U.S. patent application number 17/142825 was filed with the patent office on 2021-05-20 for diagnostic tests and methods for assessing safety, efficacy or outcome of allergen-specific immunotherapy (sit).
The applicant listed for this patent is REGENERON PHARMACEUTICALS, INC.. Invention is credited to Jamie M. ORENGO, George D. YANCOPOULOS.
Application Number | 20210148929 17/142825 |
Document ID | / |
Family ID | 1000005373976 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210148929 |
Kind Code |
A1 |
YANCOPOULOS; George D. ; et
al. |
May 20, 2021 |
DIAGNOSTIC TESTS AND METHODS FOR ASSESSING SAFETY, EFFICACY OR
OUTCOME OF ALLERGEN-SPECIFIC IMMUNOTHERAPY (SIT)
Abstract
The present invention provides in vitro and in vivo diagnostic
tests and methods for determining the safety, efficacy, or outcome
of allergen-specific immunotherapy (SIT) in a patient. The present
invention also provides for the measurement of allergen specific
IgG and IgE in a patient tissue sample, or extract thereof, or in a
biological fluid or blood sample, and determining whether the
allergen-specific immunoglobulins contained in the patient
sample(s), upon injection into an allergen-sensitized animal, will
protect the animal following challenge with the allergen. The
invention also provides methods for determining whether a patient
suffering from an allergy is responsive to therapy with one or more
therapeutic antibodies specific for the allergen.
Inventors: |
YANCOPOULOS; George D.;
(Yorktown Heights, NY) ; ORENGO; Jamie M.;
(Cortlandt Manor, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENERON PHARMACEUTICALS, INC. |
Tarrytown |
NY |
US |
|
|
Family ID: |
1000005373976 |
Appl. No.: |
17/142825 |
Filed: |
January 6, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14913645 |
Feb 22, 2016 |
10935554 |
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PCT/US2014/052295 |
Aug 22, 2014 |
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17142825 |
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61869214 |
Aug 23, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/24 20130101;
G01N 2800/52 20130101; G01N 33/5088 20130101; A61K 49/0008
20130101; G01N 33/6854 20130101; G01N 33/502 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/50 20060101 G01N033/50; A61K 49/00 20060101
A61K049/00 |
Claims
1-65. (canceled)
66. A method of determining if a patient suffering from an allergy
to an allergen is responsive to therapy with one or more antibodies
or antigen binding fragments thereof specific for the allergen, the
method comprising: (a) collecting a sample of tissue or an extract
thereof, or a biological fluid, or a blood sample from the patient;
(b) extracting an allergen-specific IgE from one or more of the
patient samples of (a); (c) mixing the IgE from the patient sample
with the allergen and with one or more antibodies or antigen
binding fragments thereof specific for the allergen; and (d)
determining if the addition of the antibodies or antigen binding
fragments thereof specific for the allergen blocks the binding of
the allergen specific IgE from step b) to the allergen, wherein the
ability of the one or more antibodies or antigen binding fragments
thereof specific for the allergen to block the binding of allergen
specific IgE from step b) to the allergen is indicative that the
patient suffering from an allergy is responsive to therapy with the
one or more antibodies specific for the allergy.
67. (canceled)
68. The method of claim 66, wherein the allergen is Fel d1.
69. The method of claim 66, wherein the one or more antibodies
specific for the allergen are human monoclonal antibodies that bind
specifically to Fel d1, and wherein the one or more antibodies
comprise the heavy chain complementary determining regions (HCDRs)
of a heavy chain variable region (HCVR) comprising the amino acid
sequence of SEQ ID NO: 2 or 18 and the light chain complementarity
determining regions (LCDRs) of a light chain variable region (LCVR)
comprising the amino acid sequence of SEQ ID NO: 10 or 26.
70. The method of claim 66, wherein the one or more human
monoclonal antibodies or antigen binding fragments that bind
specifically to Fel d1 comprise three HCDRs (HCDR1, HCDR2 and
HCDR3) and three LCDRs (LCDR1, LCDR2 and LDR3), wherein the HCDR1
comprises the amino acid sequence of SEQ ID NO: 2 or 20; the HCDR2
comprises the amino acid sequence of SEQ ID NO: 6 or 22; the HCDR3
comprises the amino acid sequence of SEQ ID NO: 8 or 24; the LCDR1
comprises the amino acid sequence of SEQ ID NO: 12 or 28; the LCDR2
comprises the amino acid sequence of SEQ ID NO: 14 or 30; and the
LCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or 32.
71. The method of claim 66, wherein the one or more human
monoclonal antibodies that bind specifically to Fel d1 comprises a
HCVR comprising the amino acid sequence of SEQ ID NO: 2 or 18 and a
LCVR comprising the amino acid sequence of SEQ ID NO: 10 or 26.
72. The method of claim 66, wherein the one or more human
monoclonal antibodies that bind specifically to Fel d1 comprise the
HCVR/LCVR amino acid sequence pairs of SEQ ID NOs: 2/10 or SEQ ID
NOs:18/26.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to diagnostic tests and assay
methods for determining the safety, efficacy, or outcome of
allergen-specific immunotherapy.
BACKGROUND
[0002] Allergies and allergic diseases are serious medical
conditions with consequences ranging from non-life threatening
responses that resolve over time to life threatening effects such
as anaphylaxis. Allergic reactions can result from contact or
exposure to a variety of products such as certain food items,
insect venom, plant-derived material (e.g., pollen), chemicals,
drugs/medications, and animal dander.
[0003] Current treatment options for allergies include avoidance of
the allergen where possible, the use of anti-allergy medications to
treat the symptoms (e.g. antihistamines and corticosteroids) and
prophylaxis using allergen-specific immunotherapy (SIT). These
current treatment strategies are often inadequate, costly,
impractical or involve significant risk. For example, avoidance of
allergen is not always possible and can negatively impact on
patient and caregiver quality of life. Medications used to treat
allergies ameliorate the symptoms, but do not stop the progression.
The only therapy that modifies progression of allergies is
allergen-specific immunotherapy (SIT). However, SIT has several
disadvantages in that it requires numerous allergen administrations
over a 3 to 5 year period and can cause severe adverse events that
range from local allergic reactions to anaphylaxis (Hedlin, et al.
(1991), J. Allergy Clin. Immunol. 87:955-64; Hedlin, et al.,
(1986), J. Allergy Clin. Immunol. 77:488-96).
[0004] Given the potential risks associated with allergen-specific
immunotherapy, it would be beneficial to be able to assess the
safety, efficacy, or outcome of allergen-specific immmunotherapy
during the treatment period and/or at the time of completion of
therapy. Such a test or assay method would provide information as
to whether the patient is responding to treatment, and as such, it
may also allow for a determination as to whether the patient will
be adequately protected by SIT. Such a test or assay method would
also aid in the determination of when a patient can initiate or
terminate maintenance therapy. Currently there are no quantitative
tests that aid in these safety or efficacy determinations.
Accordingly, an unmet need exists in the art for development of
such a diagnostic test, or an assay method for determining the
safety, efficacy, or outcome of allergen-specific
immunotherapy.
BRIEF SUMMARY OF THE INVENTION
[0005] In a first aspect the present invention provides a
diagnostic test for determining the safety, efficacy, or outcome of
allergen-specific immunotherapy (SIT) in a patient, the test
comprising: [0006] (a) obtaining a tissue sample, or an extract
thereof, a biological fluid, or a blood sample from a patient
undergoing allergen-specific immunotherapy (SIT); [0007] (b)
quantitating total IgG, allergen-specific IgG, and
allergen-specific IgE from any one or more of the samples of (a);
[0008] (c) reacting the allergen-specific IgG from step (b) with
the allergen for which the patient is undergoing SIT, plus
allergen-specific IgE; and [0009] (d) measuring either [0010] (i)
the amount of allergen-specific IgG in the tissue sample, or an
extract thereof, in the biological fluid, or in the blood sample
obtained from the patient that is bound to the allergen, or [0011]
(ii) the amount of allergen-specific IgE displaced or prevented
from binding to allergen by the allergen-specific IgG contained in
the tissue sample, or extract thereof, or the biological fluid, or
the blood sample from the patient,
[0012] wherein the amount of allergen-specific IgG in the tissue
sample, or extract thereof, or the biological fluid, or the blood
sample from the patient bound to the allergen is directly
proportional to the safety, efficacy, or outcome of
allergen-specific immunotherapy (SIT) in a patient, or
[0013] wherein the amount of allergen-specific IgE bound to the
allergen and subsequently displaced or prevented from binding to
allergen by the allergen-specific IgG contained in the tissue
sample, or extract thereof, or the biological fluid, or the blood
sample from the patient is directly proportional to the safety,
efficacy, or outcome of allergen-specific immunotherapy (SIT) in a
patient.
[0014] In one embodiment, the diagnostic test may be performed in
vitro or in vivo.
[0015] In one embodiment, the tissue sample obtained from the
patient undergoing SIT may be any tissue sample, or extract
thereof, biological fluid, or blood sample containing
immunoglobulin expressing cells.
[0016] In a related embodiment, the tissue sample obtained from the
patient undergoing SIT may be a whole blood sample, or serum, or
plasma.
[0017] In one embodiment, the binding of allergen-specific IgG from
the patient's tissue sample, or extract thereof, or biological
fluid, or blood sample, to the allergen, or the amount of
allergen-specific IgE displaced or prevented from binding to
allergen by the allergen-specific IgG in the patient's tissue
sample, or extract thereof, or biological fluid, or blood sample is
determined by an in vitro method selected from the group consisting
of an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay
(RIA), an immunoradiometric assay (IRMA), a luminescence
immunoassay (LIA), an immunoblot, FACs analysis, an IgE-facilitated
allergen binding (FAB) assay, and an assay using an engineered cell
line expressing Fc.epsilon.R1.
[0018] In one embodiment, the diagnostic test may be performed in
vivo in an allergen-specific animal model, wherein the binding of
allergen-specific IgG from the patient's tissue sample, or extract
thereof, or biological fluid, or blood sample, to the allergen, or
the amount of allergen-specific IgE displaced or prevented from
binding to allergen by the allergen-specific IgG in the patient's
tissue sample, or extract thereof, or biological fluid, or blood
sample is determined.
[0019] In one embodiment, the in vivo diagnostic test is performed
in an animal model of passive cutaneous anaphylaxis (PCA).
[0020] In a related aspect the invention provides a diagnostic test
for determining the safety, efficacy, or outcome of
allergen-specific immunotherapy (SIT) in a patient, the test
comprising: [0021] (a) a tissue sample, or extract thereof, a
biological fluid, or a blood sample from a patient who is
undergoing, or has completed allergen-specific immunotherapy;
[0022] (b) an allergen sample that corresponds to the allergen for
which the patient is undergoing SIT; [0023] (c) an
allergen-specific IgE; [0024] (d) a receptacle for mixing the
reagents of step (a) through step (c); [0025] (e) reagents for
measuring either the amount of allergen-specific IgG in the
patient's tissue sample, or extract thereof, or biological fluid,
or blood sample bound to the allergen, or for measuring the amount
of allergen-specific IgE displaced or prevented from binding to
allergen by the allergen-specific IgG contained in the patient
tissue sample, or extract thereof, or biological fluid, or blood
sample after mixing a sample from (a) with the reagents of (b) and
(c); and [0026] (f) directions for measuring the amount of
allergen-specific IgG bound to the allergen, or for measuring the
amount of allergen-specific IgE displaced or prevented from binding
to allergen by the allergen-specific IgG contained in the patient
tissue sample, or extract thereof, or biological fluid, or blood
sample,
[0027] wherein the amount of allergen-specific IgG contained in the
patient tissue sample, or extract thereof, or biological fluid, or
blood sample of (a) bound to the allergen is directly proportional
to the safety, efficacy, or outcome of allergen-specific
immunotherapy (SIT) in a patient, or
[0028] wherein the amount of allergen-specific IgE bound to the
allergen and subsequently displaced or prevented from binding to
allergen by the allergen-specific IgG contained in the patient
tissue sample, or extract thereof, or biological fluid, or blood
sample of (a) is directly proportional to the safety, efficacy, or
outcome of allergen-specific immunotherapy (SIT) in a patient.
[0029] In a related aspect the invention provides a test kit for
determining if a patient is responsive to allergen-specific
immunotherapy (SIT), the kit comprising:
[0030] (a) a first reagent containing the allergen for which
allergen-specific immunotherapy is being administered;
[0031] (b) a second reagent containing allergen-specific IgE;
[0032] (c) third reagent containing an allergen-specific IgG as a
known positive standard;
[0033] (d) reagents for measuring the amount of allergen-specific
IgG or IgE;
[0034] (e) a receptacle for collecting a tissue sample, or extract
thereof, a biological fluid, or a blood sample from a patient
undergoing SIT, or who has completed SIT; and
[0035] (f) instructions for use of the kit.
[0036] In one embodiment, the first reagent in the test kit is
provided on a solid phase support.
[0037] In one embodiment, the second reagent in the test kit is
provided on a solid phase support.
[0038] In one embodiment the first reagent in the test kit may
contain a detectable label.
[0039] In one embodiment the second reagent in the test kit may
contain a detectable label.
[0040] In one embodiment, the second reagent in the test kit is an
allergen-specific IgE containing a detectable label.
[0041] In one embodiment, IgE from a cat allergic patient is
captured on a solid support and labeled allergen is mixed together
with SIT IgG and the amount of allergen not bound by SIT IgG is
detected after binding to the IgE on the solid support.
[0042] In one embodiment, the detectable label is selected from the
group consisting of a fluorescence label, a radiolabel, an enzyme
label, a luminescent label, an electrochemical, or a visual
label.
[0043] In one embodiment, the diagnostic tests and test kits may be
useful for measuring a response in a patient undergoing SIT for an
allergen selected from the group consisting of an animal product, a
food allergen, plant pollen, mold spores, house dust mites,
cockroaches, perfume, detergents, household cleaners, latex, a drug
product, or insect venom.
[0044] In one embodiment, the animal product is selected from the
group consisting of animal fur, animal dander, wool, and mite
excretions.
[0045] In one embodiment, the animal product contains the allergen
Fel d1.
