser Met Glu Phe 1 5 10 17718PRTArtificial
SequenceGallus gallus Ovalbumin epitope 177Ile Gly Leu Phe Arg Val
Ala Ser Met Ala Ser Glu Lys Met Lys Ile 1 5 10 15 Leu Glu
17818PRTArtificial SequenceGallus gallus Ovalbumin epitope 178Ile
Lys His Ile Ala Thr Asn Ala Val Leu Phe Phe Gly Arg Cys Val 1 5 10
15 Ser Pro 17913PRTArtificial SequenceGallus gallus Ovalbumin
epitope 179Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys 1 5
10 18014PRTArtificial SequenceGallus gallus Ovomucoid precursor
epitope 180Ala Glu Val Asp Cys Ser Arg Phe Pro Asn Ala Thr Asp Lys
1 5 10 18114PRTArtificial SequenceGallus gallus Ovomucoid precursor
epitope 181Ala Thr Asp Lys Glu Gly Lys Asp Val Leu Val Cys Asn Lys
1 5 10 18217PRTArtificial SequenceGallus gallus Ovomucoid precursor
epitope 182Ala Val Val Glu Ser Asn Gly Thr Leu Thr Leu Ser His Phe
Gly Lys 1 5 10 15 Cys 18316PRTArtificial SequenceGallus gallus
Ovomucoid precursor epitope 183Cys Leu Leu Cys Ala Tyr Ser Ile Glu
Phe Gly Thr Asn Ile Ser Lys 1 5 10 15 18420PRTArtificial
SequenceGallus gallus Ovomucoid precursor epitope 184Asp Asn Glu
Cys Leu Leu Cys Ala His Lys Val Glu Gln Gly Ala Ser 1 5 10 15 Val
Asp Lys Arg 20 18516PRTArtificial SequenceMusa acuminata pectate
lyase epitope 185Gly His Ser Asp Glu Leu Thr Ser Asp Lys Ser Met
Gln Val Thr Ile 1 5 10 15 18616PRTArtificial SequenceZinnia
violacea Pectate lyase precursor epitope 186Gly His Ser Asp Ser Tyr
Thr Gln Asp Lys Asn Met Gln Val Thr Ile 1 5 10 15
18721PRTArtificial SequenceDermatophagoides farinae Peptidase 1
precursor (Major mite fecal allergen Der f 1) (Allergen Der f I)
epitope 187Asp Gly Arg Thr Ile Ile Gln His Asp Asn Gly Tyr Gln Pro
Asn Tyr 1 5 10 15 His Ala Val Asn Ile 20 18819PRTArtificial
SequenceDermatophagoides farinae Peptidase 1 precursor (Major mite
fecal allergen Der f 1) (Allergen Der f I) epitope 188Asp Leu Arg
Ser Leu Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly 1 5 10 15 Cys
Gly Ser 18919PRTArtificial SequenceDermatophagoides farinae
Peptidase 1 precursor (Major mite fecal allergen Der f 1) (Allergen
Der f I) epitope 189Gly Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala
Ala Thr Glu Ser 1 5 10 15 Ala Tyr Leu 19021PRTArtificial
SequenceDermatophagoides farinae Peptidase 1 precursor (Major mite
fecal allergen Der f 1) (Allergen Der f I) epitope 190Ile Arg Glu
Ala Leu Thr Gln Thr His Thr Ala Ile Ala Val Ile Ile 1 5 10 15 Gly
Ile Lys Asp Leu 20 19119PRTArtificial SequenceDermatophagoides
farinae Peptidase 1 precursor (Major mite fecal allergen Der f 1)
(Allergen Der f I) epitope 191Ile Arg Met Gln Gly Gly Cys Gly Ser
Cys Trp Ala Phe Ser Gly Val 1 5 10 15 Ala Ala Thr
19219PRTArtificial SequenceEuroglyphus maynei Peptidase 1 precursor
(Mite group 1 allergen Eur m 1) (Allergen Eur m I) epitope 192Phe
Arg His Tyr Asp Gly Arg Thr Ile Met Gln His Asp Asn Gly Tyr 1 5 10
15 Gln Pro Asn 19319PRTArtificial SequenceEuroglyphus maynei
Peptidase 1 precursor (Mite group 1 allergen Eur m 1) (Allergen Eur
m I) epitope 193Gly Arg Thr Ile Met Gln His Asp Asn Gly Tyr Gln Pro
Asn Tyr His 1 5 10 15 Ala Val Asn 19419PRTArtificial
SequenceEuroglyphus maynei Peptidase 1 precursor (Mite group 1
allergen Eur m 1) (Allergen Eur m I) epitope 194His Ala Val Asn Ile
Val Gly Tyr Gly Asn Thr Gln Gly Val Asp Tyr 1 5 10 15 Trp Ile Val
19519PRTArtificial SequenceEuroglyphus maynei Peptidase 1 precursor
(Mite group 1 allergen Eur m 1) (Allergen Eur m I) epitope 195Asn
Lys Ile Arg Gln Ala Leu Thr Gln Thr His Thr Ala Val Ala Val 1 5 10
15 Ile Ile Gly 19619PRTArtificial SequenceEuroglyphus maynei
Peptidase 1 precursor (Mite group 1 allergen Eur m 1) (Allergen Eur
m I) epitope 196Pro Tyr Val Ala Arg Glu Gln Ser Cys His Arg Pro Asn
Ala Gln Arg 1 5 10 15 Tyr Gly Leu 19715PRTArtificial SequencePhleum
pratense Phl p 3 allergen epitope 197Ala Val Gln Val Thr Phe Thr
Val Gln Lys Gly Ser Asp Pro Lys 1 5 10 15 19815PRTArtificial
SequencePhleum pratense Phl p 3 allergen epitope 198Glu Glu Trp Glu
Pro Leu Thr Lys Lys Gly Asn Val Trp Glu Val 1 5 10 15
19915PRTArtificial SequencePhleum pratense Phl p 3 allergen epitope
199Phe Thr Val Gln Lys Gly Ser Asp Pro Lys Lys Leu Val Leu Asp 1 5
10 15 20015PRTArtificial SequencePhleum pratense Phl p 3 allergen
epitope 200Phe Thr Val Gln Lys Gly Ser Asp Pro Lys Lys Leu Val Leu
Asn 1 5 10 15 20115PRTArtificial SequencePhleum pratense Phl p 3
allergen epitope 201Gly Ser Asp Pro Lys Lys Leu Val Leu Asp Ile Lys
Tyr Thr Arg 1 5 10 15 20215PRTArtificial SequenceApis mellifera
Phospholipase A2 precursor epitope 202Cys Asp Cys Asp Asp Lys Phe
Tyr Asp Cys Leu Lys Asn Ser Ala 1 5 10 15 20312PRTArtificial
SequenceApis mellifera Phospholipase A2 precursor epitope 203Cys
Leu His Tyr Thr Val Asp Lys Ser Lys Pro Lys 1 5 10
20415PRTArtificial SequenceApis mellifera Phospholipase A2
precursor epitope 204Cys Arg Thr His Asp Met Cys Pro Asp Val Met
Ser Ala Gly Glu 1 5 10 15 20518PRTArtificial SequenceApis mellifera
Phospholipase A2 precursor epitope 205Asp Thr Ile Ser Ser Tyr Phe
Val Gly Lys Met Tyr Phe Asn Leu Ile 1 5 10 15 Asp Thr
20618PRTArtificial SequenceApis mellifera Phospholipase A2
precursor epitope 206Glu Arg Thr Glu Gly Arg Cys Leu His Tyr Thr
Val Asp Lys Ser Lys 1 5 10 15 Pro Lys 20716PRTArtificial
SequenceSpiroplasma citri plectrovirus spv1-r8a2b orf 14
transmembrane protein epitope 207His Val Ile Glu Val Gln Gln Ile
Asn Ser Glu Arg Ser Trp Phe Phe 1 5 10 15 20820PRTArtificial
SequenceLolium perenne pollen allergen epitope 208Cys Gly Tyr Lys
Asp Val Asp Lys Ala Pro Phe Asn Gly Met Thr Gly 1 5 10 15 Cys Gly
Asn Thr 20 20920PRTArtificial SequenceLolium perenne pollen
allergen epitope 209Gly Ala Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly
Tyr Lys Asp Val 1 5 10 15 Asp Lys Ala Pro 20 21020PRTArtificial
SequenceLolium perenne pollen allergen epitope 210Ser Glu Val Glu
Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ser 1 5 10 15 Tyr Ser
Ala Lys 20 21120PRTArtificial SequenceLolium perenne pollen
allergen epitope 211Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Ile
Leu Val Lys Tyr 1 5 10 15 Val Asp Gly Asp 20 21220PRTArtificial
SequenceLolium perenne pollen allergen epitope 212Tyr Pro Asp Asp
Thr Lys Pro Thr Phe His Val Glu Lys Gly Ser Asn 1 5 10 15 Pro Asn
Tyr Leu 20 21316PRTArtificial SequenceAmbrosia artemisiifolia
Pollen allergen Amb a 1.1 precursor epitope 213Gly Ala Gly Asp Glu
Asn Ile Glu Asp Arg Gly Met Leu Ala Thr Val 1 5 10 15
21416PRTArtificial SequenceAmbrosia artemisiifolia Pollen allergen
Amb a 2 precursor epitope 214Gly Ala Ser Asp Thr His Phe Gln Asp
Leu Lys Met His Val Thr Leu 1 5 10 15 21511PRTArtificial
SequenceAmbrosia artemisiifolia var. elatior Pollen allergen Amb a
3 epitope 215Glu Glu Ala Tyr His Ala Cys Asp Ile Lys Asp 1 5 10
21615PRTArtificial SequenceAmbrosia artemisiifolia var. elatior
Pollen allergen Amb a 3 epitope 216Gly Lys Val Tyr Leu Val Gly Gly
Pro Glu Leu Gly Gly Trp Lys 1 5 10 15 21715PRTArtificial
SequenceAmbrosia artemisiifolia var. elatior Pollen allergen Amb a