[0046] In one embodiment, the animal product contains the allergen
can f1, can f2, can f3, can f4, can f5 or can f6.
[0047] In one embodiment, the food allergen is selected from the
group consisting of eggs, meat, fruit, legumes, milk or other dairy
products, seafood, sesame, soy, wheat, oat, barley, celery and
celeriac, corn or maize and tree nuts.
[0048] In one embodiment, the legumes are selected from the group
consisting of peanuts, beans, peas and soybeans.
[0049] In one embodiment, the tree nuts are selected from the group
consisting of pecans, almonds, cashews, hazelnuts (filberts),
walnuts, brazil nuts, macadamia nuts, chestnuts, pine nuts and
pistachio nuts.
[0050] In one embodiment, the plant pollen is selected from the
group consisting of grass pollen, weed pollen and tree pollen.
[0051] In one embodiment, the tree pollen is selected from the
group consisting of birch pollen, cedar pollen, oak pollen, alder
pollen, hornbeam pollen, aesculus pollen, willow pollen, poplar
pollen, plantanus pollen, tilia pollen, olea pollen, Ashe juniper
pollen, and Alstonia scholaris pollen.
[0052] In one embodiment, the birch pollen contains the allergen
Betv 1.
[0053] In one embodiment, the cedar pollen contains the allergen
Cryj1 or Cryj2.
[0054] In one embodiment, the grass pollen is ryegrass or
timothy-grass.
[0055] In one embodiment, the weed pollen is selected from the
group consisting of ragweed, plantago, nettle, Artemisia vulgaris,
Chenopodium album and sorrel.
[0056] In one embodiment, the insect venom is produced by bees,
wasps or fire ants.
[0057] In one embodiment, the allergen-specific IgE used in the
diagnostic test may be selected from the group consisting of an
allergen-specific IgE obtained from the patient undergoing SIT, an
allergen-specific IgE obtained from another allergen-injected
mammal, and a recombinant IgE specific for the allergen.
[0058] In one embodiment, the steps of any of the diagnostic tests
described above may further include injecting a sample of the
patient's tissue sample or extract thereof, or biological fluid, or
blood sample into an allergen-specific animal model to assess the
protective efficacy of the allergen-specific IgG from the patient,
wherein protection of the animal following challenge with the
allergen is indicative of the safety, efficacy or outcome of SIT in
the patient.
[0059] In one embodiment, the animal model is a mouse model of
Passive Cutaneous Anaphylaxis (PCA) and wherein the model comprises
the following steps: [0060] (a) injecting the animal with
allergen-specific IgE, or antiserum containing allergen-specific
IgE, intradermally at one skin site and injecting the animal with
non-allergen-specific IgE or antiserum containing
non-allergen-specific IgE intradermally at a second different skin
site; [0061] (b) injecting the animal systemically with the
allergen for which the patient is undergoing, or has undergone SIT
therapy, along with a dye; and [0062] (c) assessing the extent of
dye extravasation at the site of allergen injection;
[0063] wherein the amount of dye extravasated into the tissue is
directly related to the amount of mast cell activation in the
animal and wherein a decrease in the amount of dye extravasated
into the tissue is indicative of safety, efficacy, or a positive
outcome of allergen-specific immunotherapy in the patient.
[0064] A second aspect of the invention provides a method for
assessing the safety, efficacy, or outcome of allergen-specific
immunotherapy, the method comprising assessing the level of
allergen-specific IgG in a patient tissue sample, or extract
thereof, or biological fluid, or blood sample using any of the
diagnostic tests or test kits noted herein, followed by
confirmation of the in vitro results by measuring the protective
efficacy of the allergen-specific IgG from the patient in an
allergen-specific animal model, wherein elevated levels of
allergen-specific IgG from the patient's serum and protection of
the animal following challenge with the allergen is indicative of
safety, efficacy, or a positive outcome of SIT.
[0065] In a related aspect, the invention provides a direct in vivo
method for assessing the safety, efficacy, or outcome of
allergen-specific immunotherapy (SIT) in a patient, the method
comprising injecting a sample containing allergen-specific IgG from
the patient into an allergen-specific animal model to determine the
protective efficacy of the allergen-specific IgG from the patient,
without the need to assess the patient's tissue sample, or
biological fluid sample or blood sample in an in vitro assay prior
to the in vivo animal model, wherein protection of the animal
following challenge with the allergen is indicative of safety,
efficacy, or a positive outcome of allergen-specific immunotherapy
in the patient.
[0066] Any allergy animal model known to those skilled in the art
to assess the safety, efficacy or outcome of SIT may be used,
although in one embodiment, the animal model that is used herein to
assess safety, efficacy, or the outcome of SIT is a mouse model of
Passive Cutaneous Anaphylaxis (PCA) and wherein the model comprises
the following steps: [0067] (a) injecting the animal with
allergen-specific IgE, or antiserum containing allergen-specific
IgE, intradermally at one skin site and injecting the animal with
non-allergen-specific IgE or antiserum containing
non-allergen-specific IgE intradermally at a second different skin
site; [0068] (b) injecting the animal systemically with the
allergen for which the patient is undergoing, or has undergone SIT
therapy, along with a dye; and [0069] (c) assessing the extent of
dye extravasation at the site of allergen injection;
[0070] wherein the amount of dye extravasated into the tissue is
directly related to the amount of mast cell activation in the
animal and wherein a decrease in the amount of dye extravasated
into the tissue is indicative of safety, efficacy, or a positive
outcome of allergen-specific immunotherapy in the patient.
[0071] In one embodiment, the methods for assessing safety,
efficacy, or outcome of SIT may be used to assess patients
receiving such therapy for an allergen selected from the group
consisting of an animal product, a food allergen, plant pollen,
mold spores, house dust mites, cockroaches, perfume, detergents,
household cleaners, latex, a drug product, or insect venom.
[0072] In one embodiment, the animal product is selected from the
group consisting of animal fur, animal dander, wool, and mite
excretions.
[0073] In one embodiment, the animal product contains the allergen
Fel d1.
[0074] In one embodiment, the animal product contains the allergen
can f1, can f2, can f3, can f4, can f5 or can f6.
[0075] In one embodiment, the food allergen is selected from the
group consisting of eggs, meat, fruit, legumes, milk or other dairy
products, seafood, sesame, soy, wheat, oat, barley, celery and
celeriac, corn or maize and tree nuts.
[0076] In one embodiment, the legumes are selected from the group
consisting of peanuts, beans, peas and soybeans.
[0077] In one embodiment, the tree nuts are selected from the group
consisting of pecans, almonds, cashews, hazelnuts (filberts),
walnuts, brazil nuts, macadamia nuts, chestnuts, pine nuts and
pistachio nuts.
[0078] In one embodiment, the plant pollen is selected from the
group consisting of grass pollen, weed pollen and tree pollen.
[0079] In one embodiment, the tree pollen is selected from the
group consisting of birch pollen, cedar pollen, oak pollen, alder
pollen, hornbeam pollen, aesculus pollen, willow pollen, poplar
pollen, plantanus pollen, tilia pollen, olea pollen, Ashe juniper
pollen, and Alstonia scholaris pollen.
[0080] In one embodiment, the birch pollen contains the allergen
Betv 1.
[0081] In one embodiment, the cedar pollen contains the allergen
Cryj1 or Cryj2.
[0082] In one embodiment, the grass pollen is ryegrass or
timothy-grass.
[0083] In one embodiment, the weed pollen is selected from the
group consisting of ragweed, plantago, nettle, Artemisia vulgaris,
Chenopodium album and sorrel.
[0084] In one embodiment, the insect venom is produced by bees,
wasps or fire ants.
[0085] In a related aspect, the invention provides for determining
if allergen-specific immunotherapy induces allergen-specific
immunoglobulins that are protective in a mammal upon challenge of
the mammal with the allergen, the method comprising measuring the
level of allergen-specific IgG in a patient tissue sample, or
extract thereof, or biological fluid, or blood sample using any of
the diagnostic tests or test kits described herein, and assessing
the protective efficacy of the allergen-specific IgG from the
patient tissue sample, or extract thereof, or biological fluid, or
blood sample in an allergen-specific animal model, such as the PCA
model described herein and in Example 1, wherein a decrease in mast
cell degranulation as evidenced in this model indicates that the
allergen-specific immunoglobulins generated during SIT are
protective against subsequent challenge with the allergen and that
the allergen-specific immunotherapy was effective.
[0086] A third aspect of the invention provides a method of
screening a patient to determine if the patient has responded to
allergen specific immunotherapy (SIT), or will be adequately
protected by SIT, or for determining when a patient can initiate or
terminate maintenance therapy, the method comprising measuring the
level of allergen-specific IgG in a patient tissue sample, or
extract thereof, or biological fluid, or blood sample using any of
the diagnostic tests or test kits described herein, and assessing
the protective efficacy of the allergen-specific IgG from the
patient tissue sample, or extract thereof, or biological fluid, or
blood sample in an allergen-specific animal model, wherein elevated
levels of allergen-specific IgG from the patient's tissue sample,
or extract thereof, or biological fluid, or blood sample and
protection of the animal following challenge with the allergen is
indicative that the patient has responded to allergen specific
immunotherapy (SIT), or will be adequately protected by SIT, or
that the patient can initiate or terminate maintenance therapy.
[0087] A fourth aspect of the invention provides a method of
determining if a patient suffering from an allergy is responsive to
therapy with one or more antibodies or antigen binding fragments
thereof specific for the allergen, the method comprising: [0088] a)
collecting a sample of tissue or an extract thereof, or a
biological fluid, or a blood sample from a patient suffering from
an allergy; [0089] b) extracting allergen specific IgE from any one
or more of the patient samples of (a); [0090] c) mixing the IgE
from the patient sample with the allergen and with one or more
antibodies or antigen binding fragments thereof specific for the
allergen; and [0091] d) determining if the addition of the
antibodies or antigen binding fragments thereof specific for the
allergen blocks the binding of the allergen specific IgE from step
b) to the allergen,
[0092] wherein the ability of the one or more antibodies or antigen
binding fragments thereof specific for the allergen to block the
binding of allergen specific IgE from step b) to the allergen is
indicative that the patient suffering from an allergy is responsive
to therapy with the one or more antibodies specific for the
allergy.
[0093] In one embodiment, the allergen is a cat allergen.
[0094] In one embodiment, the cat allergen is Fel d1.
[0095] In one embodiment, the one or more antibodies specific for
the allergen are human monoclonal antibodies that bind specifically
to Fel d1, wherein the one or more antibodies comprise the heavy
chain complementary determining regions (HCDRs) of a heavy chain
variable region (HCVR) comprising the amino acid sequence of SEQ ID
NO: 2 or 18 and the light chain complementarity determining regions
(LCDRs) of a light chain variable region (LCVR) comprising the
amino acid sequence of SEQ ID NO: 10 or 26.
[0096] In one embodiment, the one or more human monoclonal
antibodies or antigen binding fragments that bind specifically to
Fel d1 comprise three HCDRs (HCDR1, HCDR2 and HCDR3) and three
LCDRs (LCDR1, LCDR2 and LDR3), wherein the HCDR1 comprises the
amino acid sequence of SEQ ID NO: 4 or 20; the HCDR2 comprises the
amino acid sequence of SEQ ID NO: 6 or 22; the HCDR3 comprises the
amino acid sequence of SEQ ID NO: 8 or 24; the LCDR1 comprises the
amino acid sequence of SEQ ID NO: 12 or 28; the LCDR2 comprises the
amino acid sequence of SEQ ID NO: 14 or 30; and the LCDR3 comprises
the amino acid sequence of SEQ ID NO: 16 or 32.
[0097] In one embodiment, the one or more human monoclonal
antibodies that bind specifically to Fel d1 comprises a HCVR
comprising the amino acid sequence of SEQ ID NO: 2 or 18 and a LCVR
comprising the amino acid sequence of SEQ ID NO: 10 or 26.
[0098] In one embodiment, the one or more human monoclonal
antibodies that bind specifically to Fel d1 comprise the HCVR/LCVR
amino acid sequence pairs of SEQ ID NOs: 2/10 or SEQ ID
NOs:18/26.
[0099] In one embodiment, a composition comprising two anti-Fel d1
monoclonal antibodies may be used for treating cat allergic
patients, wherein the two Fel d1 human monoclonal antibodies have
HCVR/LCVR amino acid sequence pairs of SEQ ID NOs: 2/10 and SEQ ID
NOs:18/26. The diagnostic tests and methods described herein may be
used to determine if a patient suffering from a cat allergy is
responsive to therapy with these antibodies, or with other
antibodies that have the same or a similar binding specificity.
[0100] In one embodiment, the results obtained from the in vitro
study noted above, may be further confirmed in an animal allergy
model, such as the Passive Cutaneous Anaphylaxis (PCA) model.
[0101] This model comprises the following steps: [0102] (a)
injecting the animal with allergen-specific IgE, or antiserum
containing allergen-specific IgE, intradermally at one skin site
and injecting the animal with non-allergen-specific IgE or
antiserum containing non-allergen-specific IgE intradermally at a
second different skin site; [0103] (b) injecting the animal
systemically with the allergen for which the patient is undergoing,
or has undergone SIT therapy, along with a dye; and [0104] (c)
assessing the extent of dye extravasation at the site of allergen
injection;
[0105] wherein the amount of dye extravasated into the tissue is
directly related to the amount of mast cell activation in the
animal and wherein a decrease in the amount of dye extravasated
into the tissue is indicative of safety, efficacy, or a positive
outcome of allergen-specific immunotherapy in the patient.
BRIEF DESCRIPTION OF THE FIGURES
[0106] FIG. 1. Panel (A) depicts a schematic of a peanut specific
immunotherapy mouse model that was used to generate peanut specific
antisera containing peanut specific IgG. Panel B shows the extent
of anaphylaxis in two groups of experimental mice as a means to
determine the efficacy of the antigen specific immunotherapy. This
is assessed in terms of a core temperature decrease. Mice receiving
no SIT are designated black dashes with squares; mice not
undergoing SIT are designated with grey triangles.
[0107] FIG. 2. Shows the results of a blocking assay for Fel d 1
binding to allergic IgE (donor 1) by anti-Fel d 1 IgG elicited by
SIT.
[0108] FIG. 3. Shows the results of a blocking assay for Fel d 1
binding to allergic IgE (donor 2) by anti-Fel d 1 IgG elicited by
SIT.
[0109] FIG. 4. Shows the results of a blocking assay for Fel d 1
binding to allergic IgE (mouse sera immunized with Fel d 1) by
anti-Fel d 1 IgG elicited by SIT.
[0110] FIG. 5. Shows the results of a blocking assay for Fel d 1
binding to human allergic IgE by anti-Fel d 1 mouse sera elicited
by immunization with Fel d 1.
DETAILED DESCRIPTION
[0111] Before the present invention is described, it is to be
understood that this invention is not limited to particular methods
and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
[0112] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. As used
herein, the term "about," when used in reference to a particular
recited numerical value, means that the value may vary from the
recited value by no more than 1%. For example, as used herein, the
expression "about 100" includes 99 and 101 and all values in
between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
[0113] Although any methods and materials similar or equivalent to
those described herein can be used in the practice of the present
invention, the preferred methods and materials are now described.
All patents, applications and non-patent publications mentioned in
this specification are incorporated herein by reference in their
entireties. Other embodiments will become apparent from a review of
the ensuing detailed description.
Definitions
[0114] As used herein, the expressions "allergen-specific
immunotherapy," "specific immunotherapy," "SIT," "SIT regimen," and
the like, refer to the repeated administration of an allergen to a
subject over time as means for treating or preventing allergies and
allergic reactions, or to reduce or eliminate allergic responses.
Subcutaneous immunotherapy uses a protocol of weekly injections
with gradually increasing dosages of allergen extract until
amaintenance dose is achieved. Subsequently the maintenance dose
administration is reduced to biweekly and then to monthly intervals
for a period of 3 to 5 years (See J. Allergy Clin. Immunol. (2007),
120:S25-85). More recent studies have been conducted to administer
the allergen sublingually.
[0115] To measure the "safety", "efficacy" or "outcome" of SIT,
various clinical measurements have historically been utilized. The
"safety" of SIT generally takes into account any untoward allergic
reaction to the allergen being administered. The allergic reaction
may be mild or may be severe following allergen administration. The
"efficacy" or "outcome" of SIT takes into account several clinical
parameters, including a measurement of symptoms (with emphasis on
alleviating the symptoms associated with the particular allergen
exposure) and the need for concomitant medications. Both physician
rated and patient self rated scores have been implemented in
clinical trials. The use of questionnaires for evaluating the
quality of life are generally used as secondary outcome parameters.
Skin prick tests are also utilized as a means of assessing
efficacy/outcome of therapy.
[0116] The term "allergen," as used herein, includes any substance,
chemical, particle or composition which is capable of stimulating
an allergic response in a susceptible individual. Allergens may be
contained within or derived from a food item such as, e.g., dairy
products (e.g., cow's milk), egg, wheat, soy, fish, shellfish,
peanuts and tree nuts. Alternatively, an allergen may be contained
within or derived from a non-food item such as, e.g., dust (e.g.,
containing dust mite), pollen, insect venom (e.g., venom of bees,
wasps, mosquitos, etc.), mold, animal dander, latex, medication,
drugs, ragweed, grass and birch.
[0117] As used herein, the phrases "allergic response," "allergic
reaction," "allergic symptom," and the like, include one or more
signs or symptoms selected from the group consisting of urticaria
(e.g., hives), angioedema, rhinitis, asthma, vomiting, sneezing,
runny nose, sinus inflammation, watery eyes, wheezing,
bronchospasm, reduced peak expiratory flow (PEF), gastrointestinal
distress, flushing, swollen lips, swollen tongue, reduced blood
pressure, anaphylaxis, and organ dysfunction/failure. An "allergic
response," "allergic reaction," "allergic symptom," etc., also
includes immunological responses and reactions such as, e.g.,
increased IgE production and/or increased allergen-specific
immunoglobulin production.
[0118] The term "up-dosing" means a period of treatment during
which the doses of allergen administered are gradually increased to
reach a full dose level, which is used in the following maintenance
phase, and the up-dosing phase ends when the full dose level is
reached, ie. immediately subsequent to the administration of the
first full dose.
[0119] "Maintenance phase" or "maintenance therapy" means a period
of treatment in continuation of the up-dosing phase and during
which a full dose of allergen is administered, the maintenance
phase starting immediately subsequent to the administration of the
first full dose.
[0120] "Mast cell", as used herein refers to one or more mast
cells, basophils, and other cells with IgE receptors.
[0121] "Sensitized animal" refers to an animal having adapted an
immunological state so that, when it encounters an antigen, it has
a response similar to that observed in allergic humans. This may
include a reaction to an allergen resulting in any one or more of
the following symptoms and/or reactions: cutaneous reactions,
wheezing and labored respiration, a higher percentage of
degranulated mast cells, increased histamine levels upon challenge
with allergen, an increase in the level of antigen specific IgG in
the animal after sensitization, an increase in allergen specific
IgE levels, or an anaphylactic reaction. "Desensitize" is defined
herein as to decrease the allergic-reactivity of an
allergen-sensitive individual to exposure to the allergen, (e.g.,
to a level less than that which the allergen-sensitive individual
would otherwise experience).
[0122] The "Passive Cutaneous Anaphylaxis" or "PCA" animal model is
a model used to assess in vivo mast cell degranulation (See
Bradley, et al. (1991), J. Allergy Clin. Immunol. 88:661-74). The
model relies on the passive transfer of antisera containing
allergen-specific polyclonal IgE, which binds to Fc.epsilon.R1 on
mast cells near the site of administration. The animals are
effectively "sensitized" locally to the specific antigen. When
antigen is subsequently injected systemically as an allergen
challenge along with Evan's blue dye, antigen-induced mast cell
degranulation results in rapid capillary dilation and increased
vascular permeability that can be visualized and quantitated by
measuring dye leakage into the reaction site.
[0123] As used herein, the terms "treat", "treating", or the like,
mean to alleviate symptoms, eliminate the causation of symptoms
either on a temporary or permanent basis, or to prevent or slow the
appearance of symptoms of an allergic reaction.
[0124] As used herein, the term "a subject in need thereof" means
any human or non-human animal who: (a) is prone to allergic
reactions or responses when exposed to one or more allergens; (b)
has previously exhibited an allergic response or reaction to one or
more allergens; (c) has a known history of allergies; and/or (d)
exhibits a sign or symptom of an allergic response or
anaphylaxis.
[0125] The term "patient sample" may include any tissue sample,
including both solid tissue (or extracts thereof), biological
fluids, or blood samples. The blood sample may be whole blood,
plasma or serum. The tissue sample or extract thereof, or
biological fluid may be any tissue sample or bodily fluid that
contains immunoglobulin expressing cells.
[0126] The term "Fel d1", "Fel d 1", or "FELD1", as used herein,
refers to at least one Fel d1 protein, either in natural/native
form, or recombinantly produced. The Fel d1 protein comprises, or
alternatively consists of, chain 1 (also referred to as chain A) of
Fel d1 (SEQ ID NO: 33) and chain 2 (also referred to as chain B) of
Fel d1 (SEQ ID NO: 34). The natural Fel d1 protein is an
approximately 18 kDa heterodimeric glycoprotein composed of two
chains derived from two independent genes (See Duffort, O. A. et
al., (1991), Mol. Immunol. 28:301-309; Kristensen, A. K. et al.,
(1997), Biol. Chem. 378:899-908; Kaiser L. et al. (2003), J. Biol.
Chem. 278(39):37730-37735). The amino acid sequence of chain 1 of
Fel d1 is also provided in GenBank as accession number P30438, or
as accession number NP_001041618.1, which also include the signal
peptide which is removed in the mature protein. The amino acid
sequence of chain 2 of Fel d1 is also provided in GenBank as
accession number P30440, or as accession number NP_001041619.1,
which include the signal peptide which is removed in the mature
protein. A recombinant form of Fel d1 produced with a C-terminal
myc-myc-hexahistidine tag (Fel d1mmH) is shown as SEQ ID NO: 52,
wherein the Fel d1 B chain and Fel d1 A chain are linked as a
continuous, in-line fusion with Fel d1 at the N terminus and the
myc-myc-his tag at the C terminus. Another recombinant form of Fel
d1 is produced with a C-terminal mouse Fc tag (Fel d1mFc) and is
shown as SEQ ID NO: 53. (See Fel d1 B chain at accession number
NP_001041619.1, amino acids 18-109 and Fel d1 A chain at accession
number NP_001041618.1, amino acids 19-88 with a D27G mutation).
[0127] A human Fc.epsilon.R1.alpha. was produced with a terminal
mouse Fc tag and this is shown as SEQ ID NO: 51. (See also
accession number NP_001992 (V26-E205) with the C terminal mouse
IgG2a Fc tag (98-330 of P01863).
[0128] The term "antibody", as used herein, means any
antigen-binding molecule or molecular complex comprising at least
one complementarity determining region (CDR) that specifically
binds to or interacts with a particular antigen (e.g., Fel d1). The
term "antibody", as used herein, is intended to refer to
immunoglobulin molecules comprised of four polypeptide chains, two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds (i.e., "full antibody molecules"), as well as
multimers thereof (e.g. IgM) or antigen-binding fragments thereof.
Each heavy chain is comprised of a heavy chain variable region
("HCVR" or "V.sub.H") and a heavy chain constant region (comprised
of domains C.sub.H1, C.sub.H2 and C.sub.H3). Each light chain is
comprised of a light chain variable region ("LCVR or "V.sub.L") and
a light chain constant region (C.sub.L). The V.sub.H and V.sub.L
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each V.sub.H and V.sub.L is composed of three CDRs and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments
of the invention, the FRs of the antibody (or antigen binding
fragment thereof) may be identical to the human germline sequences,
or may be naturally or artificially modified. An amino acid
consensus sequence may be defined based on a side-by-side analysis
of two or more CDRs.
[0129] Substitution of one or more CDR residues or omission of one
or more CDRs is also possible. Antibodies have been described in
the scientific literature in which one or two CDRs can be dispensed
with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed
the contact regions between antibodies and their antigens, based on
published crystal structures, and concluded that only about one
fifth to one third of CDR residues actually contact the antigen.
Padlan also found many antibodies in which one or two CDRs had no
amino acids in contact with an antigen (see also, Vajdos et al.
(2002), J Mol Biol 320:415-428).
[0130] CDR residues not contacting antigen can be identified based
on previous studies (for example residues H60-H65 in CDRH2 are
often not required), from regions of Kabat CDRs lying outside
Chothia CDRs, by molecular modeling and/or empirically. If a CDR or
residue(s) thereof is omitted, it is usually substituted with an
amino acid occupying the corresponding position in another human
antibody sequence or a consensus of such sequences. Positions for
substitution within CDRs and amino acids to substitute can also be
selected empirically. Empirical substitutions can be conservative
or non-conservative substitutions.
[0131] The fully human (recombinant, not naturally occurring)
monoclonal antibodies that specifically bind to Fel d1, as
disclosed herein, may comprise one or more amino acid
substitutions, insertions and/or deletions in the framework and/or
CDR regions of the heavy and light chain variable domains as
compared to the corresponding germline sequences. Such mutations
can be readily ascertained by comparing the amino acid sequences
disclosed herein to germline sequences available from, for example,
public antibody sequence databases. The present invention includes
antibodies, and antigen-binding fragments thereof, which are
derived from any of the amino acid sequences disclosed herein,
wherein one or more amino acids within one or more framework and/or
CDR regions are mutated to the corresponding residue(s) of the
germline sequence from which the antibody was derived, or to the
corresponding residue(s) of another human germline sequence, or to
a conservative amino acid substitution of the corresponding
germline residue(s) (such sequence changes are referred to herein
collectively as "germline mutations"). A person of ordinary skill
in the art, starting with the heavy and light chain variable region
sequences disclosed herein, can easily produce numerous antibodies
and antigen-binding fragments which comprise one or more individual
germline mutations or combinations thereof. In certain embodiments,
all of the framework and/or CDR residues within the V.sub.H and/or
V.sub.L domains are mutated back to the residues found in the
original germline sequence from which the antibody was derived. In
other embodiments, only certain residues are mutated back to the
original germline sequence, e.g., only the mutated residues found
within the first 8 amino acids of FR1 or within the last 8 amino
acids of FR4, or only the mutated residues found within CDR1, CDR2
or CDR3. In other embodiments, one or more of the framework and/or
CDR residue(s) are mutated to the corresponding residue(s) of a
different germline sequence (i.e., a germline sequence that is
different from the germline sequence from which the antibody was
originally derived). Furthermore, the antibodies of the present
invention may contain any combination of two or more germline
mutations within the framework and/or CDR regions, e.g., wherein
certain individual residues are mutated to the corresponding
residue of a particular germline sequence while certain other
residues that differ from the original germline sequence are
maintained or are mutated to the corresponding residue of a
different germline sequence. Once obtained, antibodies and
antigen-binding fragments that contain one or more germline
mutations can be easily tested for one or more desired property
such as, improved binding specificity, increased binding affinity,
improved or enhanced antagonistic or agonistic biological
properties (as the case may be), reduced immunogenicity, etc.
Antibodies and antigen-binding fragments obtained in this general
manner are encompassed within the present invention.
[0132] The present invention also includes fully human monoclonal
antibodies comprising variants of any of the HCVR, LCVR, and/or CDR
amino acid sequences disclosed herein having one or more
conservative substitutions. For example, the present invention
includes antibodies having HCVR, LCVR, and/or CDR amino acid
sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or
fewer, etc. conservative amino acid substitutions relative to any
of the HCVR, LCVR, and/or CDR amino acid sequences disclosed
herein.
[0133] The term "human antibody", as used herein, may include
antibodies having variable and constant regions derived from human
germline immunoglobulin sequences. The human mAbs of the invention
may include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo),
for example in the CDRs and in particular CDR3. However, the term
"human antibody", as used herein, is not intended to include mAbs
in which CDR sequences derived from the germline of another
mammalian species (e.g., mouse), have been grafted onto human FR
sequences.