3 epitope 217Leu Gly Gly Trp Lys Leu Gln Ser Asp Pro Arg Ala Tyr
Ala Leu 1 5 10 15 21815PRTArtificial SequenceAmbrosia
artemisiifolia var. elatior Pollen allergen Amb a 3 epitope 218Pro
Gly Gly Pro Asp Arg Phe Thr Leu Leu Thr Pro Gly Ser His 1 5 10 15
21915PRTArtificial SequenceAmbrosia artemisiifolia var. elatior
Pollen allergen Amb a 5 epitope 219Ala Tyr Cys Cys Ser Asp Pro Gly
Arg Tyr Cys Pro Trp Gln Val 1 5 10 15 22020PRTArtificial
SequenceAmbrosia artemisiifolia var. elatior Pollen allergen Amb a
5 epitope 220Cys Gly Glu Lys Arg Ala Tyr Cys Cys Ser Asp Pro Gly
Arg Tyr Cys 1 5 10 15 Pro Trp Gln Val 20 22117PRTArtificial
SequenceAmbrosia artemisiifolia var. elatior Pollen allergen Amb a
5 epitope 221Asp Pro Gly Arg Tyr Cys Pro Trp Gln Val Val Cys Tyr
Glu Ser Ser 1 5 10 15 Glu 22220PRTArtificial SequenceAmbrosia
artemisiifolia var. elatior Pollen allergen Amb a 5 epitope 222Asp
Pro Gly Arg Tyr Cys Pro Trp Gln Val Val Cys Tyr Glu Ser Ser 1 5 10
15 Glu Ile Cys Ser 20 22315PRTArtificial SequenceAmbrosia
artemisiifolia var. elatior Pollen allergen Amb a 5 epitope 223Gly
Asn Val Cys Gly Glu Lys Arg Ala Tyr Cys Cys Ser Asp Pro 1 5 10 15
22415PRTArtificial SequenceAmbrosia artemisiifolia var. elatior
Pollen allergen Amb a 5 epitope 224Leu Val Pro Cys Ala Trp Ala Gly
Asn Val Cys Gly Glu Lys Arg 1 5 10 15 22520PRTArtificial
SequenceAmbrosia artemisiifolia var. elatior Pollen allergen Amb a
5 epitope 225Leu Val Pro Cys Ala Trp Ala Gly Asn Val Cys Gly Glu
Lys Arg Ala 1 5 10 15 Tyr Cys Cys Ser 20 22615PRTArtificial
SequenceAmbrosia artemisiifolia var. elatior Pollen allergen Amb a
5 epitope 226Val Cys Tyr Glu Ser Ser Glu Ile Cys Ser Lys Lys Cys
Gly Lys 1 5 10 15 22720PRTArtificial SequenceAmbrosia trifida
Pollen allergen Amb t 5 precursor epitope 227Cys Gly Lys Val Gly
Lys Tyr Cys Cys Ser Pro Ile Gly Lys Tyr Cys 1 5 10 15 Val Cys Tyr
Asp 20 22820PRTArtificial SequenceAmbrosia trifida Pollen allergen
Amb t 5 precursor epitope 228Asp Asp Gly Leu Cys Tyr Glu Gly Thr
Asn Cys Gly Lys Val Gly Lys 1 5 10 15 Tyr Cys Cys Ser 20
22912PRTArtificial SequenceAmbrosia trifida Pollen allergen Amb t 5
precursor epitope 229Gly Lys Tyr Cys Val Cys Tyr Asp Ser Lys Ala
Ile 1 5 10 23014PRTArtificial SequenceAmbrosia trifida Pollen
allergen Amb t 5 precursor epitope 230Pro Ile Gly Lys Tyr Cys Val
Cys Tyr Asp Ser Lys Ala Ile 1 5 10 23120PRTArtificial
SequenceAmbrosia trifida Pollen allergen Amb t 5 precursor epitope
231Pro Ile Gly Lys Tyr Cys Val Cys Tyr Asp Ser Lys Ala Ile Cys Asn
1 5 10 15 Lys Asn Cys Thr 20 23214PRTArtificial SequenceAmbrosia
trifida Pollen allergen Amb t 5 precursor epitope 232Val Cys Tyr
Asp Ser Lys Ala Ile Cys Asn Lys Asn Cys Thr 1 5 10
23315PRTArtificial SequenceBetula pendula pollen allergen Bet v 1
epitope 233His Glu Val Lys Ala Glu Gln Val Lys Ala Thr Lys Glu Met
Gly 1 5 10 15 23420PRTArtificial SequencePoa pratensis Pollen
allergen KBG 60 precursor epitope 234Ala Ala Asn Lys Tyr Lys Thr
Phe Val Ala Thr Phe Gly Ala Ala Ser 1 5 10 15 Asn Lys Ala Phe 20
23520PRTArtificial SequencePoa pratensis Pollen allergen KBG 60
precursor epitope 235Ala Ala Pro Ala Asn Asp Lys Phe Thr Val Phe
Glu Ala Ala Phe Asn 1 5 10 15 Asp Ala Ile Lys 20 23620PRTArtificial
SequencePoa pratensis Pollen allergen KBG 60 precursor epitope
236Ala Ala Val Asp Ser Ser Lys Ala Ala Leu Thr Ser Lys Leu Asp Ala
1 5 10 15 Ala Tyr Lys Leu 20 23720PRTArtificial SequencePoa
pratensis Pollen allergen KBG 60 precursor epitope 237Ala Glu Glu
Val Lys Ala Thr Pro Ala Gly Glu Leu Gln Val Ile Asp 1 5 10 15 Lys
Val Asp Ala 20 23820PRTArtificial SequencePoa pratensis Pollen
allergen KBG 60 precursor epitope 238Ala Phe Lys Val Ala Ala Thr
Ala Ala Asn Ala Ala Pro Ala Asn Asp 1 5 10 15 Lys Phe Thr Val 20
23920PRTArtificial SequenceLolium perenne Pollen allergen Lol p 1
precursor epitope 239Ala Phe Gly Ser Met Ala Lys Lys Gly Glu Glu
Gln Asn Val Arg Ser 1 5 10 15 Ala Gly Glu Leu 20 24020PRTArtificial
SequenceLolium perenne Pollen allergen Lol p 1 precursor epitope
240Ala Gly Glu Leu Glu Leu Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro
1 5 10 15 Asp Asp Thr Lys 20 24120PRTArtificial SequenceLolium
perenne Pollen allergen Lol p 1 precursor epitope 241Ala Lys Ser
Thr Trp Tyr Gly Lys Pro Thr Gly Ala Gly Pro Lys Asp 1 5 10 15 Asn
Gly Gly Ala 20 24220PRTArtificial SequenceLolium perenne Pollen
allergen Lol p 1 precursor epitope 242Ala Pro Tyr His Phe Asp Leu
Ser Gly His Ala Phe Gly Ser Met Ala 1 5 10 15 Lys Lys Gly Glu 20
24312PRTArtificial SequenceLolium perenne Pollen allergen Lol p 1
precursor epitope 243Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His
Ala 1 5 10 24420PRTArtificial SequenceLolium perenne Pollen
allergen Lol p VA precursor epitope 244Ala Ala Leu Thr Lys Ala Ile
Thr Ala Met Thr Gln
Ala Gln Lys Ala 1 5 10 15 Gly Lys Pro Ala 20 24520PRTArtificial
SequenceLolium perenne Pollen allergen Lol p VA precursor epitope
245Ala Ala Asn Ala Ala Pro Thr Asn Asp Lys Phe Thr Val Phe Glu Ser
1 5 10 15 Ala Phe Asn Lys 20 24620PRTArtificial SequenceLolium
perenne Pollen allergen Lol p VA precursor epitope 246Ala Asp Lys
Phe Lys Ile Phe Glu Ala Ala Phe Ser Glu Ser Ser Lys 1 5 10 15 Gly
Leu Leu Ala 20 24720PRTArtificial SequenceLolium perenne Pollen
allergen Lol p VA precursor epitope 247Ala Phe Ser Glu Ser Ser Lys
Gly Leu Leu Ala Thr Ser Ala Ala Lys 1 5 10 15 Ala Pro Gly Leu 20
24820PRTArtificial SequenceLolium perenne Pollen allergen Lol p VA
precursor epitope 248Ala Tyr Ala Ala Thr Val Ala Ala Ala Pro Glu
Val Lys Tyr Ala Val 1 5 10 15 Phe Glu Ala Ala 20 24912PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 epitope 249Ala Cys
Ser Gly Glu Pro Val Val Val His Ile Thr 1 5 10 25012PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 epitope 250Ala Glu
Asp Val Ile Pro Glu Gly Trp Lys Ala Asp 1 5 10 25112PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 epitope 251Ala Gly
Glu Leu Glu Leu Gln Phe Arg Arg Val Lys 1 5 10 25212PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 epitope 252Asp Lys
Trp Ile Glu Leu Lys Glu Ser Trp Gly Ala 1 5 10 25312PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 epitope 253Asp Lys
Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly 1 5 10 25412PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 precursor epitope
254Phe Glu Ile Lys Cys Thr Lys Pro Glu Ala Cys Ser 1 5 10
25512PRTArtificial SequencePhleum pratense Pollen allergen Phl p 1
precursor epitope 255Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly
Ala 1 5 10 25615PRTArtificial SequencePhleum pratense Pollen
allergen Phl p 1 precursor epitope 256Glu Leu Lys Glu Ser Trp Gly
Ala Ile Trp Arg Ile Asp Thr Pro 1 5 10 15 25715PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 precursor epitope
257Glu Pro Ile Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe 1 5
10 15 25815PRTArtificial SequencePhleum pratense Pollen allergen
Phl p 1 precursor epitope 258Phe Glu Ile Lys Cys Thr Lys Pro Glu
Ala Cys Ser Gly Glu Pro 1 5 10 15 25915PRTArtificial SequencePhleum
pratense Pollen allergen Phl p 1 precursor epitope 259Trp Gly Ala
Ile Trp Arg Ile Asp Thr Pro Asp Lys Leu Thr Gly 1 5 10 15
26015PRTArtificial SequencePhleum pratense Pollen allergen Phl p 11
epitope 260Arg Tyr Ala Asn Pro Ile Ala Phe Phe Arg Lys Glu Pro Leu
Lys 1 5 10 15 26115PRTArtificial SequencePhleum pratense Pollen
allergen Phl p 2 epitope 261Glu His Gly Ser Asp Glu Trp Val Ala Met
Thr Lys Gly Glu Gly 1 5 10 15 26215PRTArtificial SequencePhleum
pratense Pollen allergen Phl p 2 epitope 262Glu Trp Val Ala Met Thr
Lys Gly Glu Gly Gly Val Trp Thr Phe 1 5 10 15 26315PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 2 epitope 263Gly Val
Trp Thr Phe Asp Ser Glu Glu Pro Leu Gln Gly Pro Phe 1 5 10 15
26415PRTArtificial SequencePhleum pratense Pollen allergen Phl p 2
epitope 264Lys Asn Val Phe Asp Asp Val Val Pro Glu Lys Tyr Thr Ile
Gly 1 5 10 15 26515PRTArtificial SequencePhleum pratense Pollen
allergen Phl p 2 epitope 265Leu Gln Gly Pro Phe Asn Phe Arg Phe Leu
Thr Glu Lys Gly Met 1 5 10 15 26615PRTArtificial SequencePhleum
pratense Pollen allergen Phl p 4 epitope 266Phe Lys Pro Phe Ala Glu
Tyr Lys Ser Asp Tyr Val Tyr Glu Pro 1 5 10 15 26715PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 4 epitope 267Phe Pro
Lys Glu Val Trp Glu Gln Ile Phe Ser Thr Trp Leu Leu 1 5 10 15
26815PRTArtificial SequencePhleum pratense Pollen allergen Phl p 4
epitope 268Phe Val His Leu Gly His Arg Asp Asn Ile Glu Asp Asp Leu
Leu 1 5 10 15 26915PRTArtificial SequencePhleum pratense Pollen
allergen Phl p 4 epitope 269Gly Ile Val Val Ala Trp Lys Val Arg Leu
Leu Pro Val Pro Pro 1 5 10 15 27015PRTArtificial SequencePhleum
pratense Pollen allergen Phl p 4 epitope 270Asn Arg Asn Asn Thr Phe
Lys Pro Phe Ala Glu Tyr Lys Ser Asp 1 5 10 15 27112PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 5a epitope 271Glu Val
Lys Tyr Thr Val Phe Glu Thr Ala Leu Lys 1 5 10 27219PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 5a epitope 272Asn Ala
Gly Phe Lys Ala Ala Leu Ala Gly Ala Gly Val Gln Pro Ala 1 5 10 15
Asp Lys Tyr 27327PRTArtificial SequencePhleum pratense Pollen
allergen Phl p 5b precursor epitope 273Ala Ala Gly Lys Ala Thr Thr
Glu Glu Gln Lys Leu Ile Glu Asp Ile 1 5 10 15 Asn Val Gly Phe Lys
Ala Ala Val Ala Ala Ala 20 25 27433PRTArtificial SequencePhleum
pratense Pollen allergen Phl p 5b precursor epitope 274Ala Ala Gly
Lys Ala Thr Thr Glu Glu Gln Lys Leu Ile Glu Asp Ile 1 5 10 15 Asn
Val Gly Phe Lys Ala Ala Val Ala Ala Ala Ala Ser Val Pro Ala 20 25
30 Ala 27519PRTArtificial SequencePhleum pratense Pollen allergen
Phl p 5b precursor epitope 275Ala Ala Val Ala Ala Ala Ala Ser Val
Pro Ala Ala Asp Lys Phe Lys 1 5 10 15 Thr Phe Glu
27627PRTArtificial SequencePhleum pratense Pollen allergen Phl p 5b
precursor epitope 276Ala Lys Phe Asp Ser Phe Val Ala Ser Leu Thr
Glu Ala Leu Arg Val 1 5 10 15 Ile Ala Gly Ala Leu Glu Val His Ala
Val Lys 20 25 27719PRTArtificial SequencePhleum pratense Pollen
allergen Phl p 5b precursor epitope 277Ala Met Ser Glu Val Gln Lys
Val Ser Gln Pro Ala Thr Gly Ala Ala 1 5 10 15 Thr Val Ala
27820PRTArtificial SequenceChamaecyparis obtusa Polygalacturonase
epitope 278Ala Arg Trp Lys Asn Ser Lys Ile Trp Leu Gln Phe Ala Gln
Leu Thr 1 5 10 15 Asp Phe Asn Leu 20 27920PRTArtificial
SequenceChamaecyparis obtusa Polygalacturonase epitope 279Ala Val
Leu Leu Val Pro Ala Asn Lys Lys Phe Phe Val Asn Asn Leu 1 5 10 15
Val Phe Arg Gly 20 28020PRTArtificial SequenceChamaecyparis obtusa
Polygalacturonase epitope 280Asp Gly Thr Ile Val Ala Gln Pro Asp
Pro Ala Arg Trp Lys Asn Ser 1 5 10 15 Lys Ile Trp Leu 20
28120PRTArtificial SequenceChamaecyparis obtusa Polygalacturonase
epitope 281Phe Phe Val Asn Asn Leu Val Phe Arg Gly Pro Cys Gln Pro
His Leu 1 5 10 15 Ser Phe Lys Val 20 28220PRTArtificial
SequenceChamaecyparis obtusa Polygalacturonase epitope 282Phe Gly
Glu Cys Glu Gly Val Lys Ile Gln Gly Leu Lys Ile Lys Ala 1 5 10 15
Pro Arg Asp Ser 20 28315PRTArtificial SequenceCryptomeria japonica
Polygalacturonase precursor epitope 283Ala Ala Tyr Gln Asn Pro Ala
Ser Trp Lys Asn Asn Arg Ile Trp 1 5 10 15 28415PRTArtificial
SequenceCryptomeria japonica Polygalacturonase precursor epitope
284Ala Cys Lys Lys Pro Ser Ala Met Leu Leu Val Pro Gly Asn Lys 1 5
10 15 28515PRTArtificial SequenceCryptomeria japonica
Polygalacturonase precursor epitope 285Ala Ile Lys Phe Asp Phe Ser
Thr Gly Leu Ile Ile Gln Gly Leu 1 5 10 15 28615PRTArtificial
SequenceCryptomeria japonica Polygalacturonase precursor epitope
286Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr Gly Ala Val Gly Asp 1 5
10 15 28715PRTArtificial SequenceCryptomeria japonica
Polygalacturonase precursor epitope 287Ala Asn Gly Tyr Phe Ser Gly
His Val Ile Pro Ala Cys Lys Asn 1 5 10 15 28816PRTArtificial
SequenceArabidopsis thaliana Probable pectate lyase 18 precursor
epitope 288Gly His Ser Asp Thr Tyr Ser Arg Asp Lys Asn Met Gln Val
Thr Ile 1 5 10 15 28915PRTArtificial SequencePhleum pratense
Profilin-2/4 epitope 289Leu Gly His Asp Gly Thr Val Trp Ala Gln Ser
Ala Asp Phe Pro 1 5 10 15 29020PRTArtificial SequenceHevea
brasiliensis Pro-hevein precursor epitope 290Asp Glu Tyr Cys Ser
Pro Asp His Asn Cys Gln Ser Asn Cys Lys Asp 1 5 10 15 Ser Gly Glu
Gly 20 29120PRTArtificial SequenceHevea brasiliensis Pro-hevein
precursor epitope 291Glu Gln Cys Gly Arg Gln Ala Gly Gly Lys Leu
Cys Pro Asn Asn Leu 1 5 10 15 Cys Cys Ser Gln 20 29243PRTArtificial
SequenceHevea brasiliensis Pro-hevein precursor epitope 292Glu Gln
Cys Gly Arg Gln Ala Gly Gly Lys Leu Cys Pro Asn Asn Leu 1 5 10 15
Cys Cys Ser Gln Trp Gly Trp Cys Gly Ser Thr Asp Glu Tyr Cys Ser 20
25 30 Pro Asp His Asn Cys Gln Ser Asn Cys Lys Asp 35 40
29320PRTArtificial SequenceHevea brasiliensis Pro-hevein precursor
epitope 293Lys Leu Cys Pro Asn Asn Leu Cys Cys Ser Gln Trp Gly Trp
Cys Gly 1 5 10 15 Ser Thr Asp Glu 20 29420PRTArtificial
SequenceHevea brasiliensis Pro-hevein precursor epitope 294Asn Gly
Gly Leu Asp Leu Asp Val Asn Val Phe Arg Gln Leu Asp Thr 1 5 10 15
Asp Gly Lys Gly 20 29510PRTArtificial SequencePrunus persica pru p
1 epitope 295Gly Lys Cys Gly Val Ser Ile Pro Tyr Lys 1 5 10
29610PRTArtificial SequencePrunus persica pru p 1 epitope 296Ile
Thr Cys Gly Gln Val Ser Ser Ser Leu 1 5 10 29710PRTArtificial
SequencePrunus persica pru p 1 epitope 297Ser Ile Pro Tyr Lys Ile
Ser Ala Ser Thr 1 5 10 29815PRTArtificial SequencePrunus persica
pru p 1 epitope 298Asp Arg Gln Ala Ala Cys Asn Cys Leu Lys Gln Leu
Ser Ala Ser 1 5 10 15 29915PRTArtificial SequencePrunus persica pru
p 1 epitope 299Val Asn Pro Asn Asn Ala Ala Ala Leu Pro Gly Lys Cys
Gly Val 1 5 10 15 30016PRTArtificial SequenceArabidopsis thaliana
Putative pectate lyase 17 precursor epitope 300Gly His Asn Asp Asn
Phe Val Lys Asp Val Lys Met Lys Val Thr Val 1 5 10 15
30116PRTArtificial SequenceHomo sapiens RAD51-like 1 isoform 1
epitope 301Thr Arg Leu Ile Leu Gln Tyr Leu Asp Ser Glu Arg Arg Gln