[0134] The antibodies used in the methods of the present invention
may be recombinant, non-naturally occurring human antibodies. The
term "recombinant human antibody," as used herein, is intended to
include all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as antibodies expressed
using a recombinant expression vector transfected into a host cell
(described further below), antibodies isolated from a recombinant,
combinatorial human antibody library (described further below),
antibodies isolated from an animal (e.g., a mouse) that is
transgenic for human immunoglobulin genes (see e.g., Taylor et al.
(1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared,
expressed, created or isolated by any other means that involves
splicing of human immunoglobulin gene sequences to other DNA
sequences. Such recombinant human antibodies have variable and
constant regions derived from human germline immunoglobulin
sequences. In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the V.sub.H and
V.sub.L regions of the recombinant antibodies are sequences that,
while derived from and related to human germline V.sub.H and
V.sub.L sequences, may not naturally exist within the human
antibody germline repertoire in vivo.
Allergen-Specific Immunotherapy (SIT)
[0135] As used herein, the expressions "allergen-specific
immunotherapy," "specific immunotherapy," "SIT," "SIT regimen," and
the like, refer to the repeated administration of an allergen to a
subject over time as means for treating or preventing allergies and
allergic reactions, or to reduce or eliminate allergic responses.
In a typical SIT regimen, small amounts of allergen are initially
administered to an allergic subject, followed by administration of
increased amounts of allergen. In certain instances, the SIT
regimen comprises at least two consecutive phases: (1) an up-dosing
phase, and (2) a maintenance phase. In the up-dosing phase,
increasing doses of allergen are administered until an effective
and safe dose is achieved. The dose that is established at the end
of the up-dosing phase is then administered to the subject
throughout the course of the maintenance phase. The duration of the
up-dosing phase can be several weeks or several months. In certain
embodiments, however, the up-dosing phase is of substantially
shorter duration (e.g., less than one week, less than 6 days, less
than 5 days, less than 4 days, less than 3 days, or less than 2
days). SIT regimens comprising an up-dosing phase of less than 5
days are sometimes referred to as "Rush" immunotherapy or "Rush
SIT." The maintenance phase of an SIT regimen can last several
weeks, several months, several years, or indefinitely.
Methods for Determining the Safety, Efficacy, or Outcome Following
Allergen-Specific Immunotherapy (SIT)
[0136] Historically, variability in safety and clinical efficacy
has limited the widespread application of SIT. However, more recent
studies have attempted to better characterize the active
ingredients by measurement of protein content, determination of
biological activity, estimation of the predominant allergen that
makes up the composition and the production of pure allergen
molecules with the aid of recombinant DNA technology. Safety
concerns using the subcutaneous route of allergen injection
resulted in more recent studies to assess the sublingual route of
allergen delivery and to characterize the immunological response to
the allergen delivered via this route. While it appears that
sublingual SIT is clearly efficacious and is associated with a
favorable safety profile, efficacy appears to be lower than that of
subcutaneous SIT. The better safety profile may be attributed to
the presence of fewer mast cells in the oral mucosa, or to the
delivery of smaller immunologically active doses of allergen,
despite the larger dose that is administered. However, if larger
doses are delivered via the sublingual route, this may alter the
safety profile such that it is similar to SIT using the
subcutaneous route of allergen injection. So the improvement in
clinical efficacy may also result in a reduction in the safety
profile. Because of the safety concerns, as well as non-compliance
associate with SIT, it would be beneficial to be able to assess if
the patient is responding to therapy early in the course of SIT,
and if so, whether the patient may initiate or terminate
maintenance therapy sooner rather than later.
[0137] As a general rule, in order to measure the "safety",
"efficacy" or "outcome" of SIT, various clinical assessments have
historically been utilized. The "safety" of SIT generally takes
into account any untoward allergic reaction to the allergen being
administered. The allergic reaction may be mild after allergen
administration, or it may be severe following allergen
administration. To measure the "efficacy" or "outcome" of SIT,
several clinical parameters are generally taken into account,
including a measurement of symptoms (with an emphasis on
alleviating the symptoms associated with the particular allergen
exposure after immunotherapy is completed) and the need for
concomitant medications. In addition, both physician rated and
patient self rated scores have been implemented in clinical trials.
The use of questionnaires for evaluating the quality of life are
generally used as secondary outcome parameters. Skin prick tests
are also utilized as a means of assessing efficacy/outcome of
therapy. None of the methods employed take into account a
quantitative means of assessing whether the patient is actually
responding to the particular therapy, or if they have responded,
whether the protection attributed to SIT is sufficient or adequate
to confer protection against future allergen exposure.
[0138] One aspect of the present invention provides methods for
determining the safety, efficacy, or outcome of allergen-specific
immunotherapy. The methods comprise obtaining a tissue sample, or
extract thereof, or a biological fluid, or a blood sample from a
patient who is undergoing SIT and using various in vitro and in
vivo assays for assessing whether the patient is responding, or has
responded to such therapy.
[0139] One embodiment of the invention provides a diagnostic test
for determining the safety, efficacy, or outcome of
allergen-specific immunotherapy (SIT) in a patient, the test
comprising: [0140] (a) obtaining a tissue sample, or an extract
thereof, a biological fluid, or a blood sample from a patient
undergoing allergen-specific immunotherapy (SIT); [0141] (b)
quantitating total IgG, allergen-specific IgG, and
allergen-specific IgE from any one or more of the samples of (a);
[0142] (c) reacting the allergen-specific IgG from step (b) with
the allergen for which the patient is undergoing SIT, plus
allergen-specific IgE; and [0143] (d) measuring either [0144] (i)
the amount of allergen-specific IgG in the tissue sample, or an
extract thereof, in the biological fluid, or in the blood sample
obtained from the patient that is bound to the allergen, or [0145]
(ii) the amount of allergen-specific IgE displaced or prevented
from binding to allergen by the allergen-specific IgG contained in
the tissue sample, or extract thereof, or the biological fluid, or
the blood sample from the patient,
[0146] wherein the amount of allergen-specific IgG in the tissue
sample, or extract thereof, or the biological fluid, or the blood
sample from the patient bound to the allergen is directly
proportional to the safety, efficacy, or outcome of
allergen-specific immunotherapy (SIT) in a patient, or
[0147] wherein the amount of allergen-specific IgE bound to the
allergen and subsequently displaced or prevented from binding to
allergen by the allergen-specific IgG contained in the tissue
sample, or extract thereof, or the biological fluid, or the blood
sample from the patient is directly proportional to the safety,
efficacy, or outcome of allergen-specific immunotherapy (SIT) in a
patient.
[0148] The tissue sample obtained for the diagnostic test may be a
solid tissue sample, or an extract thereof, or it may be a
biological/bodily fluid. The sample may also be a blood sample.
Whole blood may be used, or serum or plasma may be used in the
assay. In one embodiment, the tissue sample contains immunoglobulin
containing cells.
[0149] The binding of allergen-specific IgG from the patient's
tissue sample, or extract thereof, or biological fluid, or blood
sample, to the allergen, or the amount of allergen-specific IgE
displaced or prevented from binding to allergen by the
allergen-specific IgG in the patient's tissue sample, or extract
thereof, or biological fluid, or blood sample may be determined by
an in vitro method selected from the group consisting of an
enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay
(RIA), an immunoradiometric assay (IRMA), a luminescence
immunoassay (LIA), an immunoblot., FACs analysis, an
IgE-facilitated allergen binding (FAB) assay (See Shamji, et al.
(2006), J. Immunol. Methods, 317(1-2):71-79), and an assay using an
engineered cell line expressing Fc.epsilon.R1.
[0150] Alternatively, the binding of allergen-specific IgG from the
patient's tissue sample, or extract thereof, or biological fluid,
or blood sample, to the allergen, or the amount of
allergen-specific IgE displaced or prevented from binding to
allergen by the allergen-specific IgG in the patient's tissue
sample, or extract thereof, or biological fluid, or blood sample is
determined in vivo using an allergen-specific animal model.
[0151] In one embodiment, the allergen-specific animal model is a
mouse model of Passive Cutaneous Anaphylaxis (PCA), wherein the
model comprises the following steps: [0152] (a) injecting the
animal with allergen-specific IgE, or antiserum containing
allergen-specific IgE, intradermally at one skin site and injecting
the animal with non-allergen-specific IgE or antiserum containing
non-allergen-specific IgE intradermally at a second different skin
site; [0153] (b) injecting the animal systemically with the
allergen for which the patient is undergoing, or has undergone SIT
therapy, along with a dye; and [0154] (c) assessing the extent of
dye extravasation at the site of allergen injection;
[0155] wherein the amount of dye extravasated into the tissue is
directly related to the amount of mast cell activation in the
animal and wherein a decrease in the amount of dye extravasated
into the tissue is indicative of safety, efficacy, or a positive
outcome of allergen-specific immunotherapy in the patient.
[0156] In certain embodiments, the patient sample may be analyzed
first in vitro using the methods and diagnostic tests described
above, followed by a confirmatory analysis in the in vivo animal
model, such as the PCA model described herein.
[0157] Alternatively, each assay (in vitro and in vivo) may be used
as a stand-alone analytical tool for quantitating the patient
response to SIT.
[0158] In one embodiment, the invention provides a diagnostic test
for determining the safety, efficacy, or outcome of
allergen-specific immunotherapy (SIT) in a patient, the test
comprising: [0159] (a) a tissue sample, or extract thereof, a
biological fluid, or a blood sample from a patient who is
undergoing, or has completed allergen-specific immunotherapy;
[0160] (b) an allergen sample that corresponds to the allergen for
which the patient is undergoing SIT; [0161] (c) an
allergen-specific IgE; [0162] (d) a receptacle for mixing the
reagents of step (a) through step (c); [0163] (e) reagents for
measuring either the amount of allergen-specific IgG in the
patient's tissue sample, or extract thereof, or biological fluid,
or blood sample bound to the allergen, or for measuring the amount
of allergen-specific IgE displaced or prevented from binding to
allergen by the allergen-specific IgG contained in the patient
tissue sample, or extract thereof, or biological fluid, or blood
sample after mixing a sample from (a) with the reagents of (b) and
(c); and [0164] (f) directions for measuring the amount of
allergen-specific IgG bound to the allergen, or for measuring the
amount of allergen-specific IgE displaced or prevented from binding
to allergen by the allergen-specific IgG contained in the patient
tissue sample, or extract thereof, or biological fluid, or blood
sample,
[0165] wherein the amount of allergen-specific IgG contained in the
patient tissue sample, or extract thereof, or biological fluid, or
blood sample of (a) bound to the allergen is directly proportional
to the safety, efficacy, or outcome of allergen-specific
immunotherapy (SIT) in a patient, or
[0166] wherein the amount of allergen-specific IgE bound to the
allergen and subsequently displaced or prevented from binding to
allergen by the allergen-specific IgG contained in the patient
tissue sample, or extract thereof, or biological fluid, or blood
sample of (a) is directly proportional to the safety, efficacy, or
outcome of allergen-specific immunotherapy (SIT) in a patient.
[0167] In a related embodiment, the invention provides a test kit
for determining if a patient is responsive to allergen-specific
immunotherapy (SIT), the kit comprising: [0168] (a) a first reagent
containing the allergen for which allergen-specific immunotherapy
is being administered; [0169] (b) a second reagent containing
allergen-specific IgE; [0170] (c) a third reagent containing an
allergen-specific IgG as a known positive standard; [0171] (d)
reagents for measuring the amount of allergen-specific IgG or IgE;
[0172] (e) a receptacle for collecting a tissue sample, or extract
thereof, a biological fluid, or a blood sample from a patient
undergoing SIT, or who has completed SIT; and [0173] (f)
instructions for use of the kit.
[0174] In one embodiment, the first reagent is provided on a solid
phase support.
[0175] In one embodiment, the second reagent is provided on a solid
support.
[0176] In one embodiment, the first reagent contains a detectable
label.
[0177] In one embodiment, the second reagent contains a detectable
label.
[0178] In one embodiment, the second reagent is an
allergen-specific IgE containing a detectable label.
[0179] The detectable label may be selected from the group
consisting of a fluorescence label, a radiolabel, an enzyme label,
a luminescent label, an electrochemical, or a visual label.
[0180] The diagnostic tests described herein may be used to measure
a response in a patient undergoing SIT for an allergen selected
from the group consisting of an animal product, a food allergen,
plant pollen, mold spores, house dust mites, cockroaches, perfume,
detergents, household cleaners, latex, a drug product, or insect
venom.
[0181] In one embodiment, the animal product is selected from the
group consisting of animal fur, animal dander, wool, and mite
excretions.
[0182] In one embodiment, the animal product contains the allergen
Fel d1.
[0183] In one embodiment, the animal product contains the allergen
can f1, can f2, can f3, can f4, can f5 or can f6.
[0184] In one embodiment, the food allergen is selected from the
group consisting of eggs, meat, fruit, legumes, milk or other dairy
products, seafood, sesame, soy, wheat, oat, barley, celery and
celeriac, corn or maize and tree nuts.
[0185] In one embodiment, the legumes are selected from the group
consisting of peanuts, beans, peas and soybeans.
[0186] In one embodiment, the tree nuts are selected from the group
consisting of pecans, almonds, cashews, hazelnuts (filberts),
walnuts, brazil nuts, macadamia nuts, chestnuts, pine nuts and
pistachio nuts.
[0187] In one embodiment, the plant pollen is selected from the
group consisting of grass pollen, weed pollen and tree pollen.
[0188] In one embodiment, the tree pollen is selected from the
group consisting of birch pollen, cedar pollen, oak pollen, alder
pollen, hornbeam pollen, aesculus pollen, willow pollen, poplar
pollen, plantanus pollen, tilia pollen, olea pollen, Ashe juniper
pollen, and Alstonia scholaris pollen.
[0189] In one embodiment, the birch pollen contains the allergen
Betv 1.
[0190] In one embodiment, the cedar pollen contains the allergen
Cryj1 or Cryj2
[0191] In one embodiment, the grass pollen is ryegrass or
timothy-grass.
[0192] In one embodiment, the weed pollen is selected from the
group consisting of ragweed, plantago, nettle, Artemisia vulgaris,
Chenopodium album and sorrel.
[0193] In one embodiment, the insect venom is produced by bees,
wasps or fire ants.