Ile Leu 1 5 10 15 30216PRTArtificial SequenceAspergillus fumigatus
Ribonuclease mitogillin precursor epitope 302Asp Pro Gly Pro Ala
Arg Val Ile Tyr Thr Tyr Pro Asn Lys Val Phe 1 5 10 15
30320PRTArtificial SequenceAspergillus fumigatus Ribonuclease
mitogillin precursor epitope 303Ala Thr Trp Thr Cys Ile Asn Gln Gln
Leu Asn Pro Lys Thr Asn Lys 1 5 10 15 Trp Glu Asp Lys 20
30420PRTArtificial SequenceAspergillus fumigatus Ribonuclease
mitogillin precursor epitope 304His Tyr Leu Leu Glu Phe Pro Thr Phe
Pro Asp Gly His Asp Tyr Lys 1 5 10 15 Phe Asp Ser Lys 20
30520PRTArtificial SequenceAspergillus fumigatus Ribonuclease
mitogillin precursor epitope 305Lys Phe Asp Ser Lys Lys Pro Lys Glu
Asp Pro Gly Pro Ala Arg Val 1 5 10 15 Ile Tyr Thr Tyr 20
30620PRTArtificial SequenceAspergillus fumigatus Ribonuclease
mitogillin precursor epitope 306Leu Ile Lys Gly Arg Thr Pro Ile Lys
Phe Gly Lys Ala Asp Cys Asp 1 5 10 15 Arg Pro Pro Lys 20
30720PRTArtificial SequenceAspergillus fumigatus Ribonuclease
mitogillin precursor epitope 307Ser Tyr Pro His Trp Phe Thr Asn Gly
Tyr Asp Gly Asn Gly Lys Leu 1 5 10 15 Ile Lys Gly Arg 20
30819PRTArtificial SequenceHevea brasiliensis Rubber elongation
factor protein epitope 308Ala Glu Asp Glu Asp Asn Gln Gln Gly Gln
Gly Glu Gly Leu Lys Tyr 1 5 10 15 Leu Gly Phe 30919PRTArtificial
SequenceHevea brasiliensis Rubber elongation factor protein epitope
309Phe Ser Asn Val Tyr Leu Phe Ala Lys Asp Lys Ser Gly Pro Leu Gln
1 5 10 15 Pro Gly Val 31019PRTArtificial SequenceHevea brasiliensis
Rubber elongation factor protein epitope 310Lys Phe Val Asp Ser Thr
Val Val Ala Ser Val Thr Ile Ile Asp Arg 1 5 10 15 Ser Leu Pro
31119PRTArtificial SequenceHevea brasiliensis Rubber elongation
factor protein epitope 311Gln Pro Gly Val Asp Ile Ile Glu Gly Pro
Val Lys Asn Val Ala Val 1 5 10 15 Pro Leu Tyr 31219PRTArtificial
SequenceHevea brasiliensis Rubber elongation factor protein epitope
312Arg Ser Leu Pro Pro Ile Val Lys Asp Ala Ser Ile Gln Val Val Ser
1 5 10 15 Ala Ile Arg 31317PRTArtificial SequenceBos taurus Serum
albumin precursor epitope 313Asp Asp Ser Pro Asp Leu Pro Lys Leu
Lys Pro Asp Pro Asn Thr Leu 1 5 10 15 Cys 31420PRTArtificial
SequenceBos taurus Serum albumin precursor epitope 314Glu Lys Asp
Ala Ile Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe 1 5 10 15 Ala
Glu Asp Lys 20 3159PRTArtificial SequenceBos taurus Serum albumin
precursor epitope 315Glu Ser His Ala Gly Cys Glu Lys Ser 1 5
31610PRTArtificial SequenceBos taurus Serum albumin precursor
epitope 316His Pro Glu Tyr Ala Val Ser Val Leu Leu 1 5 10
3179PRTArtificial SequenceBos taurus Serum albumin precursor
epitope 317Leu Ser Leu Ile Leu Asn Arg Leu Cys 1 5
31812PRTArtificial SequenceHevea brasiliensis Small rubber particle
protein epitope 318Asp Phe Val Arg Ala Ala Gly Val Tyr Ala Val Asp
1 5 10 31912PRTArtificial SequenceHevea brasiliensis Small rubber
particle protein epitope 319Lys Tyr Leu Asp Phe Val Arg Ala Ala Gly
Val Tyr 1 5 10 32012PRTArtificial SequenceHevea brasiliensis Small
rubber particle protein epitope 320Asn Val Val Lys Thr Val Val Thr
Pro Val Tyr Tyr 1 5 10 32112PRTArtificial SequenceHevea
brasiliensis Small rubber particle protein epitope 321Pro Arg Ile
Val Leu Asp Val Ala Ser Ser Val Phe 1 5 10 32212PRTArtificial
SequenceHevea brasiliensis Small rubber particle protein epitope
322Gln Gly Tyr Arg Val Ser Ser Tyr Leu Pro Leu Leu 1 5 10
32315PRTArtificial SequenceGlycine max Stress-induced protein SAM22
epitope 323Ala Leu Phe Lys Ala Ile Glu Ala Tyr Leu Leu Ala His Pro
Asp 1 5 10 15 32415PRTArtificial SequenceCryptomeria japonica Sugi
basic protein precursor epitope 324Ala Phe Asn Val Glu Asn Gly Asn
Ala Thr Pro Gln Leu Thr Lys 1 5 10 15 32515PRTArtificial
SequenceCryptomeria japonica Sugi basic protein precursor epitope
325Ala Asn Asn Asn Tyr Asp Pro Trp Thr Ile Tyr Ala Ile Gly Gly 1 5
10 15 32615PRTArtificial SequenceCryptomeria japonica Sugi basic
protein precursor epitope 326Ala Tyr Ser Asp Asp Lys Ser Met Lys
Val Thr Val Ala
Phe Asn 1 5 10 15 32715PRTArtificial SequenceCryptomeria japonica
Sugi basic protein precursor epitope 327Cys Gly Gln Arg Met Pro Arg
Ala Arg Tyr Gly Leu Val His Val 1 5 10 15 32815PRTArtificial
SequenceCryptomeria japonica Sugi basic protein precursor epitope
328Cys Ser Asn Trp Val Trp Gln Ser Thr Gln Asp Val Phe Tyr Asn 1 5
10 15 32920PRTArtificial SequenceTrichophyton rubrum Tri r 2
allergen epitope 329Ala Asp Phe Ser Asn Tyr Gly Ala Val Val Asp Val
Tyr Ala Pro Gly 1 5 10 15 Lys Asp Ile Thr 20 33020PRTArtificial
SequenceTrichophyton rubrum Tri r 2 allergen epitope 330Ala Lys Gly
Val Ser Leu Val Ala Val Lys Val Leu Asp Cys Asp Gly 1 5 10 15 Ser
Gly Ser Asn 20 33120PRTArtificial SequenceTrichophyton rubrum Tri r
2 allergen epitope 331Ala Ser Asn Gln Ala Ala Lys Ala Ile Ser Asp
Ala Gly Ile Phe Met 1 5 10 15 Ala Val Ala Ala 20 33220PRTArtificial
SequenceTrichophyton rubrum Tri r 2 allergen epitope 332Asp Cys Asn
Gly His Gly Thr His Val Ala Gly Thr Val Gly Gly Thr 1 5 10 15 Lys
Tyr Gly Leu 20 33320PRTArtificial SequenceTrichophyton rubrum Tri r
2 allergen epitope 333Asp Pro Ser Ala Gly Lys Gly Val Thr Ala Tyr
Ile Ile Asp Thr Gly 1 5 10 15 Ile Asp Ile Asp 20 33412PRTArtificial
SequenceVespula vulgaris Venom allergen 5 precursor epitope 334Ala
Cys Lys Tyr Gly Ser Leu Lys Pro Asn Cys Gly 1 5 10
33512PRTArtificial SequenceVespula vulgaris Venom allergen 5
precursor epitope 335Cys Asn Tyr Gly Pro Ser Gly Asn Phe Met Asn
Glu 1 5 10 33612PRTArtificial SequenceVespula vulgaris Venom
allergen 5 precursor epitope 336Asp Val Ala Lys Tyr Gln Val Gly Gln
Asn Val Ala 1 5 10 33712PRTArtificial SequenceVespula vulgaris
Venom allergen 5 precursor epitope 337Glu Lys Trp His Lys His Tyr
Leu Val Cys Asn Tyr 1 5 10 33812PRTArtificial SequenceVespula
vulgaris Venom allergen 5 precursor epitope 338Glu Leu Ala Tyr Val
Ala Gln Val Trp Ala Asn Gln 1 5 10 33915PRTArtificial
SequenceCorylus avellana 11S globulin-like protein epitope 339Ala
Phe Gln Ile Ser Arg Glu Glu Ala Arg Arg Leu Lys Tyr Asn 1 5 10 15
34012PRTArtificial SequenceCarya illinoinensis 11S legumin protein
epitope 340Glu Glu Ser Gln Arg Gln Ser Gln Gln Gly Gln Arg 1 5 10
34118PRTArtificial SequenceFagopyrum esculentum 13S globulin
epitope 341Asp Ala His Gln Pro Thr Arg Arg Val Arg Lys Gly Asp Val
Val Ala 1 5 10 15 Leu Pro 34212PRTArtificial SequenceFagopyrum
esculentum 13S globulin seed storage protein 1 precursor
(Legumin-like protein 1) epitope 342Phe Lys Gln Asn Val Asn Arg Pro
Ser Arg Ala Asp 1 5 10 34312PRTArtificial SequenceFagopyrum
esculentum 13S globulin seed storage protein 3 precursor
(Legumin-like protein 3) (Allergen Fag e 1) epitope 343Asp Ile Ser
Thr Lys Glu Ala Phe Arg Leu Lys Asn 1 5 10 34412PRTArtificial
SequenceAnacardium occidentale 2s albumin epitope 344Cys Gln Arg
Gln Phe Glu Glu Gln Gln Arg Phe Arg 1 5 10 34510PRTArtificial
SequenceSesamum indicum 2S seed storage protein 1 epitope 345His
Phe Arg Glu Cys Cys Asn Glu Ile Arg 1 5 10 34610PRTArtificial
SequenceSesamum indicum 2S seed storage protein 1 precursor epitope
346Cys Met Gln Trp Met Arg Ser Met Arg Gly 1 5 10
34714PRTArtificial SequenceBertholletia excelsa 2S sulfur-rich seed
storage protein precursor (Allergen Ber e 1) epitope 347Cys Arg Cys
Glu Gly Leu Arg Met Met Met Met Arg Met Gln 1 5 10
34821PRTArtificial SequenceCynodon dactylon acidic Cyn d 1
isoallergen isoform 1 precursor epitope 348Gln Asp Asp Val Ile Pro
Glu Asp Trp Lys Pro Asp Thr Val Tyr Lys 1 5 10 15 Ser Lys Ile Gln
Phe 20 34950PRTArtificial SequenceCynodon dactylon acidic Cyn d 1
isoallergen isoform 3 precursor epitope 349Glu Glu Asp Lys Leu Arg
Lys Ala Gly Glu Leu Met Leu Gln Phe Arg 1 5 10 15 Arg Val Lys Cys
Glu Tyr Pro Ser Asp Thr Lys Ile Thr Phe His Val 20 25 30 Glu Lys
Gly Ser Ser Pro Asn Tyr Leu Ala Leu Leu Val Lys Tyr Ala 35 40 45
Ala Gly 50 3507PRTArtificial SequenceBos taurus albumin epitope
350Pro Val Glu Ser Lys Val Thr 1 5 35113PRTArtificial
SequenceJuglans regia Albumin seed storage protein epitope 351Gly
Leu Arg Gly Glu Glu Met Glu Glu Met Val Gln Ser 1 5 10
35218PRTArtificial SequenceCochliobolus lunatus alcohol
dehydrogenase epitope 352Ala Val Asn Gly Asp Trp Pro Leu Pro Thr
Lys Leu Pro Leu Val Gly 1 5 10 15 Gly His 35337PRTArtificial
SequencePenicillium chrysogenum alkaline serine protease epitope
353Ala Asn Val Val Gln Arg Asn Ala Pro Ser Trp Gly Leu Ser Arg Ile
1 5 10 15 Ser Ser Lys Lys Ser Gly Ala Thr Asp Tyr Val Tyr Asp Ser
Thr Ala 20 25 30 Gly Glu Gly Ile Val 35 35410PRTArtificial
SequenceArachis hypogaea allergen epitope 354Asp Asp Gln Cys Gln
Arg Gln Leu Gln Arg 1 5 10 35515PRTArtificial SequenceAnacardium
occidentale allergen Ana o 2 epitope 355Glu Glu Ser Glu Asp Glu Lys
Arg Arg Trp Gly Gln Arg Asp Asn 1 5 10 15 35610PRTArtificial
SequenceArachis hypogaea Allergen Ara h 1, clone P41B precursor
epitope 356Ala Lys Ser Ser Pro Tyr Gln Lys Lys Thr 1 5 10
35715PRTArtificial SequenceArachis hypogaea allergen Arah3/Arah4
epitope 357Ala Gly Val Ala Leu Ser Arg Leu Val Leu Arg Arg Asn Ala
Leu 1 5 10 15 35810PRTArtificial SequenceArachis hypogaea allergen
Arah6 epitope 358Asp Arg Gln Met Val Gln His Phe Lys Arg 1 5 10
35911PRTArtificial SequencePeriplaneta americana Allergen Cr-PI
epitope 359Ile Pro Lys Gly Lys Lys Gly Gly Gln Ala Tyr 1 5 10
3608PRTArtificial SequenceAspergillus fumigatus allergen I/a; Asp f
I/a epitope 360Ile Asn Gln Gln Leu Asn Pro Lys 1 5
36110PRTArtificial SequenceArachis hypogaea Allergen II epitope
361Asp Arg Leu Gln Gly Arg Gln Gln Glu Gln 1 5 10
36215PRTArtificial SequenceLens culinaris allergen Len c 1.0101
epitope 362Ala Ile Asn Ala Ser Ser Asp Leu Asn Leu Ile Gly Phe Gly
Ile 1 5 10 15 36312PRTArtificial SequenceDermatophagoides farinae
Allergen Mag epitope 363Asp Val Glu Leu Ser Leu Arg Ser Ser Asp Ile
Ala 1 5 10 36431PRTArtificial SequencePenicillium chrysogenum
Allergen Pen n 18 epitope 364Ala His Ile Lys Lys Ser Lys Lys Gly
Asp Lys Lys Phe Lys Gly Ser 1 5 10 15 Val Ala Asn Met Ser Leu Gly
Gly Gly Ser Ser Arg Thr Leu Asp 20 25 30 36514PRTArtificial
SequenceSinapis alba Allergen Sin a 1 epitope 365Gln Gly Pro His
Val Ile Ser Arg Ile Tyr Gln Thr Ala Thr 1 5 10 36612PRTArtificial
SequenceZiziphus mauritiana allergen Ziz m 1 epitope 366Lys Thr Asn
Tyr Ser Ser Ser Ile Ile Leu Glu Tyr 1 5 10 36737PRTArtificial
SequenceFagopyrum tataricum allergenic protein epitope 367Asp Ile
Ser Thr Glu Glu Ala Tyr Lys Leu Lys Asn Gly Arg Gln Glu 1 5 10 15
Val Glu Val Phe Arg Pro Phe Gln Ser Arg Tyr Glu Lys Glu Glu Glu 20
25 30 Lys Glu Arg Glu Arg 35 36830PRTArtificial SequenceBos taurus
alpha S1 casein epitope 368Glu Asp Gln Ala Met Glu Asp Ile Lys Gln
Met Glu Ala Glu Ser Ile 1 5 10 15 Ser Ser Ser Glu Glu Ile Val Pro
Asn Ser Val Glu Gln Lys 20 25 30 36910PRTArtificial
SequenceTriticum aestivum Alpha/beta-gliadin A-II precursor epitope
369Gln Val Ser Phe Gln Gln Pro Gln Gln Gln 1 5 10
37010PRTArtificial SequenceTriticum aestivum Alpha/beta-gliadin A-V
epitope 370Leu Ala Leu Gln Thr Leu Pro Ala Met Cys 1 5 10
37120PRTArtificial SequenceBos taurus alpha2(I) collagen epitope
371Leu Pro Gly Leu Lys Gly His Asn Gly Leu Gln Gly Leu Pro Gly Leu
1 5 10 15 Ala Gly His His 20 3725PRTArtificial SequenceTriticum
aestivum Alpha-amylase inhibitor 0.28 precursor (CIII) (WMAI-1)
epitope 372Ala Tyr Pro Asp Val 1 5 37312PRTArtificial
SequenceTriticum aestivum Alpha-gliadin epitope 373Leu Gly Gln Gly
Ser Phe Arg Pro Ser Gln Gln Asn 1 5 10 37410PRTArtificial
SequenceBos taurus Alpha-lactalbumin epitope 374Lys Asp Leu Lys Gly
Tyr Gly Gly Val Ser 1 5 10 37514PRTArtificial SequenceBos taurus
Alpha-lactalbumin precursor epitope 375Lys Cys Glu Val Phe Arg Glu
Leu Lys Asp Leu Lys Gly Tyr 1 5 10 37620PRTArtificial SequenceBos
taurus alpha-S1-casein epitope 376Leu Asn Glu Asn Leu Leu Arg Phe
Phe Val Ala Pro Phe Pro Gln Val 1 5 10 15 Phe Gly Lys Glu 20
37710PRTArtificial SequenceBos taurus Alpha-S1-casein precursor
epitope 377Ala Met Glu Asp Ile Lys Gln Met Glu Ala 1 5 10
37810PRTArtificial SequenceBos taurus Alpha-S2-casein precursor
epitope 378Glu Asn Leu Cys Ser Thr Phe Cys Lys Glu 1 5 10
37915PRTArtificial SequenceArachis hypogaea Ara h 2.01 allergen
epitope 379Cys Cys Asn Glu Leu Asn Glu Phe Glu Asn Asn Gln Arg Cys
Met 1 5 10 15 38015PRTArtificial SequenceGlycine max Bd 30K (34 kDa
maturing seed protein) epitope 380Glu Asp Trp Gly Glu Asp Gly Tyr
Ile Trp Ile Gln Arg Asn Thr 1 5 10 15 3818PRTArtificial
SequenceBetula pendula Bet v 4 epitope 381Phe Ala Arg Ala Asn Arg
Gly Leu 1 5 3829PRTArtificial SequenceMusa acuminata beta-1,
3-glucananse epitope 382Gly Leu Phe Tyr Pro Asn Lys Gln Pro 1 5
38315PRTArtificial SequenceHevea brasiliensis beta-1,3-glucanase
epitope 383Gly Leu Phe Phe Pro Asp Lys Arg Pro Lys Tyr Asn Leu Asn
Phe 1 5 10 15 38412PRTArtificial SequenceOlea europaea
beta-1,3-glucanase-like protein epitope 384Ala Gly Arg Asn Ser Trp
Asn Cys Asp Phe Ser Gln 1 5 10 38513PRTArtificial SequenceBos
taurus beta-casein epitope 385Gln Ser Lys Val Leu Pro Val Pro Gln
Lys Ala Val Pro 1 5 10 38612PRTArtificial SequenceBos taurus
Beta-casein precursor epitope 386Asp Glu Leu Gln Asp Lys Ile His
Pro Phe Ala Gln 1 5 10 38710PRTArtificial SequenceBos taurus
Beta-lactoglobulin epitope 387Ala Gln Lys Lys Ile Ile Ala Glu Lys
Thr 1 5 10 38816PRTArtificial SequenceBos taurus Beta-lactoglobulin
precursor epitope 388Ala Ala Ser Asp Ile Ser Leu Leu Asp Ala Gln
Ser Ala Pro Leu Arg 1 5 10 15 3898PRTArtificial SequenceFagopyrum
esculentum BW 16kDa allergen epitope 389Glu Gly Val Arg Asp Leu Lys
Glu 1 5 39017PRTArtificial SequenceBetula pendula Chain A, Birch
Pollen Profilin epitope 390Ala Gln Ser Ser Ser Phe Pro Gln Phe Lys
Pro Gln Glu Ile Thr Gly 1 5 10 15 Ile 39120PRTArtificial
SequenceOncorhynchus mykiss collagen a2(I) epitope 391Met Lys Gly
Leu Arg Gly His Gly Gly Leu Gln Gly Met Pro Gly Pro 1 5 10 15 Asn
Gly Pro Ser 20 39246PRTArtificial SequenceBos taurus collagen, type
I, alpha 2 epitope 392Ala Pro Gly Pro Asp Gly Asn Asn Gly Ala Gln