[0194] Accordingly, the methods for assessing the safety, efficacy
or outcome of SIT may be done solely by an in vitro assay, as
described herein using any of the diagnostic tests or kits
described.
[0195] Alternatively, the methods for assessing the safety,
efficacy or outcome of SIT may be done solely by an in vivo assay,
such as the allergy model described herein (the PCA model), or any
other animal allergy model known to those skilled in the art, which
measures, for example, a decrease in any one or more allergy
symptoms or allergic reactions, such as mast cell
degranulation.
[0196] In one embodiment the in vitro assay and the in vivo allergy
model (e.g., PCA model) may be run concurrently.
[0197] In one embodiment, the assessment of safety, efficacy, or
outcome of SIT may be done as a two step process, the first
measurement done by using one of the in vitro diagnostic tests
described herein, and then confirmed using an in vivo allergy mode,
e.g. the PCA model described in Example 1, whereby the patient's
immunoglobulins after SIT are purified and injected directly into
the animal model to see if they confer protection to the animal
when the animal is challenged with the allergen.
[0198] In a related embodiment, the invention provides a method of
screening a patient to determine if the patient has responded to
allergen specific immunotherapy (SIT), or will be adequately
protected by SIT, or for determining when a patient can initiate or
terminate maintenance therapy, the method comprising measuring the
level of allergen-specific IgG in a patient tissue sample, or
extract thereof, or biological fluid, or blood sample using any of
the diagnostic tests or test kits described above, and assessing
the protective efficacy of the allergen-specific IgG from the
patient tissue sample, or extract thereof, or biological fluid, or
blood sample in an allergen-specific animal model, wherein elevated
levels of allergen-specific IgG from the patient's tissue sample,
or extract thereof, or biological fluid, or blood sample and
protection of the animal following challenge with the allergen is
indicative that the patient has responded to allergen specific
immunotherapy (SIT), or will be adequately protected by SIT, or
that the patient can initiate or terminate maintenance therapy.
Comparison of SIT with the Use of Therapeutic Antibodies Specific
for the Allergen
[0199] While allergen-specific immunotherapy is the only method
available which actually alters the disease state in allergy prone
individuals, there are times when such therapy may not be adequate
or appropriate, such as in patients who cannot build up an adequate
immune response to the allergen due to health issues, such as
patients who are immunocompromised due to disease, illness, or
immunosuppressive drug therapies. In those situations, it may be
beneficial to utilize a therapeutic antibody approach to treat the
allergy.
[0200] The approach of passively targeting allergens with
allergen-specific monoclonal antibodies to block mast cell
degranulation may prove to be an efficacious alternative to SIT.
The study described herein utilized two such fully human monoclonal
antibodies that bind specifically to the cat allergen, Fel d1.
These two human monoclonal antibodies specific for Fel d1 were
tested in the PCA model along with Fel d1 specific IgG purified
from patients who had undergone SIT for cat allergy (See Example
1).
[0201] While the Fel d1 IgG from SIT patients was efficacious in
this model, the ranges of inhibition of mast cell degranulation
varied depending on the length of time SIT was administered. On the
other hand, Fel d1 specific human monoclonal antibodies
significantly inhibited mast cell degranulation in this model. More
specifically, the Fel d1 monoclonal antibody cocktail was about
5-fold more potent than the SIT patient derived Fel d1 specific IgG
based on total antibody mass dosage.
[0202] The fact that both the human monoclonal anti-Fel d1
antibodies and the Fel d1 specific immunoglobulin isolated from SIT
patients demonstrated protection in the PCA model indicates that
allergen-specific immunoglobulins play a role in altering a
patient's response to allergen challenge. However, as noted
previously, while both methods confer protection, the
immunoglobulin derived from the SIT patients took years to develop
in the patient in need of such therapy, while the human monoclonal
antibodies can be administered immediately and thus may prove to
have an added benefit over SIT, since they can be used on an
emergency basis.
[0203] Furthermore, the testing of either immunoglobulins from SIT
patients or the human monoclonals specific for the allergen, e.g.
Fel d1, may be tested in any one of the in vitro diagnostic tests
or assays described herein, or may be tested directly in the PCA
model.
[0204] Thus, the present invention provides a method of determining
if a patient suffering from an allergy is responsive to therapy
with one or more antibodies or antigen binding fragments thereof
specific for the allergen, the method comprising: [0205] a)
collecting a sample of tissue or an extract thereof, or a
biological fluid, or a blood sample from a patient suffering from
an allergy; [0206] b) extracting allergen specific IgE from any one
or more of the patient samples of (a); [0207] c) mixing the IgE
from the patient sample with the allergen and with one or more
antibodies or antigen binding fragments thereof specific for the
allergen; and [0208] d) determining if the addition of the
antibodies or antigen binding fragments thereof specific for the
allergen blocks the binding of the allergen specific IgE from step
b) to the allergen,
[0209] wherein the ability of the one or more antibodies or antigen
binding fragments thereof specific for the allergen to block the
binding of allergen specific IgE from step b) to the allergen is
indicative that the patient suffering from an allergy is responsive
to therapy with the one or more antibodies specific for the
allergen.
Therapeutic Antibodies for use in Treating Allergies
Preparation of Human Antibodies
[0210] Methods for generating human antibodies in transgenic mice
are known in the art. Any such known methods can be used in the
context of the present invention to make human antibodies that
specifically bind to any antigen.
[0211] Using VELOCIMMUNE.TM. technology (see, for example, U.S.
Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE.RTM.) or
any other known method for generating monoclonal antibodies, high
affinity chimeric antibodies to an allergen are initially isolated
having a human variable region and a mouse constant region. The
VELOCIMMUNE.RTM. technology involves generation of a transgenic
mouse having a genome comprising human heavy and light chain
variable regions operably linked to endogenous mouse constant
region loci such that the mouse produces an antibody comprising a
human variable region and a mouse constant region in response to
antigenic stimulation. The DNA encoding the variable regions of the
heavy and light chains of the antibody are isolated and operably
linked to DNA encoding the human heavy and light chain constant
regions. The DNA is then expressed in a cell capable of expressing
the fully human antibody.
[0212] Generally, a VELOCIMMUNE.RTM. mouse is challenged with the
antigen of interest, and lymphatic cells (such as B-cells) are
recovered from the mice that express antibodies. The lymphatic
cells may be fused with a myeloma cell line to prepare immortal
hybridoma cell lines, and such hybridoma cell lines are screened
and selected to identify hybridoma cell lines that produce
antibodies specific to the antigen of interest. DNA encoding the
variable regions of the heavy chain and light chain may be isolated
and linked to desirable isotypic constant regions of the heavy
chain and light chain. Such an antibody protein may be produced in
a cell, such as a CHO cell. Alternatively, DNA encoding the
antigen-specific chimeric antibodies or the variable domains of the
light and heavy chains may be isolated directly from
antigen-specific lymphocytes.
[0213] Initially, high affinity chimeric antibodies are isolated
having a human variable region and a mouse constant region. As in
the experimental section below, the antibodies are characterized
and selected for desirable characteristics, including affinity,
selectivity, epitope, etc. The mouse constant regions are replaced
with a desired human constant region to generate the fully human
antibody of the invention, for example wild-type or modified IgG1
or IgG4. While the constant region selected may vary according to
specific use, high affinity antigen-binding and target specificity
characteristics reside in the variable region.
[0214] In general, the antibodies of the instant invention possess
very high affinities, typically possessing K.sub.D of from about
10.sup.-12 through about 10.sup.-9 M, when measured by binding to
antigen either immobilized on solid phase or in solution phase. The
mouse constant regions are replaced with desired human constant
regions to generate the fully human antibodies of the invention.
While the constant region selected may vary according to specific
use, high affinity antigen-binding and target specificity
characteristics reside in the variable region.
[0215] According to certain embodiments, the antibodies used in the
methods of the present invention to confer protection in the PCA
model are human monoclonal antibodies that specifically bind Fel
d1. The term "specifically binds," or the like, means that an
antibody or antigen-binding fragment thereof forms a complex with
an antigen that is relatively stable under physiologic conditions.
Methods for determining whether an antibody specifically binds to
an antigen are well known in the art and include, for example,
equilibrium dialysis, surface plasmon resonance, and the like. For
example, an antibody that "specifically binds" Fel d1, as used in
the context of the present invention, includes antibodies that bind
Fel d1 or portion thereof with a K.sub.D of less than about 1000
nM, less than about 500 nM, less than about 300 nM, less than about
200 nM, less than about 100 nM, less than about 90 nM, less than
about 80 nM, less than about 70 nM, less than about 60 nM, less
than about 50 nM, less than about 40 nM, less than about 30 nM,
less than about 20 nM, less than about 10 nM, less than about 5 nM,
less than about 4 nM, less than about 3 nM, less than about 2 nM,
less than about 1 nM or less than about 0.5 nM, as measured in a
surface plasmon resonance assay.
[0216] According to certain exemplary embodiments of the present
invention, the Fel d1 monoclonal antibodies, or antigen-binding
fragments thereof that can be used in the context of the methods of
the present invention comprise the heavy chain complementarity
determining regions (HCDRs) of a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 2 or 18 and the
light chain complementarity determining regions (LCDRs) of a light
chain variable region (LCVR) comprising the amino acid sequence of
SEQ ID NO: 10 or 26. According to certain embodiments, the anti-Fel
d1 antibody or antigen-binding fragment thereof comprises three
HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and
LCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ
ID NO:4 or 20; the HCDR2 comprises the amino acid sequence of SEQ
ID NO:6 or 22; the HCDR3 comprises the amino acid sequence of SEQ
ID NO:8 or 24; the LCDR1 comprises the amino acid sequence of SEQ
ID NO:12 or 28; the LCDR2 comprises the amino acid sequence of SEQ
ID NO:14 or 30; and the LCDR3 comprises the amino acid sequence of
SEQ ID NO:16 or 32. In yet other embodiments, the anti-Fel d1
antibody or antigen-binding fragment thereof comprises an HCVR
comprising SEQ ID NO:2 or 18 and an LCVR comprising SEQ ID NO:10 or
26. In certain embodiments, the methods of the invention provide
for the use of a composition comprising two anti-Fel d1 monoclonal
antibodies having the HCVR/LCVR amino acid sequence pairs as set
forth in SEQ ID NOs: 2/10 and 18/26.
Pharmaceutical Compositions
[0217] The present invention may include methods, which comprise
administering an allergen-specific antibody, e.g. a Fel d1
antibody, to a subject wherein the antibody is contained within a
pharmaceutical composition. The pharmaceutical compositions of the
invention may be formulated with suitable carriers, excipients, and
other agents that provide suitable transfer, delivery, tolerance,
and the like. A multitude of appropriate formulations can be found
in the formulary known to all pharmaceutical chemists: Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These
formulations include, for example, powders, pastes, ointments,
jellies, waxes, oils, lipids, lipid (cationic or anionic)
containing vesicles (such as LIPOFECTIN.TM.), DNA conjugates,
anhydrous absorption pastes, oil-in-water and water-in-oil
emulsions, emulsions carbowax (polyethylene glycols of various
molecular weights), semi-solid gels, and semi-solid mixtures
containing carbowax. See also Powell et al. "Compendium of
excipients for parenteral formulations" PDA (1998) J Pharm Sci
Technol 52:238-311.
[0218] Various delivery systems are known and can be used to
administer the pharmaceutical composition of the invention, e.g.,
encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the mutant viruses,
receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol.
Chem. 262:4429-4432). Methods of administration include, but are
not limited to, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The composition may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents.
[0219] A pharmaceutical composition of the present invention can be
delivered subcutaneously or intravenously with a standard needle
and syringe. In addition, with respect to subcutaneous delivery, a
pen delivery device readily has applications in delivering a
pharmaceutical composition of the present invention. Such a pen
delivery device can be reusable or disposable. A reusable pen
delivery device generally utilizes a replaceable cartridge that
contains a pharmaceutical composition. Once all of the
pharmaceutical composition within the cartridge has been
administered and the cartridge is empty, the empty cartridge can
readily be discarded and replaced with a new cartridge that
contains the pharmaceutical composition. The pen delivery device
can then be reused. In a disposable pen delivery device, there is
no replaceable cartridge. Rather, the disposable pen delivery
device comes prefilled with the pharmaceutical composition held in
a reservoir within the device. Once the reservoir is emptied of the
pharmaceutical composition, the entire device is discarded.
[0220] In certain situations, the pharmaceutical composition can be
delivered in a controlled release system. In one embodiment, a pump
may be used. In another embodiment, polymeric materials can be
used; see, Medical Applications of Controlled Release, Langer and
Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another
embodiment, a controlled release system can be placed in proximity
of the composition's target, thus requiring only a fraction of the
systemic dose (see, e.g., Goodson, 1984, in Medical Applications of
Controlled Release, supra, vol. 2, pp. 115-138). Other controlled
release systems are discussed in the review by Langer, 1990,
Science 249:1527-1533.
[0221] The injectable preparations may include dosage forms for
intravenous, subcutaneous, intracutaneous and intramuscular
injections, drip infusions, etc. These injectable preparations may
be prepared by known methods. For example, the injectable
preparations may be prepared, e.g., by dissolving, suspending or
emulsifying the antibody or its salt described above in a sterile
aqueous medium or an oily medium conventionally used for
injections. As the aqueous medium for injections, there are, for
example, physiological saline, an isotonic solution containing
glucose and other auxiliary agents, etc., which may be used in
combination with an appropriate solubilizing agent such as an
alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,
polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,
HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor
oil)], etc. As the oily medium, there are employed, e.g., sesame
oil, soybean oil, etc., which may be used in combination with a
solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
The injection thus prepared is preferably filled in an appropriate
ampoule.
[0222] Advantageously, the pharmaceutical compositions for oral or
parenteral use described above are prepared into dosage forms in a
unit dose suited to fit a dose of the active ingredients. Such
dosage forms in a unit dose include, for example, tablets, pills,
capsules, injections (ampoules), suppositories, etc.
[0223] Exemplary pharmaceutical compositions comprising an
anti-IL-4R antibody that can be used in the context of the present
invention are disclosed, e.g., in US Patent Application Publication
No. 2012/0097565.