Gly Pro Pro Gly Leu 1 5 10 15 Gln Gly Val Gln Gly Gly Lys Gly Glu
Gln Gly Pro Ala Gly Pro Pro 20 25 30 Gly Phe Gln Gly Leu Pro Gly
Pro Ala Gly Thr Ala Gly Glu 35 40 45 39315PRTArtificial
SequenceArachis hypogaea Conglutin-7 precursor epitope 393Ala Ala
His Ala Ser Ala Arg Gln Gln Trp Glu Leu Gln Gly Asp 1 5 10 15
3948PRTArtificial SequencePeriplaneta americana Cr-PII allergen
epitope 394Ile Arg Ser Trp Phe Gly Leu Pro 1 5 39511PRTArtificial
SequenceCochliobolus lunatus Cytochrome c epitope 395Glu Asn Pro
Lys Lys Tyr Ile Pro Gly Thr Lys 1 5 10 39610PRTArtificial
SequenceRattus norvegicus Cytochrome P450 3A1 epitope 396Asp Met
Val Leu Asn Glu Thr Leu Arg Leu 1 5 10 3979PRTArtificial
SequenceDermatophagoides farinae Der f 2 epitope 397Ile Ala Thr His
Ala Lys Ile Arg Asp 1 5 39815PRTArtificial SequenceDermatophagoides
farinae Der f 7 allergen epitope 398His Ile Gly Gly Leu Ser Ile Leu
Asp Pro Ile Phe Gly Val Leu 1 5 10 15 39943PRTArtificial
SequenceDermatophagoides pteronyssinus Der p 1 allergen epitope
399Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly Ile
1 5 10 15 Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Ala Asn Lys Ile
Arg Glu 20 25 30 Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val 35 40
40015PRTArtificial SequenceDermatophagoides pteronyssinus Der p 7
allergen polypeptide epitope 400His Ile Gly Gly Leu Ser Ile Leu Asp
Pro Ile Phe Ala Val Leu 1 5 10 15 40139PRTArtificial
SequenceCandida albicans Enolase 1 (2-phosphoglycerate dehydratase)
(2-phospho-D-glycerate hydro-lyase) epitope 401Gln Ala Ala Asn Asp
Ser Tyr Ala Ala Gly Trp Gly Val Met Val Ser 1 5 10 15 His Arg Ser
Gly Glu Thr Glu Asp Thr Phe Ile Ala Asp Leu Ser Val 20 25 30 Gly
Leu Arg Ser Gly Gln Ile 35 40221PRTArtificial SequenceHevea
brasiliensis ENSP-like protein epitope 402Phe Pro Leu Ile Thr Cys
Cys Gly Tyr Gly Gly Lys Tyr Asn Phe Ser 1 5 10 15 Val Thr Ala Pro
Cys 20 40312PRTArtificial SequenceFagopyrum esculentum Fag e 1
epitope 403Ala Val Val Leu Lys Ala Gly Asn Glu Gly Leu Glu 1 5 10
40410PRTArtificial SequenceTriticum aestivum Gamma-gliadin
precursor epitope 404Leu Gln Pro Gln Gln Pro Phe Pro Gln Gln 1 5 10
40521PRTArtificial SequenceChironomus thummi thummi Globin CTT-III
epitope 405Ala His Thr Asp Phe Ala Gly Ala Glu Ala Ala Trp Gly Ala
Thr Leu 1 5 10 15 Asp Thr Phe Phe Gly 20 40611PRTArtificial
SequenceChironomus thummi thummi Globin CTT-III precursor epitope
406Gly Val Thr His Asp Gln Leu Asn Asn Phe Arg 1 5 10
40723PRTArtificial SequenceChironomus thummi thummi Globin CTT-IV
precursor epitope 407Lys Ala His Thr Asp Phe Ala Gly Ala Glu Ala
Ala Trp Gly Ala Thr 1 5 10 15 Leu Asp Ala Phe Phe Gly Met 20
40835PRTArtificial SequenceChironomus thummi thummi Globin CTT-VI
precursor epitope 408Ile Val Ser Phe Leu Ser Glu Val Ile Ser Leu
Ala Gly Ser Asp Ala 1 5 10 15 Asn Ile Pro Ala Ile Gln Asn Leu Ala
Lys Glu Leu Ala Thr Ser His 20 25 30 Lys Pro Arg 35
40935PRTArtificial SequenceChironomus thummi thummi Globin CTT-VIII
epitope 409Ile Val Gly Phe Phe Ser Glu Val Ile Gly Leu Ile Gly Asn
Pro Glu 1 5 10 15 Asn Arg Pro Ala Leu Lys Thr Leu Ile Asp Gly Leu
Ala Ser Ser His 20 25 30 Lys Ala Arg 35 4109PRTArtificial
SequenceHevea brasiliensis Glucan endo-1,3-beta- glucosidase, basic
vacuolar
isoform epitope 410Ala Trp Leu Ala Gln Phe Val Leu Pro 1 5
41114PRTArtificial SequenceTriticum aestivum Glutenin, high
molecular weight subunit DX5 epitope 411Ala Gln Gly Gln Gln Pro Gly
Gln Gly Gln Gln Gly Gln Gln 1 5 10 4125PRTArtificial
SequenceTriticum aestivum Glutenin, high molecular weight subunit
DX5 precursor epitope 412Gln Gln Pro Gly Gln 1 5 4135PRTArtificial
SequenceTriticum aestivum Glutenin, low molecular weight subunit
precursor epitope 413Gln Gln Gln Pro Pro 1 5 41415PRTArtificial
SequencePhaseolus vulgaris Glycine-rich cell wall structural
protein 1.8 precursor epitope 414Gly Gly Tyr Gly Asp Gly Gly Ala
His Gly Gly Gly Tyr Gly Gly 1 5 10 15 41515PRTArtificial
SequenceArachis hypogaea Glycinin epitope 415Ala Leu Ser Arg Leu
Val Leu Arg Arg Asn Ala Leu Arg Arg Pro 1 5 10 15
41613PRTArtificial SequenceGlycine max Glycinin G1 precursor
epitope 416Gly Ala Ile Val Thr Val Lys Gly Gly Leu Ser Val Ile 1 5
10 41715PRTArtificial SequenceGlycine max Glycinin G2 precursor
epitope 417Ala Leu Ser Arg Cys Thr Leu Asn Arg Asn Ala Leu Arg Arg
Pro 1 5 10 15 41815PRTArtificial SequenceHolcus lanatus group V
allergen epitope 418Ala Asn Val Pro Pro Ala Asp Lys Tyr Lys Thr Phe
Glu Ala Ala 1 5 10 15 41912PRTArtificial SequenceOryza sativa
Japonica Group hypothetical protein epitope 419Ala Phe Asn His Phe
Gly Ile Gln Leu Val Gln Arg 1 5 10 42014PRTArtificial SequenceBos
taurus Kappa-casein precursor epitope 420Ala Lys Tyr Ile Pro Ile
Gln Tyr Val Leu Ser Arg Tyr Pro 1 5 10 42120PRTArtificial
SequenceAlternaria alternata Major allergen Alt a 1 precursor
epitope 421Ala Asp Pro Val Thr Thr Glu Gly Asp Tyr Val Val Lys Ile
Ser Glu 1 5 10 15 Phe Tyr Gly Arg 20 42215PRTArtificial
SequenceAnisakis simplex Major allergen Ani s 1 epitope 422Cys Lys
Met Pro Asp Arg Gly Ala Cys Ala Leu Gly Lys Lys Pro 1 5 10 15
42313PRTArtificial SequenceAspergillus fumigatus Major allergen Asp
f 1 epitope 423Leu Asn Pro Lys Thr Asn Lys Trp Glu Asp Lys Arg Tyr
1 5 10 42410PRTArtificial SequenceAspergillus fumigatus Major
allergen Asp f 2 epitope 424Ala His Ile Leu Arg Trp Gly Asn Glu Ser
1 5 10 42520PRTArtificial SequenceBos taurus major allergen
beta-lactoglobulin epitope 425Leu Gln Lys Trp Glu Asn Asp Glu Cys
Ala Gln Lys Lys Ile Ile Ala 1 5 10 15 Glu Lys Thr Lys 20
42614PRTArtificial SequenceFelis catus Major allergen I polypeptide
chain 1 precursor epitope 426Asp Ala Lys Met Thr Glu Glu Asp Lys
Glu Asn Ala Leu Ser 1 5 10 42714PRTArtificial SequenceFelis catus
Major allergen I polypeptide chain 2 precursor epitope 427Glu Pro
Glu Arg Thr Ala Met Lys Lys Ile Gln Asp Cys Tyr 1 5 10
42811PRTArtificial SequenceFelis catus major allergen I,
polypeptide chain 1 epitope 428Leu Leu Asp Lys Ile Tyr Thr Ser Pro
Leu Cys 1 5 10 42929PRTArtificial SequenceTurbo cornutus major
allergen Tur c1 - Turbo cornutus epitope 429Leu Glu Asp Glu Leu Leu
Ala Glu Lys Glu Lys Tyr Lys Ala Ile Ser 1 5 10 15 Asp Glu Leu Asp
Gln Thr Phe Ala Glu Leu Ala Gly Tyr 20 25 43025PRTArtificial
SequenceDermatophagoides pteronyssinus major house dust allergen
epitope 430Leu Ala His Arg Asn Gln Ser Leu Asp Leu Ala Glu Gln Glu
Leu Val 1 5 10 15 Asp Cys Ala Ser Gln His Gly Cys His 20 25
4319PRTArtificial SequenceHevea brasiliensis Major latex allergen
Hev b 5 epitope 431Ala Pro Pro Ala Ser Glu Gln Glu Thr 1 5
43243PRTArtificial SequenceDermatophagoides pteronyssinus Major
mite fecal allergen Der p 1 epitope 432Ala Arg Glu Gln Ser Cys Arg
Arg Pro Asn Ala Gln Arg Phe Gly Ile 1 5 10 15 Ser Asn Tyr Cys Gln
Ile Tyr Pro Pro Asn Ala Asn Lys Ile Arg Glu 20 25 30 Ala Leu Ala
Gln Pro Gln Arg Tyr Cys Arg His 35 40 43312PRTArtificial
SequenceOlea europaea Major pollen allergen epitope 433Phe Thr Glu