Dosage
[0224] The amount of Fel d1 antibody administered to a subject
according to the methods of the present invention is, generally, a
therapeutically effective amount. As used herein, the phrase
"therapeutically effective amount" means an amount of the antibody
that results in one or more of: (a) a reduction in the severity or
duration of an allergic reaction; (b) the alleviation of one or
more symptoms or indicia of an allergic reaction; (c) prevention or
alleviation of anaphylaxis; (d) a reduction in serum IgE level; (e)
a reduction in the use or need for conventional allergy therapy
(e.g., reduced or eliminated use of antihistamines, decongestants,
nasal or inhaled steroids, anti-IgE treatment, epinephrine,
etc.).
[0225] In the case of an anti-Fel d1 antibody, a therapeutically
effective amount can be from about 0.05 mg to about 600 mg, e.g.,
about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0
mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50
mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100
mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about
150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg,
about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240
mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about
290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg,
about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380
mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about
430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg,
about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520
mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about
570 mg, about 580 mg, about 590 mg, or about 600 mg, of the
anti-IL-4R antibody. In certain embodiments, 300 mg of an
anti-IL-4R antibody is administered.
[0226] The amount of Fel d1 antibody contained within the
individual doses may be expressed in terms of milligrams of
antibody per kilogram of patient body weight (i.e., mg/kg). For
example, the antibody may be administered to a patient at a dose of
about 0.0001 to about 10 mg/kg of patient body weight.
EXAMPLES
[0227] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1. Blocking Mast Cell Degranulation by Polyclonal
Antibodies Generated During Allergen-Specific Immunotherapy Against
the Cat Allergen Fel d1 and with Human Monoclonal Antibodies that
Specifically Bind Fel d1
[0228] The passive cutaneous anaphylaxis (PCA) in vivo model is
used to assess in vivo mast cell degranulation. The model relies on
the passive transfer of antisera containing allergen-specific mouse
polyclonal IgE, which binds to Fc.epsilon.R1 on mast cells near the
site of administration. The mice are effectively "sensitized"
locally to the specific antigen(s) (either Fel d 1 or peanut as a
control) used to generate the antiserum. When antigen is
subsequently injected systemically as an allergen challenge along
with Evan's blue dye, antigen-induced mast cell degranulation
results in rapid capillary dilation and increased vascular
permeability that can be visualized and quantitated by measuring
dye leakage into the reaction site. In this model, the
peanut-sensitized ear provides an internal control for the
specificity of the effector cell response when Fel d 1 is injected
as the challenge. Mast cell activation has several measurable
downstream effects. For instance, mucosal-associated mast cell
release of histamine and other molecules stimulates the dilation of
blood vessels, irritation of nerve endings and an increase in fluid
accumulation. These events cause local swelling, increased mucus
production and itching, which together lead to the symptoms
characteristic of an ocular allergic response and allergic rhinitis
in humans. The PCA model, which measures mast cell-induced vascular
permeability, has been used to study hypersensitivity in various
settings (Bradley, et al. 1991, J Allergy Clin Immunol; 88:661-74).
In particular, the PCA model is a sensitive model that has been
used to test the ability of Fel d 1-specific antibodies, both alone
and as multi-antibody cocktails, to block mast cell degranulation
induced by Fel d 1.
[0229] SIT is a disease modifying therapy that introduces a regimen
of increasing doses of allergen with the goal of stimulating immune
tolerance through repeated exposure, a process that can take years
to confer protection. SIT has been shown to induce the production
of allergen-specific polyclonal immunoglobulins (IgGs) that, when
passively transferred, inhibit immediate inflammatory responses in
allergic patients that have not undergone SIT (Cooke, et al, 1935.
62:733-50).
[0230] The PCA model can be used to assess the degree to which
polyclonal IgG antibodies generated during SIT block mast cell
degranulation induced by Fel d 1. The PCA model can also be used to
compare the protective effect of SIT-induced polyclonal IgG
antibodies to the protective effect of Fel d 1-specific monoclonal
antibodies, administered passively as either individual or as
multi-antibody cocktails. While the present study describes the
results obtained using blood samples obtained from patients
undergoing allergen-specific immunotherapy (SIT) for the cat
allergen Fel d1, it is feasible that similar results may be
observed using samples from patients after undergoing SIT for other
allergens. Moreover, the method described herein can be used to
determine if a patient has responded to SIT by assessing the amount
and/or capability of allergen-specific IgG in a patient sample to
prevent mast cell degranulation.
Methods and Results
[0231] For generating antisera used in the PCA model, Balb/c mice
were sensitized by intraperitoneal injection on day 0 with 5 .mu.g
natural Fel d1 protein purified from cat hair extract (Indoor
Biotechnologies, #LTN-FD1-1) or with 5 .mu.g of crude peanut
allergen extract (Greer Laboratories, #XPF171D3A25) in a solution
of 1 mg/ml of alum (Pierce, #77161) in 1.times. phosphate buffered
saline. Two weeks later (day 14) sensitized mice were boosted with
doses of allergen identical to those used for the initial
immunization. Two weeks after the boost (day 28), mice were
sacrificed and serum was collected. Total IgE concentration in the
isolated antisera was determined by ELISA. The final concentration
of antiserum was diluted to 2500 ng/mL total IgE in 1.times.
phosphate buffered saline.
[0232] For generating antibodies to Fel d1, an immunogen comprising
any one of the following can be used. In certain embodiments, the
antibodies of the invention are obtained from mice immunized with a
primary immunogen, such as full length natural Fel d1 (nFel d1),
which may be purchased commercially (e.g., from Indoor
Biotechnologies, #LTN-FD1-1), or isolated from cat hair or dander
by multi-step column chromatography (See, for example, Chapman M D,
et al. (1988), J. Immunol. 140:812-818), or which may be produced
recombinantly (See GenBank accession numbers P30438, or
NP_001041618.1 for the full length amino acid sequence of chain 1
of Fel d1, also referred to as chain A or FELD1 A; also see SEQ ID
NO: 33) and GenBank accession number P30440, or NP_001041619.1 for
the full length amino acid sequence of chain 2 of Fel d1 (also
referred to as chain B or FELD B; also see SEQ ID NO: 34), or
fragments of either chain 1 or chain 2, or fragments from both
chain 1 and chain 2 of the Fel d1 protein, followed by immunization
with a secondary immunogen, or with an immunogenically active
fragment of the natural protein. Animals may be immunized with
either chain 1 protein alone or chain 2 protein alone, or with both
chain 1 and chain 2 proteins, administered sequentially, or
concurrently. Various constructs may be prepared using portions of
chain 1 and chain 2 along with various linking or spacer strategies
known to those skilled in the art. These constructs may be used
alone, or in various combinations to elicit antibody responses in
vivo.
[0233] Fully human Fel d 1-specific monoclonal antibodies H4H1232N,
H4H2636P and H4H1238 were generated as described herein (See also
U.S. Ser. No. 13/875,401) and the amino acid sequences of the heavy
chain and light chain variable regions and their respective heavy
chain and light chain complementary determining regions are
included herein and are shown in Table 1 below.
TABLE-US-00001 TABLE 1 Ab Protein Identification Amino Acid SEQ ID
NOs No. HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H4H1232N 2 4
6 8 10 12 14 16 H4H2636P 18 20 22 24 26 28 30 32 H4H1238N 36 38 40
42 44 46 48 50
[0234] Sera were collected from cat allergic patients who had
either undergone or not undergone specific immunotherapy for cat
allergy. The concentration of total IgG (and therefore of Fel d
1-specific IgG) contained in these samples was then increased to
support administration in vivo in the PCA model. This was achieved
by isolation of total IgG using protein G affinity resin, dialysis
of the eluted IgG into PBS, and then concentration of the total IgG
using a molecular weight cut-off membrane. Total and Fel d
1-specific IgG levels were quantitated in sera samples and purified
IgG from cat allergic patients using an ELISA. Microtiter plates
were coated with 2 .mu.g/mL of either natural Fel d 1 (LoTox nFel d
1, #LTN-FD1) or anti-human IgG (Jackson Immunoresearch,
#109-005-098) in phosphate-buffered saline (PBS) overnight at
4.degree. C. The plates were blocked with 0.5% bovine serum albumin
(BSA). Sera or purified IgG were serially diluted in 0.5% BSA-PBS
and added to the plates and incubated for 1 hour at RT. For total
IgG quantitation, a standard curve was generated using human IgG
(Thermo Scientific, #31154), while for Fel d 1-specific IgG
quantitation, a standard curve was generated using a Fel d 1 human
kappa monoclonal antibody (H4H1238N). Goat anti-human IgG-Fc-Horse
Radish Peroxidase (HRP) conjugated secondary antibody (Jackson
Immunoresearch, Catalogue No. 109-035-098) was added (1:5000
dilution), followed by addition of TMB/H.sub.2O.sub.2 substrate to
detect IgG. Concentrations of total and Fel d 1 specific IgG were
computed from the respective standard curve plots and are shown in
Table 2.
TABLE-US-00002 TABLE 2 Characterization of Sera Obtained from Cat
Allergic Patients Total Fel d Total Fel d 1-specific SIT Total
Human Purified and 1-specific IgG in Purified Sample Immuno-
Duration IgG in Sera concentrated IgG in Sera and concentrated Name
therapy (months) (mg/ml) IgG (mg/ml) (mg/ml) Sera (mg/ml) AMC-003
NO 0 14.7 107 Not 0.008 detectable AMC-004 NO 0 11.1 112 Not 0.007
detectable AMC-005 YES 14 18.1 131 0.014 0.059 AMC-007 YES 20 14.8
146 0.013 0.119 AMC-010 YES 37 15.8 161 0.050 0.463 AMC-001 NO 0
12.6 61.7 0.005 0.025 AMC-002 YES 27 8.9 61.7 0.013 0.078 AMC-006
NO 0 13.7 60.6 Not 0.010 detectable AMC-008 YES 20 13.1 67.1 Not
0.015 detectable AMC-009 NO 0 10.4 62.7 Not 0.015 detectable
[0235] The concentration of Fel d 1-specific IgG was highest in a
serum sample from the patient who had undergone SIT for the longest
duration (sample AMC-010 exhibited serum levels of 0.050 mg/mL
after SIT treatment spanning 37 months). For sera from cat allergic
patients who did not undergo SIT (see patients AMC-003, AMC-004,
AMC-001, AMC-006 and AMC-009), Fel d 1-specific IgG levels were
either 0.005 mg/ml or below the detection limit in initial sera but
could be measured after the protein G isolation and concentration
procedure. For the four sera samples from patients subjected to SIT
with measurable Fel d 1 specific IgG in the initial sera (AMC-005,
AMC-007, AMC-010, AMC-002), the protein G isolation and
concentration procedure resulted in increasing the Fel d 1-specific
IgG concentrations by factors of 4.2, 9.2, 9.3, and 6.0
respectively, relative to the concentration in the initial serum
(calculated based on the numbers in Table 2).
[0236] To evaluate the protective effect in the PCA model from
administration of the patient-derived IgG and from a cocktail of
two Fel d 1-specific antibodies that do not cross-compete for
binding to Fel d 1 (See US2013/0295097), groups of Balb/c mice were
injected subcutaneously with either of four different treatments:
i) a human IgG4 isotype control antibody, ii) a combination of Fel
d 1-specific antibodies H4H1232N/H4H2636P, iii) IgG isolated from
cat allergic patients (AMC-003 and AMC-004) who had not undergone
SIT, or iv) IgG isolated from cat allergic patients (AMC-005,
AMC-007 and AMC-010) who had undergone SIT for cat allergy. The
IgG4 isotype control antibody and the combination of two Fel d 1
antibodies were injected subcutaneously at doses of 1 mg/kg total
antibody (0.5 mg/kg of each antibody for the H4H1232N/H4H2636P
combination treatment). The isolated and concentrated IgG samples
from cat allergic patients (either with or without SIT therapy)
were injected at volumes approximately one-tenth the blood volume
of the mice used in these studies (i.e., 200 uL volumes were
injected, and the expected blood volume is approximately 2.0 mL for
a 20 g mouse). Given the concentration factors described above for
the SIT patient serum samples, this dosing procedure was expected
to reconstitute approximately 42%, 92%, and 93%, respectively, of
the original serum levels of Fel d 1-specific IgG for samples
AMC-005, AMC-007, and AMC-010, respectively. Three days after
pre-treatment with antibodies, mice were sensitized by intradermal
injection with 10 .mu.l of natural Fel d 1-derived antiserum or 10
.mu.l of peanut-derived antiserum (negative control) into the right
and left ears, respectively, of each mouse. Twenty-four hours after
sensitization, mice were challenged by intravenous injection (100
.mu.L per mouse) of a solution of 1 .mu.g/mL natural Fel d 1
(Indoor Biotechnologies, #LTN-FD1-1) dissolved in 1.times.
phosphate buffered saline containing 0.5% (w/v) Evan's blue dye
(Sigma, #E2129). One hour after antigen challenge, mice were
sacrificed, ears were excised and placed in 1 mL formamide and
incubated for 3 days at 56.degree. C. to extract the Evan's blue
dye from the tissue. Ear tissue was then removed from the
formamide, blotted to remove excess liquid and weighed. Two hundred
microliter aliquots of each formamide extract were transferred to
96 well plates in duplicate. Absorbance of the resulting
supernatants was measured at 620 nm. The OD was converted to Evan's
blue dye concentration using a standard curve. The average
concentration of Evan's blue dye extravasated into the tissue of
the antisera-sensitized ear (normalized by ear tissue weight) was
calculated for the group treated with the isotype control antibody
and defined as F(isotype,avg). The reduction in Evan's blue dye
extravasation resulting from antibody pre-treatment was calculated
per mouse by subtracting the amount of Evan's blue dye for the
antibody-treated group's Fel d 1 extract sensitized ear, defined as
F(mAb,i), from F(isotype,avg). This number was then divided by the
difference between F(isotype,avg) and the dye amount for the
antibody-treated group's peanut sensitized ear [P(mAb,i)] and
multiplied by 100 to give the overall percent reduction in dye
extravasation for each mouse (% Reduction).
% Reduction (per
mouse)=100*[F(isotype,avg)-F(mAb,i)]/[F(isotype,avg)-P(mAb,i)]
[0237] The average percent reduction in dye leakage was then
calculated for each group. Results, expressed as (mean.+-.SEM) of
percent Evan's blue reduction are shown in Table 3. All reductions
that were statistically significant (p<0.05) compared to isotype
control as determined by two-way ANOVA with Bonferroni's post-test
are noted with an asterisk (*). The number of mice used per group
(n) is noted within parentheses in the tables.
TABLE-US-00003 TABLE 3 Percent reduction in dye extravasation in
the PCA model % Reduction in Dye Treatment Extravasation (.+-.SEM)
H4H1232N/H4H2636P (n = 10) 63 .+-. 4* AMC-005 (SIT IgG) (n = 9) 20
.+-. 4 AMC-007 (SIT IgG) (n = 10) 50 .+-. 5* AMC-010 (SIT IgG) (n =
10) 67 .+-. 4* AMC-003 (non-SIT IgG) (n = 10) 13 .+-. 2 AMC-004
(non-SIT IgG)(n = 10) 5 .+-. 6
[0238] As shown in Table 3, groups of mice when treated with the
Fel d 1 antibody cocktail H4H1232N/H4H2636P exhibit a 63% reduction
in dye extravasation compared to mice receiving control antibody.
Mice treated with cat allergic sera from patients who underwent SIT
exhibit 20% (AMC-005), 50% (AMC-007) and 67% (AMC-010) reductions
in dye extravasation compared to mice receiving isotype control
antibody demonstrating that purified IgG from cat allergic patients
who underwent SIT are efficacious in this model. The percent
reduction in dye extravasation correlates with the duration of SIT
therapy and also the amount of Fel d 1 specific IgG present in the
sera (Table 2). Mice treated with cat allergic sera from patients
who did not undergo SIT exhibited 13% (AMC-003) and 5% (AMC-004)
reductions in dye extravasation compared to mice receiving control
antibody, which did not achieve statistical significance in this
study compared to treatment with isotype control.
[0239] As shown in Table 3 the H4H1232N/H4H2636P antibody cocktail
at its dose of 1 mg/kg (0.5 mg/kg each mAb) achieved similar
potency (63.+-.4% reduction in dye extravasation) as treatment with
the SIT patient-derived sample with the highest Fel d 1-specific
IgG concentration (sample AMC-010; 67.+-.4% reduction in dye
extravasation). The approximate dosing level for AMC-010 can be
estimated as .about.4.6 mg/kg since 0.093 mg of Fel d 1-specific
IgG was injected per mouse (200 uL per injection*0.46 mg/mL of Fel
d 1-specific IgG in this sample), and the approximate weight per
mouse was 0.020 kg. This demonstrates that the H4H1232N/H4H2636P
treatment was about 5-fold more potent than the SIT patient derived
Fel d 1-specific IgG based on total antibody mass dosage.
Example 2. Peanut Specific Antibodies Generated Through Allergen
Specific Immunotherapy Block Mast Cell Degranulation in a Mouse
Peanut Allergy Model
Methods/Experimental Design
[0240] A mouse peanut specific immunotherapy model was developed
based in part on the model delineated in Kulis et al., J. Allergy
Clin Immunol. 127(1):81-88(2011). An outline of the experimental
protocol is shown in FIG. 1a. Two groups of mice were used in these
experiments. One group (`SIT`) was subjected to a peanut specific
immunotherapy regimen comprising a Sensitization Phase, a Build-up
Phase, and a Challenge. The Sensitization Phase consisted of
administration of 0.5 mg peanut extract (Greer laboratories, Part
Number XPF171D3A25)+2 mg Alum administered intraperitoneally on
Days 0, 7 and 28. The Build-up Phase consisted of twelve separate
intraperitoneal administrations of various increasing doses of
peanut extract shown in FIG. 1a without Alum on Days 49, 51, 53,
56, 58, 60, 63, 65, 67, 70, 72 and 74. The Challenge consisted of
intraperitoneal administration of 1 mg of peanut extract on Day 98.
The other group of mice (No SIT') was subjected to a Sensitization
phase and a challenge phase.
[0241] To assess the extent of anaphylaxis in this system and the
efficacy of the specific immunotherapy, mouse core temperature was
measured over the course of 180 minutes following the challenge
injection. A decrease in core temperature is a measure of
anaphylaxis. Mice subjected to the peanut specific immunotherapy
regimen were protected from the peanut induced decrease in core
temperature (FIG. 1B). Sera from these mice that had either
undergone or not undergone peanut specific immunotherapy were
collected at the completion of the experiment (day 98) for
immunoglobulin measurements. Total IgE, peanut-specific IgG1 and
peanut specific IgG2a at day 96 are shown in Table 4. Total IgE was
determined in the samples using a mouse IgE ELISA kit (BD OpT EIA,
#555248) according to the manufacturers instructions. Peanut
specific IgG1 and peanut specific IgG2a were determined by sandwich
ELISAs using coated peanut extract as the capture surface and
anti-mouse IgG2 antibody (HRP-rat anti-mouse IgG2a from BD
biosciences, Cat. No. 553391) or anti-mouse IgG1 antibody (HRP-rat
anti-mouse IgG1 from BD biosciences, Cat. No. 559626) for
detection. Total IgE, peanut specific IgG1 and peanut IgG2a levels
are higher in serum samples from mice that received SIT (Table
4).
[0242] To evaluate the protective effect of SIT, the PCA model was
conducted in groups of Balb/c mice that were subcutaneously
administered 200 .mu.l of either PBS, sera from mice that received
SIT or sera from mice that were sensitized and did not receive SIT.
Three days after the treatment with sera, mice were sensitized by
either intradermal injection with 10 .mu.l of
peanut-extract-derived antiserum or 10 .mu.l of Fel d 1 derived
antiserum (negative control) into the right and left ears,
respectively, of each mouse. Twenty-four hours after sensitization,
mice were challenged by intravenous injection (100 .mu.l per mouse)
of a solution comprised of 250 .mu.g of peanut extract dissolved in
1.times.PBS containing 0.5% Evans blue dye (Sigma, #E2129).
[0243] One hour after antigen challenge, mice were sacrificed, ears
were excised and placed in 1 mL formamide and incubated for 3 days
at 56.degree. C. to extract the Evans blue dye from the tissue. Ear
tissue was then removed from the formamide, blotted to remove
excess liquid and weighed. Two hundred microliter aliquots of each
formamide extract were transferred to 96 well plates in duplicate.
Fluorescence of the resulting supernatants was measured using an
excitation measurement of 625 nm and emission measurement of 680
nm. The fluorescence was converted to Evans blue dye concentration
using a standard curve. The average concentration of Evans blue dye
extravasated into the tissue of the antisera-sensitized ear
(normalized by ear tissue weight) was calculated for the group
treated with Control PBS treated and defined as F(control,avg). The
reduction in Evans blue dye extravasation resulting from antibody
pre-treatment was calculated per mouse by subtracting the amount of
Evans blue dye for the Sera-treated group's peanut extract
sensitized ear, defined as F(sera,i), from F(control,avg). This
number was then divided by the difference between F(control,avg)
and the dye amount for the antibody-treated group's peanut
sensitized ear [P(sera,i)] and multiplied by 100 to give the
overall percent reduction in dye extravasation for each mouse (%
Reduction).
[0244] The average percent reduction in dye leakage was then
calculated for each group. Results, expressed as (mean.+-.SD) of
percent Evans blue reduction are shown in Table 5. All reductions
that were statistically significant (p<0.05) compared to isotype
control as determined by two-way ANOVA with Bonferroni's post-test
are noted with an asterisk (*). The number of mice used per group
(n) is noted within parentheses in the tables.
Results
[0245] As shown in table 5, groups of mice when treated with peanut
SIT antisera exhibit a 97% reduction in dye extravasation compared
to mice that received PBS alone. The percent reduction in dye
extravasation correlates with peanut specific IgG present in the
sera, demonstrating the protective effects of peanut specific IgG
that is present in the SIT antisera in this model.
TABLE-US-00004 TABLE 4 Characterization of sera obtained from mice
in a peanut specific immunotherapy model No SIT (n = 5) SIT (n = 4)
Total IgE (ng/mL) 1656 7896 Peanut specific IgG1 (titer) 60596
8287003 Peanut specific IgG2a (titer) 764 26821
TABLE-US-00005 TABLE 5 Percent reduction in dye extravasation in
the PCA model % Reduction in Dye extravasation (.+-.SD) Treatment
as compared with PBS control No SIT (n = 5) 0 SIT (n = 5) 97 .+-.
5
Example 3. An In Vitro Assay/Method to Assess the Ability of
Antibodies to Block the Binding of Allergen-Specific Polyclonal IgE
to Allergen
[0246] Below is described an assay to evaluate the blockade of Fel
d 1 binding to cat allergic human IgE by Fel d 1-specific IgGs
isolated from subjects treated by allergen specific immunotherapy
(SIT).
Blocking Fel d 1 Binding to Allergen Specific IgE by Antibodies
Isolated from Patients Undergoing SIT
[0247] The ability of IgG antibodies from a cat allergic SIT
patient serum to block Fel d 1 binding to plate-captured IgE from
cat allergic human donor plasma/sera was determined using a
blocking ELISA. Microtiter plates were coated overnight at
4.degree. C. with a human Fc.epsilon.R1.alpha. (the high affinity
receptor for IgE) extracellular domain protein that was produced
with a C-terminal mouse Fc tag (hFc.epsilon.R1.alpha.-mFc; SEQ ID
NO: 51). Plates were blocked with 0.5% BSA (w/v) for 1 hour at room
temperature (RT). Plasma from Fel d 1 cat positive allergic donors
(Plasma Lab International, Everett, Wash.) was diluted 5-fold and
total IgE was captured over the receptor-coated surface. A constant
amount of recombinant Fel d 1 produced with a C-terminal
myc-myc-hexahistidine tag (Fel d 1-mmH; SEQ ID NO: 52) (0.7 nM) was
pre-mixed with serial dilutions of Fel d 1 antibodies (isolated by
protein G affinity purification from sera from human patients
undergoing cat allergen specific immunotherapy) ranging from 66.67
nM to 1.13 pM and incubated for 1 hour at RT. The antibody-Fel d 1
mixture was then added to the IgE-coated plate for 1 hour. Plates
were subsequently washed and the amount of free Fel d 1-mmH bound
to plate was detected using an anti-myc antibody derived from clone
9E10 (Evan G I, et al. Isolation of monoclonal antibodies specific
for human c-myc proto-oncogene product. Mol. Cell. Biol. 5:
3610-3616, 1985) that was produced in-house as a human IgG1
isotype, conjugated to HRP, and incubated at a final concentration
of 0.102 ug/mL for 1 hour at RT. Plates were washed with PBS-T in
between each step of the ELISA protocol described above. To develop
the colorimetric reaction, TMB/H.sub.2O.sub.2 substrate was added
to the plates and incubated for 20 minutes at RT. The reaction was
stopped using 2N sulfuric acid (H.sub.2SO.sub.4; VWR, #BDH3500-1).
Absorbance was subsequently measured on a spectrophotometer
(Victor, Perkin Elmer) at 450 nm. The concentration of antibody
required to inhibit the signal of a constant concentration of Fel d
1 by 50% (IC.sub.50) was determined using Prism software (data not
shown).
[0248] Representative results from the blocking assay are shown in
FIGS. 2-3 for two different cat allergic human donor IgE samples.
Purified IgG isolated from patients undergoing SIT with detectable
levels of Fel d 1 specific IgG blocked Fel d 1 binding to
Fc.epsilon.R1.alpha.-captured allergic IgE to the assay baseline
(complete blocking), while an irrelevant control antibody did not
block. Similar complete blocking in this assay was observed for SIT
IgG from 4 additional allergic donors that were tested. When sera
from patients undergoing SIT were collected at different time
points (once in 2013 and once in 2014), the blocking activity was
maintained (data not shown). IgG from a patient not undergoing SIT
exhibited a low degree of blocking, but only at the highest
antibody concentration tested in the assay (FIGS. 2-3, non SIT
IgG). This may be attributable to the low, but detectable, levels
of Fel d 1 specific IgG in this sample (See Table 2).
[0249] Similar results as described above and in FIG. 2 using
captured Fel d 1 allergic human IgE were also observed using
captured polyclonal mouse IgE obtained from the sera of mice
immunized with Fel d 1 protein. The capture surface was effective
in this assay because human Fc.epsilon.R1.alpha. binds to mouse
IgE. The ability of Fel d 1-specific IgG isolated from three
different human SIT sera samples to block the binding of Fel d1-mmH
to captured mouse IgE from Fel d 1-immunized mice is shown in FIG.
4.
Example 4: Blocking Fel d 1 Binding to Allergen Specific IgE by
Anti-Fel d 1 Polyclonal Serum from Mice Immunized with Fel d 1
[0250] Polyclonal serum from a mouse immunized with Fel d 1 mFc
(REGN573; SEQ ID NO: 53) was used as a source of Fel d 1 specific
IgG to mimic the IgG from allergy patients undergoing immunotherapy
in the blocking assay described above. The immune mouse serum was
serially diluted starting from 1:50 and diluted 3-fold. The immune
serum blocked Fel d 1 binding to Fc.epsilon.R1.alpha.-captured
allergy IgE to the assay baseline, while pooled pre-immune sera did
not block (FIG. 5).