Val Gly Tyr Thr Arg Ala Glu Gly Leu 1 5 10 43410PRTArtificial
SequenceBetula pendula Major pollen allergen Bet v 1-A epitope
434Asp Gly Asp Asn Leu Phe Pro Lys Val Ala 1 5 10
43511PRTArtificial SequenceChamaecyparis obtusa Major pollen
allergen Cha o 1 precursor epitope 435Trp Arg Ser Thr Gln Asp Ser
Phe Asn Asn Gly 1 5 10 43617PRTArtificial SequenceCorylus avellana
Major pollen allergen Cor a 1 epitope 436Tyr Val Leu Asp Gly Asp
Lys Leu Leu Pro Lys Val Ala Pro Gln Ala 1 5 10 15 Leu
43727PRTArtificial SequenceHolcus lanatus Major pollen allergen Hol
l 1 precursor epitope 437Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr
Gly Ala Gly Pro Lys Asp 1 5 10 15 Asn Gly Gly Ala Cys Gly Tyr Lys
Asp Val Asp 20 25 43812PRTArtificial SequenceJuniperus ashei Major
pollen allergen Jun a 1 precursor epitope 438Ala Phe Asn Gln Phe
Gly Pro Asn Ala Gly Gln Arg 1 5 10 43934PRTArtificial SequenceOlea
europaea major pollen allergen Ole e 1 epitope 439Ser Gly Arg Lys
Asp Cys Asn Glu Ile Pro Thr Glu Gly Trp Val Lys 1 5 10 15 Pro Ser
Leu Lys Phe Ile Leu Asn Thr Val Asn Gly Thr Thr Arg Thr 20 25 30
Val Asn 4409PRTArtificial SequenceMalus x domestica mal d 3 epitope
440Arg Thr Thr Ala Asp Arg Gln Thr Ala 1 5 44116PRTArtificial
SequenceBlomia tropicalis Mite allergen Blo t 5 epitope 441Glu Glu
Ala Gln Thr Leu Ser Lys Ile Leu Leu Lys Asp Leu Lys Glu 1 5 10 15
4425PRTArtificial SequenceDermatophagoides farinae Mite group 2
allergen Der f 2 precursor epitope 442Asp Pro Cys Ile Ile 1 5
44315PRTArtificial SequenceDermatophagoides pteronyssinus Mite
group 2 allergen Der p 2 precursor epitope 443Asp Gln Val Asp Val
Lys Asp Cys Ala Asn His Glu Ile Lys Lys 1 5 10 15
44433PRTArtificial SequenceLepidoglyphus destructor Mite group 2
allergen Lep d 2 precursor epitope 444Ala Ala Asn Gln Asp Thr Ala
Lys Val Thr Ile Lys Val Leu Ala Lys 1 5 10 15 Val Ala Gly Thr Thr
Ile Gln Val Pro Gly Leu Glu Thr Asp Gly Cys 20 25 30 Lys
4456PRTArtificial SequenceTriticum aestivum monomeric alpha-amylase
inhibitor epitope 445Ala Ala Ser Val Pro Glu 1 5 4469PRTArtificial
SequencePrunus armeniaca Non-specific lipid-transfer protein 1
epitope 446Val Asn Pro Asn Asn Ala Ala Ala Leu 1 5
44715PRTArtificial SequencePrunus armeniaca Non-specific
lipid-transfer protein 1 (LTP 1) (Major allergen Pru ar 3) epitope
447Leu Ala Arg Thr Thr Pro Asp Arg Arg Thr Ala Cys Asn Cys Leu 1 5
10 15 44818PRTArtificial SequencePrunus domestica Non-specific
lipid-transfer protein 1 (LTP 1) (Major allergen Pru d 3) epitope
448Leu Ala Arg Thr Thr Ala Asp Arg Arg Ala Ala Cys Asn Cys Leu Lys
1 5 10 15 Gln Leu 44915PRTArtificial SequenceMalus x domestica
Non-specific lipid-transfer protein precursor (LTP) (Allergen Mal d
3) epitope 449Ala Asp Arg Gln Thr Ala Cys Asn Cys Leu Lys Asn Leu
Ala Gly 1 5 10 15 45012PRTArtificial SequenceOlea europaea Ole e 1
protein epitope 450Glu Asp Val Pro Gln Pro Pro Val Ser Gln Phe His
1 5 10 45125PRTArtificial SequenceOlea europaea Ole e 1.0102
protein epitope 451Glu Asp Val Pro Gln Pro Pro Val Ser Gln Phe His
Ile Gln Gly Gln 1 5 10 15 Val Tyr Cys Asp Thr Cys Arg Ala Gly 20 25
45210PRTArtificial SequenceTriticum aestivum Omega gliadin storage
protein epitope 452Gln Gln Pro Gln Gln Ser Phe Pro Gln Gln 1 5 10
4537PRTArtificial SequenceTriticum aestivum omega-5 gliadin epitope
453Gln Gln Phe His Gln Gln Gln 1 5 45435PRTArtificial
SequenceAspergillus fumigatus Oryzin precursor epitope 454Ala Ser
Asn Thr Ser Pro Ala Ser Ala Pro Asn Ala Leu Thr Val Ala 1 5 10 15
Ala Ile Asn Lys Ser Asn Ala Arg Ala Ser Phe Ser Asn Tyr Gly Ser 20
25 30 Val Val Asp 35 4559PRTArtificial SequenceGallus gallus
Ovalbumin epitope 455Cys Phe Asp Val Phe Lys Glu Leu Lys 1 5
45610PRTArtificial SequenceGallus gallus Ovomucoid epitope 456Cys
Asn Phe Cys Asn Ala Val Val Glu Ser 1 5 10 45714PRTArtificial
SequenceGallus gallus Ovomucoid precursor epitope 457Ala Glu Val
Asp Cys Ser Arg Phe Pro Asn Ala Thr Asp Lys 1 5 10
45810PRTArtificial SequenceGlycine max P34 probable thiol protease
precursor epitope 458Ala Ser Trp Asp Trp Arg Lys Lys Gly Val 1 5 10
45910PRTArtificial SequenceGlycine max P34 probable thiol protease
precursor; Gly m 1 epitope 459Pro Gln Glu Phe Ser Lys Lys Thr Tyr
Gln 1 5 10 4609PRTArtificial SequenceParietaria judaica Par j
epitope 460Gly Thr Ser Ser Cys Arg Leu Val Pro 1 5
46147PRTArtificial SequenceBlomia tropicalis Paramyosin epitope
461Glu Lys Leu Arg Asp Gln Lys Glu Ala Leu Ala Arg Glu Asn Lys Lys
1 5 10 15 Leu Ala Asp Asp Leu Ala Glu Ala Lys Ser Gln Leu Asn Asp
Ala His 20 25 30 Arg Arg Ile His Glu Gln Glu Ile Glu Ile Lys Arg
Leu Glu Asn 35 40 45 4628PRTArtificial SequenceGadus morhua
callarias Parvalbumin beta epitope 462Ala Ala Glu Ala Ala Cys Phe
Lys 1 5 46315PRTArtificial SequenceSalmo salar parvalbumin like 1
epitope 463Ala Asp Ile Lys Thr Ala Leu Glu Ala Arg Lys Ala Ala Asp
Thr 1 5 10 15 46412PRTArtificial SequenceJuniperus ashei
Pathogenesis-related protein precursor epitope 464Ala Asp Ile Asn
Ala Val Cys Pro Ser Glu Leu Lys 1 5 10 46512PRTArtificial
SequenceNicotiana tabacum Pectate lyase epitope 465Ala Tyr Asn His
Phe Gly Lys Arg Leu Asp Gln Arg 1 5 10 46612PRTArtificial
SequenceMusa acuminata AAA Group pectate lyase 2 epitope 466Ala Phe
Asn His Phe Gly Glu Gly Leu Ile Gln Arg 1 5 10 46715PRTArtificial
SequenceFarfantepenaeus aztecus Pen a 1 allergen epitope 467Ala Asn
Ile Gln Leu Val Glu Lys Asp Lys Ala Leu Ser Asn Ala 1 5 10 15
46843PRTArtificial SequenceDermatophagoides pteronyssinus Peptidase
1 precursor (Major mite fecal allergen Der p 1) (Allergen Der p I)
epitope 468Ala Arg Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe
Gly Ile 1 5 10 15 Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Val Asn
Lys Ile Arg Glu 20 25 30 Ala Leu Ala Gln Thr His Ser Ala Ile Ala
Val 35 40 46945PRTArtificial SequenceApis mellifera Phospholipase
A2 precursor epitope 469Leu Ile Asp Thr Lys Cys Tyr Lys Leu Glu His
Pro Val Thr Gly Cys 1 5 10 15 Gly Glu Arg Thr Glu Gly Arg Cys Leu
His Tyr Thr Val Asp Lys Ser 20 25 30 Lys Pro Lys Val Tyr Gln Trp
Phe Asp Leu Arg Lys Tyr 35 40 45 47014PRTArtificial
SequenceMyrmecia pilosula Pilosulin-1 precursor (Major allergen Myr
p 1) (Myr p I) epitope 470Lys Glu Ala Ile Pro Met Ala Val Glu Met
Ala Lys Ser Gln 1 5 10 4718PRTArtificial SequenceBetula pendula
Polcalcin Bet v 4 epitope 471Phe Gly Arg Ala Asn Arg Gly Leu 1 5
47236PRTArtificial SequencePhleum pratense Polcalcin Phl p 7
(Calcium- binding pollen allergen Phl p 7) (P7) epitope 472Ala Asp
Asp Met Glu Arg Ile Phe Lys Arg Phe Asp Thr Asn Gly Asp 1 5 10 15
Gly Lys Ile Ser Leu Ser Glu Leu Thr Asp Ala Leu Arg Thr Leu Gly 20
25 30 Ser Thr Ser Ala 35 47325PRTArtificial SequenceLolium perenne
pollen allergen epitope 473Glu Gly Gly Thr Lys Ser Glu Val Glu Asp
Val Ile Pro Glu Gly Trp 1 5 10 15 Lys Ala Asp Thr Ser Tyr Ser Ala
Lys 20 25 47412PRTArtificial SequenceAmbrosia artemisiifolia Pollen
allergen Amb a 1.4 epitope 474Ala Phe Asn Lys Phe Thr Asp Asn Val
Asp Gln Arg 1 5 10 4758PRTArtificial SequenceAmbrosia
artemisiifolia Pollen allergen Amb a 2 precursor epitope 475Met Pro
Arg Cys Arg Phe Gly Phe 1 5 47615PRTArtificial SequenceAmbrosia
artemisiifolia var. elatior Pollen allergen Amb a 3 epitope 476Cys
Asp Ile Lys Asp Pro Ile Arg Leu Glu Pro Gly Gly Pro Asp 1 5 10 15
47731PRTArtificial SequenceBetula pendula pollen allergen Bet v 1
epitope 477Lys Ala Glu Gln Val Lys Ala Ser Lys Glu Met Gly Glu Thr
Leu Leu 1 5 10 15 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp
Ala Tyr Asn 20 25 30 47820PRTArtificial SequencePoa pratensis
Pollen allergen KBG 60 precursor epitope 478Ala Ala Asn Lys Tyr Lys
Thr Phe Val Ala Thr Phe Gly Ala Ala Ser 1 5 10 15 Asn Lys Ala Phe
20 47925PRTArtificial SequenceLolium perenne Pollen allergen Lol p
2-A (Lol p II-A) epitope 479Glu Lys Gly Met Arg Asn Val Phe Asp Asp
Val Val Pro Ala Asp Phe 1 5 10 15 Lys Val Gly Thr Thr Tyr Lys Pro
Glu 20 25 48027PRTArtificial SequenceLolium perenne Pollen allergen
Lol p 3 (Lol p III) epitope 480Lys Gly Gly Met Lys Asn Val Phe Asp
Glu Val Ile Pro Thr Ala Phe 1 5 10 15 Thr Val Gly Lys Thr Tyr Thr
Pro Glu Tyr Asn 20 25 48112PRTArtificial SequenceLolium perenne
Pollen allergen Lol p VA precursor epitope 481Ala Ala Glu Gly Ala
Thr Pro Glu Ala Lys Tyr Asp 1 5 10 48215PRTArtificial
SequencePhleum pratense Pollen allergen Phl p 1 precursor epitope
482Ala Pro Tyr His Phe Asp Leu Ser Gly His Ala Phe Gly Ala Met 1 5
10 15 4838PRTArtificial SequenceZea mays pollen allergen Phl p 11
epitope 483Arg Asp Arg Ala Arg Val Pro Leu 1 5 48412PRTArtificial
SequencePhleum pratense pollen allergen Phl pI epitope 484Ile Pro
Lys Val Pro Pro Gly Pro Asn Ile Thr Ala 1 5 10 48520PRTArtificial
SequenceCryptomeria japonica Polygalacturonase precursor epitope
485Gly Gln Cys Lys Trp Val Asn Gly Arg Glu Ile Cys Asn Asp Arg Asp
1 5 10 15 Arg Pro Thr Ala 20 48630PRTArtificial SequenceParietaria
judaica Probable non-specific lipid- transfer protein epitope
486Gln Glu Thr Cys Gly Thr Met Val Arg Ala Leu Met Pro Cys Leu Pro
1 5 10 15 Phe Val Gln Gly Lys Glu Lys Glu Pro Ser Lys Gly Cys Cys
20 25 30 48710PRTArtificial SequenceParietaria judaica Probable
non-specific lipid- transfer protein 2 epitope 487Ala Glu Val Pro
Lys Lys Cys Asp Ile Lys 1 5 10 48830PRTArtificial
SequenceParietaria judaica Probable non-specific lipid- transfer
protein 2 precursor epitope 488Glu Ala Cys Gly Lys Val Val Gln Asp
Ile Met Pro Cys Leu His Phe 1 5 10 15 Val Lys Gly Glu Glu Lys Glu
Pro Ser Lys Glu Cys Cys Ser
20 25 30 48912PRTArtificial SequenceSolanum lycopersicum Probable
pectate lyase P59 epitope 489Ala Phe Asn His Phe Gly Lys Arg Leu
Ile Gln Arg 1 5 10 49010PRTArtificial SequenceCucumis melo profilin
epitope 490Ala Phe Arg Leu Glu Glu Ile Ala Ala Ile 1 5 10
49156PRTArtificial SequenceGlycine max Profilin-1 epitope 491Trp
Ala Gln Ser Thr Asp Phe Pro Gln Phe Lys Pro Glu Glu Ile Thr 1 5 10
15 Ala Ile Met Asn Asp Phe Asn Glu Pro Gly Ser Leu Ala Pro Thr Gly
20 25 30 Leu Tyr Leu Gly Gly Thr Lys Tyr Met Val Ile Gln Gly Glu
Pro Gly 35 40 45 Ala Val Ile Arg Gly Lys Lys Gly 50 55
49243PRTArtificial SequenceHevea brasiliensis Pro-hevein precursor
epitope 492Glu Gln Cys Gly Arg Gln Ala Gly Gly Lys Leu Cys Pro Asn
Asn Leu 1 5 10 15 Cys Cys Ser Gln Trp Gly Trp Cys Gly Ser Thr Asp
Glu Tyr Cys Ser 20 25 30 Pro Asp His Asn Cys Gln Ser Asn Cys Lys
Asp 35 40 49310PRTArtificial SequencePrunus persica pru p 1 epitope
493Gly Lys Cys Gly Val Ser Ile Pro Tyr Lys 1 5 10
49415PRTArtificial SequencePrunus dulcis prunin 1 precursor epitope
494Glu Glu Ser Gln Gln Ser Ser Gln Gln Gly Arg Gln Gln Glu Gln 1 5
10 15 49515PRTArtificial SequencePrunus dulcis prunin 2 precursor
epitope 495Asp Ser Gln Pro Gln Gln Phe Gln Gln Gln Gln Gln Gln Gln
Gln 1 5 10 15 49611PRTArtificial SequenceHesperocyparis arizonica
putative allergen Cup a 1 epitope 496Trp Arg Phe Thr Arg Asp Ala
Phe Thr Asn Gly 1 5 10 49710PRTArtificial SequenceAspergillus
fumigatus Ribonuclease mitogillin precursor epitope 497Phe Pro Thr
Phe Pro Asp Gly His Asp Tyr 1 5 10 49812PRTArtificial
SequenceMangifera indica ripening-related pectate lyase epitope
498Ala Tyr Asn His Phe Gly Glu Gly Leu Ile Gln Arg 1 5 10
49910PRTArtificial SequenceHevea brasiliensis Rubber elongation
factor protein epitope 499Ala Glu Asp Glu Asp Asn Gln Gln Gly Gln 1
5 10 50015PRTArtificial SequenceJuglans regia seed storage protein
epitope 500Asp Asp Asn Gly Leu Glu Glu Thr Ile Cys Thr Leu Arg Leu
Arg 1 5 10 15 50115PRTArtificial SequenceArachis hypogaea seed
storage protein SSP2 epitope 501Cys Gly Leu Arg Ala Pro Gln Arg Cys
Asp Leu Asp Val Glu Ser 1 5 10 15 5028PRTArtificial SequenceGallus
gallus serine (or cysteine) proteinase inhibitor, clade B
(ovalbumin), member 3 epitope 502Arg Pro Asn Ala Thr Tyr Ser Leu 1
5 5038PRTArtificial SequenceGallus gallus Serum albumin epitope
503Gln Ser Arg Ala Thr Leu Gly Ile 1 5 50417PRTArtificial
SequenceBos taurus Serum albumin precursor epitope 504Asp Asp Ser
Pro Asp Leu Pro Lys Leu Lys Pro Asp Pro Asn Thr Leu 1 5 10 15 Cys
50510PRTArtificial SequenceHevea brasiliensis Small rubber particle
protein epitope 505Ala Glu Glu Val Glu Glu Glu Arg Leu Lys 1 5 10
50612PRTArtificial SequenceCryptomeria japonica Sugi basic protein
precursor epitope 506Asp Ala Leu Thr Leu Arg Thr Ala Thr Asn Ile
Trp 1 5 10 50748PRTArtificial SequenceAspergillus fumigatus
Superoxide dismutase epitope 507Tyr Thr Leu Pro Pro Leu Pro Tyr Pro
Tyr Asp Ala Leu Gln Pro Tyr 1 5 10 15 Ile Ser Gln Gln Ile Met Glu
Leu His His Lys Lys His His Gln Thr 20 25 30 Tyr Val Asn Gly Leu
Asn Ala Ala Leu Glu Ala Gln Lys Lys Ala Ala 35 40 45
50820PRTArtificial SequenceTrichophyton rubrum Tri r 2 allergen
epitope 508Asp Cys Asn Gly His Gly Thr His Val Ala Gly Thr Val Gly
Gly Thr 1 5 10 15 Lys Tyr Gly Leu 20 50910PRTArtificial
SequenceTriticum aestivum Triticum aestivum proteins epitope 509Leu
Pro Gln Gln Gln Ile Pro Gln Gln Pro 1 5 10 5109PRTArtificial
SequencePenaeus tropomyosin epitope 510Phe Leu Ala Glu Glu Ala Asp
Arg Lys 1 5 51120PRTArtificial SequenceParalichthys olivaceus type
1 collagen alpha 2 epitope 511Met Lys Gly Leu Arg Gly His Pro Gly
Leu Gln Gly Met Pro Gly Pro 1 5 10 15 Ser Gly Pro Ser 20
51210PRTArtificial SequenceTriticum aestivum type 1 non-specific
lipid transfer protein precursor epitope 512Ala Arg Gly Thr Pro Leu
Lys Cys Gly Val 1 5 10 51318PRTArtificial SequenceAnisakis simplex
UA3-recognized allergen epitope 513Met Cys Gln Cys Val Gln Lys Tyr
Gly Thr Glu Phe Cys Lys Lys Arg 1 5 10 15 Leu Ala 5148PRTArtificial
SequenceJuglans nigra vicilin seed storage protein epitope 514Ser
Phe Glu Asp Gln Gly Arg Arg 1 5 51515PRTArtificial
SequenceAnacardium occidentale Vicilin-like protein epitope 515Ala
Ile Met Gly Pro Pro Thr Lys Phe Ser Phe Ser Leu Phe Leu 1 5 10 15
51610PRTArtificial SequenceJuglans regia vicilin-like protein
precursor epitope 516Asp Gln Arg Ser Gln Glu Glu Arg Glu Arg 1 5
10
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