Sequence CWU 1
1
531357DNAArtificial SequenceSynthetic 1gaggtgcagc tggtggagtc
tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcaga aactataaca taaactgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcactc atcagtggta gtagtagtta catatattac
180gcagactcag tgaagggccg attcaccgtc tccagagaca acgccaagaa
ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gaggcggaca 300ttaagctact acgttatgga cgtctggggc
caagggacca cggtcaccgt ctcctca 3572119PRTArtificial
SequenceSynthetic 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Arg Asn Tyr 20 25 30Asn Ile Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ser Leu Ile Ser Gly Ser Ser Ser Tyr
Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Thr Leu
Ser Tyr Tyr Val Met Asp Val Trp Gly Gln Gly 100 105 110Thr Thr Val
Thr Val Ser Ser 115324DNAArtificial SequenceSynthetic 3ggattcacct
tcagaaacta taac 2448PRTArtificial SequenceSynthetic 4Gly Phe Thr
Phe Arg Asn Tyr Asn1 5524DNAArtificial SequenceSynthetic
5atcagtggta gtagtagtta cata 2468PRTArtificial SequenceSynthetic
6Ile Ser Gly Ser Ser Ser Tyr Ile1 5736DNAArtificial
SequenceSynthetic 7gcgaggcgga cattaagcta ctacgttatg gacgtc
36812PRTArtificial SequenceSynthetic 8Ala Arg Arg Thr Leu Ser Tyr
Tyr Val Met Asp Val1 5 109321DNAArtificial SequenceSynthetic
9gacatccagg tgacccagtc tccatccccc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggcgagtca gggcattagc aattatttag cctggtatca gcagaaacca
120gggagagttc ctcagctcct gatctatgct gcatccactt tgcaatcagg
ggtcccatct 180cggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagatgttg caacttatta ctgtcaaaag
tataacagtg ccccgtacac ttttggccag 300gggaccaagc tggagatcaa a
32110107PRTArtificial SequenceSynthetic 10Asp Ile Gln Val Thr Gln
Ser Pro Ser Pro Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Arg Val Pro Gln Leu Leu Ile 35 40 45Tyr Ala Ala
Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
1051118DNAArtificial SequenceSynthetic 11cagggcatta gcaattat
18126PRTArtificial SequenceSynthetic 12Gln Gly Ile Ser Asn Tyr1
5139DNAArtificial SequenceSynthetic 13gctgcatcc 9143PRTArtificial
SequenceSynthetic 14Ala Ala Ser11527DNAArtificial SequenceSynthetic
15caaaagtata acagtgcccc gtacact 27169PRTArtificial
SequenceSynthetic 16Gln Lys Tyr Asn Ser Ala Pro Tyr Thr1
517345DNAArtificial SequenceSynthetic 17gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agttatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggtc gtggttataa cgcagactac
180gcagactccg tgaagggccg gttcaccatc tccagggaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaagac acggccgtat
attactgtgc gaaattggaa 300tactttgact actggggcca gggaaccctg
gtcactgtct cctca 34518115PRTArtificial SequenceSynthetic 18Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Ser Gly Arg Gly Tyr Asn Ala Asp Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Leu Glu Tyr Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110Val Ser Ser 1151924DNAArtificial
SequenceSynthetic 19ggattcacct ttagcagtta tgcc 24208PRTArtificial
SequenceSynthetic 20Gly Phe Thr Phe Ser Ser Tyr Ala1
52124DNAArtificial SequenceSynthetic 21attagtggtc gtggttataa cgca
24228PRTArtificial SequenceSynthetic 22Ile Ser Gly Arg Gly Tyr Asn
Ala1 52324DNAArtificial SequenceSynthetic 23gcgaaattgg aatactttga
ctac 24248PRTArtificial SequenceSynthetic 24Ala Lys Leu Glu Tyr Phe
Asp Tyr1 525321DNAArtificial SequenceSynthetic 25gacatccaga
tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgaggcct 240gaagattttg caacttatta ctgccaacag
tataatagtt accctctgac tttcggcgga 300gggaccaagg tggagatcaa a
32126107PRTArtificial SequenceSynthetic 26Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Arg Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
1052718DNAArtificial SequenceSynthetic 27cagagtatta gtagctgg
18286PRTArtificial SequenceSynthetic 28Gln Ser Ile Ser Ser Trp1
5299DNAArtificial SequenceSynthetic 29aaggcgtct 9303PRTArtificial
SequenceSynthetic 30Lys Ala Ser13127DNAArtificial SequenceSynthetic
31caacagtata atagttaccc tctgact 27329PRTArtificial
SequenceSynthetic 32Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1
53370PRTArtificial SequenceSynthetic 33Glu Ile Cys Pro Ala Val Lys
Arg Asp Val Asp Leu Phe Leu Thr Gly1 5 10 15Thr Pro Asp Glu Tyr Val
Glu Gln Val Ala Gln Tyr Lys Ala Leu Pro 20 25 30Val Val Leu Glu Asn
Ala Arg Ile Leu Lys Asn Cys Val Asp Ala Lys 35 40 45Met Thr Glu Glu
Asp Lys Glu Asn Ala Leu Ser Leu Leu Asp Lys Ile 50 55 60Tyr Thr Ser
Pro Leu Cys65 703492PRTArtificial SequenceSynthetic 34Val Lys Met
Ala Glu Thr Cys Pro Ile Phe Tyr Asp Val Phe Phe Ala1 5 10 15Val Ala
Asn Gly Asn Glu Leu Leu Leu Asp Leu Ser Leu Thr Lys Val 20 25 30Asn
Ala Thr Glu Pro Glu Arg Thr Ala Met Lys Lys Ile Gln Asp Cys 35 40
45Tyr Val Glu Asn Gly Leu Ile Ser Arg Val Leu Asp Gly Leu Val Met
50 55 60Thr Thr Ile Ser Ser Ser Lys Asp Cys Met Gly Glu Ala Val Gln
Asn65 70 75 80Thr Val Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg 85
9035360DNAArtificial Sequencesynthetic 35caggtgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg
ctccatcagt agttactact ggagctggat ccggcagccc 120ccagggaagg
gactggagtg gattggatat atctattaca gtgggagaac caactacaac
180ccctccctca agagtcgagt caccatatca gtggacacgt ccaagaacca
gttctccctg 240aaactgagct ctgtgaccgc cgcagacacg gccatttatt
actgtgcgag acatcgtgta 300actagaactg cggactcctt tgactactgg
ggccagggaa ccctggtcac cgtctcctca 36036120PRTArtificial
Sequencesynthetic 36Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly
Ser Ile Ser Ser Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Tyr Tyr Ser Gly Arg Thr
Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp
Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Arg His Arg Val Thr
Arg Thr Ala Asp Ser Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu
Val Thr Val Ser Ser 115 1203724DNAArtificial Sequencesynthetic
37ggtggctcca tcagtagtta ctac 24388PRTArtificial Sequencesynthetic
38Gly Gly Ser Ile Ser Ser Tyr Tyr1 53921DNAArtificial
Sequencesynthetic 39atctattaca gtgggagaac c 21407PRTArtificial
Sequencesynthetic 40Ile Tyr Tyr Ser Gly Arg Thr1 54142DNAArtificial
Sequencesynthetic 41gcgagacatc gtgtaactag aactgcggac tcctttgact ac
424214PRTArtificial Sequencesynthetic 42Ala Arg His Arg Val Thr Arg
Thr Ala Asp Ser Phe Asp Tyr1 5 1043321DNAArtificial
Sequencesynthetic 43gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc aggcgagtca ggacattaac aactatttaa
attggtatca gcagaaaaca 120gggaaagccc ctaagttcct gatctacgat
gcatccaatt tggaaacagg ggtctcatca 180aggttcagtg gaagtggatc
tgggacagat tttactttca ccatcagcag cctgcagcct 240gaagatgttg
gaacatatta ctgtcaccag tatggtgatc tcccgtacac ttttggccag
300gggaccaagc tggagatcaa a 32144107PRTArtificial Sequencesynthetic
44Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Asn
Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Thr Gly Lys Ala Pro Lys Phe
Leu Ile 35 40 45Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Ser Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Val Gly Thr Tyr Tyr Cys His Gln
Tyr Gly Asp Leu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 1054518DNAArtificial Sequencesynthetic 45caggacatta
acaactat 18466PRTArtificial Sequencesynthetic 46Gln Asp Ile Asn Asn
Tyr1 5479DNAArtificial Sequencesynthetic 47gatgcatcc
9483PRTArtificial Sequencesynthetic 48Asp Ala Ser14927DNAArtificial
Sequencesynthetic 49caccagtatg gtgatctccc gtacact
27509PRTArtificial Sequencesynthetic 50His Gln Tyr Gly Asp Leu Pro
Tyr Thr1 551413PRTArtificial SequencehFceR1a-mFc 51Val Pro Gln Lys
Pro Lys Val Ser Leu Asn Pro Pro Trp Asn Arg Ile1 5 10 15Phe Lys Gly
Glu Asn Val Thr Leu Thr Cys Asn Gly Asn Asn Phe Phe 20 25 30Glu Val
Ser Ser Thr Lys Trp Phe His Asn Gly Ser Leu Ser Glu Glu 35 40 45Thr
Asn Ser Ser Leu Asn Ile Val Asn Ala Lys Phe Glu Asp Ser Gly 50 55
60Glu Tyr Lys Cys Gln His Gln Gln Val Asn Glu Ser Glu Pro Val Tyr65
70 75 80Leu Glu Val Phe Ser Asp Trp Leu Leu Leu Gln Ala Ser Ala Glu
Val 85 90 95Val Met Glu Gly Gln Pro Leu Phe Leu Arg Cys His Gly Trp
Arg Asn 100 105 110Trp Asp Val Tyr Lys Val Ile Tyr Tyr Lys Asp Gly
Glu Ala Leu Lys 115 120 125Tyr Trp Tyr Glu Asn His Asn Ile Ser Ile
Thr Asn Ala Thr Val Glu 130 135 140Asp Ser Gly Thr Tyr Tyr Cys Thr
Gly Lys Val Trp Gln Leu Asp Tyr145 150 155 160Glu Ser Glu Pro Leu
Asn Ile Thr Val Ile Lys Ala Pro Arg Glu Lys 165 170 175Tyr Trp Leu
Gln Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro 180 185 190Cys
Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile 195 200
205Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile
210 215 220Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp
Val Gln225 230 235 240Ile Ser Trp Phe Val Asn Asn Val Glu Val His
Thr Ala Gln Thr Gln 245 250 255Thr His Arg Glu Asp Tyr Asn Ser Thr
Leu Arg Val Val Ser Ala Leu 260 265 270Pro Ile Gln His Gln Asp Trp
Met Ser Gly Lys Glu Phe Lys Cys Lys 275 280 285Val Asn Asn Lys Asp
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys 290 295 300Pro Lys Gly
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro305 310 315
320Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr
325 330 335Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn
Gly Lys 340 345 350Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu
Asp Ser Asp Gly 355 360 365Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val
Glu Lys Lys Asn Trp Val 370 375 380Glu Arg Asn Ser Tyr Ser Cys Ser
Val Val His Glu Gly Leu His Asn385 390 395 400His His Thr Thr Lys
Ser Phe Ser Arg Thr Pro Gly Lys 405 41052199PRTArtificial
SequenceFeld1-mmH 52Val Lys Met Ala Glu Thr Cys Pro Ile Phe Tyr Asp
Val Phe Phe Ala1 5 10 15Val Ala Asn Gly Asn Glu Leu Leu Leu Asp Leu
Ser Leu Thr Lys Val 20 25 30Asn Ala Thr Glu Pro Glu Arg Thr Ala Met
Lys Lys Ile Gln Asp Cys 35 40 45Tyr Val Glu Asn Gly Leu Ile Ser Arg
Val Leu Asp Gly Leu Val Met 50 55 60Thr Thr Ile Ser Ser Ser Lys Asp
Cys Met Gly Glu Ala Val Gln Asn65 70 75 80Thr Val Glu Asp Leu Lys
Leu Asn Thr Leu Gly Arg Glu Ile Cys Pro 85 90 95Ala Val Lys Arg Gly
Val Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu 100 105 110Tyr Val Glu
Gln Val Ala Gln Tyr Lys Ala Leu Pro Val Val Leu Glu 115 120 125Asn
Ala Arg Ile Leu Lys Asn Cys Val Asp Ala Lys Met Thr Glu Glu 130 135
140Asp Lys Glu Asn Ala Leu Ser Leu Leu Asp Lys Ile Tyr Thr Ser
Pro145 150 155 160Leu Cys Gly Pro Gly Gly Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu 165 170 175Gly Gly Glu Gln Lys Leu Ile Ser Glu Glu
Asp Leu Ser Gly His His 180 185 190His His His His Ser Ser Gly
19553398PRTArtificial SequenceFeld1-mFc 53Val Lys Met Ala Glu Thr
Cys Pro Ile Phe Tyr Asp Val Phe Phe Ala1 5 10 15Val Ala Asn Gly Asn
Glu Leu Leu Leu Asp Leu Ser Leu Thr Lys Val 20 25 30Asn Ala Thr Glu
Pro Glu Arg Thr Ala Met Lys Lys Ile Gln Asp Cys 35 40 45Tyr Val Glu
Asn Gly Leu Ile Ser Arg Val Leu Asp Gly Leu Val Met 50 55 60Thr Thr
Ile Ser Ser Ser Lys Asp Cys Met Gly Glu Ala Val Gln Asn65 70 75
80Thr Val Glu Asp Leu Lys Leu Asn Thr Leu Gly Arg Glu Ile Cys Pro
85 90 95Ala Val Lys Arg Asp Val
Asp Leu Phe Leu Thr Gly Thr Pro Asp Glu 100 105 110Tyr Val Glu Gln
Val Ala Gln Tyr Lys Ala Leu Pro Val Val Leu Glu 115 120 125Asn Ala
Arg Ile Leu Lys Asn Cys Val Asp Ala Lys Met Thr Glu Glu 130 135
140Asp Lys Glu Asn Ala Leu Ser Leu Leu Asp Lys Ile Tyr Thr Ser
Pro145 150 155 160Leu Cys Leu Ile Asn Glu Pro Arg Gly Pro Thr Ile
Lys Pro Cys Pro 165 170 175Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu
Gly Gly Pro Ser Val Phe 180 185 190Ile Phe Pro Pro Lys Ile Lys Asp
Val Leu Met Ile Ser Leu Ser Pro 195 200 205Ile Val Thr Cys Val Val
Val Asp Val Ser Glu Asp Asp Pro Asp Val 210 215 220Gln Ile Ser Trp
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr225 230 235 240Gln
Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala 245 250
255Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
260 265 270Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr
Ile Ser 275 280 285Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
Val Leu Pro Pro 290 295 300Pro Glu Glu Glu Met Thr Lys Lys Gln Val
Thr Leu Thr Cys Met Val305 310 315 320Thr Asp Phe Met Pro Glu Asp
Ile Tyr Val Glu Trp Thr Asn Asn Gly 325 330 335Lys Thr Glu Leu Asn
Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp 340 345 350Gly Ser Tyr
Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 355 360 365Val
Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His 370 375
380Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys385 390
395
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