U.S. patent application number 14/017333 was filed with the patent office on 2014-03-13 for methods for treating atopic dermatitis by administering an il-4r antagonist.
This patent application is currently assigned to Regeneron Pharmaceuticals, Inc.. The applicant listed for this patent is Regeneron Pharmaceuticals, Inc.. Invention is credited to Marius ARDELEANU, Neil GRAHAM, Jennifer Davidson HAMILTON, Stephane C. KIRKESSELI, Sudeep KUNDU, Jeffrey MING, Allen RADIN, Ross E. ROCKLIN, Steven Paul WEINSTEIN.
Application Number | 20140072583 14/017333 |
Document ID | / |
Family ID | 50233497 |
Filed Date | 2014-03-13 |
United States Patent
Application |
20140072583 |
Kind Code |
A1 |
ARDELEANU; Marius ; et
al. |
March 13, 2014 |
METHODS FOR TREATING ATOPIC DERMATITIS BY ADMINISTERING AN IL-4R
ANTAGONIST
Abstract
The present invention provides methods for treating atopic
dermatitis (AD). Also provided are methods for improving one or
more AD-associated parameter(s), and methods for decreasing the
level of at least one AD-associated biomarker in a subject in need
thereof. The methods of the present invention comprise
administering to a subject in need thereof a pharmaceutical
composition comprising an interleukin-4 receptor (IL-4R) antagonist
such as an anti-IL-4R antibody.
Inventors: |
ARDELEANU; Marius; (White
Plains, NY) ; GRAHAM; Neil; (Croton-on-Hudson,
NY) ; HAMILTON; Jennifer Davidson; (Hopewell
Junction, NY) ; KIRKESSELI; Stephane C.; (Paris,
FR) ; KUNDU; Sudeep; (New York, NY) ; MING;
Jeffrey; (Bridgewater, NJ) ; RADIN; Allen;
(New York, NY) ; ROCKLIN; Ross E.; (Basking Ridge,
NJ) ; WEINSTEIN; Steven Paul; (Hartsdale,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regeneron Pharmaceuticals, Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Regeneron Pharmaceuticals,
Inc.
Tarrytown
NY
|
Family ID: |
50233497 |
Appl. No.: |
14/017333 |
Filed: |
September 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61697972 |
Sep 7, 2012 |
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61738715 |
Dec 18, 2012 |
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61748588 |
Jan 3, 2013 |
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61764624 |
Feb 14, 2013 |
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61768229 |
Feb 22, 2013 |
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61770091 |
Feb 27, 2013 |
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61782420 |
Mar 14, 2013 |
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61816191 |
Apr 26, 2013 |
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Current U.S.
Class: |
424/172.1 |
Current CPC
Class: |
A61K 31/573 20130101;
G01N 2800/202 20130101; A61P 17/02 20180101; A61P 17/04 20180101;
A61K 31/58 20130101; A61P 17/00 20180101; C07K 2317/21 20130101;
G01N 33/53 20130101; A61P 43/00 20180101; A61K 39/3955 20130101;
A61K 2039/54 20130101; A61K 45/06 20130101; A61P 37/00 20180101;
C07K 16/2866 20130101; A61P 35/00 20180101; A61P 37/08
20180101 |
Class at
Publication: |
424/172.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/58 20060101 A61K031/58; A61K 31/573 20060101
A61K031/573; A61K 45/06 20060101 A61K045/06; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 10, 2013 |
FR |
1356759 |
Claims
1-149. (canceled)
150. A method of treating moderate-to-severe atopic dermatitis (AD)
in a patient, the method comprising administering to the patient a
pharmaceutical composition comprising a therapeutically effective
amount of an antibody or antigen-binding fragment thereof that
specifically binds interleukin-4-receptor (IL-4R).
151. The method of claim 150, wherein the antibody or
antigen-binding fragment thereof that binds IL-4R comprises
complementarity determining regions (CDRs) in a heavy chain
variable region (HCVR)/light chain variable region (LCVR) sequence
pair of SEQ ID NOs: 162/164.
152. The method of claim 150, wherein the antibody or
antigen-binding fragment that binds IL-4R comprises three heavy
chain complementarity determining region (HCDR) sequences
comprising SEQ ID NOs: 148, 150, 152, respectively, and three light
chain complementarity determining (LCDR) sequences comprising SEQ
ID NOs: 156, 158 and 160, respectively.
153. The method of claim 152, wherein the antibody or
antigen-binding fragment that binds IL-4R comprises an HCVR having
the amino acid sequence of SEQ ID NO: 162 and an LCVR having the
amino acid sequence of SEQ ID NO: 164.
154. The method of claim 150, wherein the patient is resistant,
non-responsive or inadequately responsive to treatment by either a
topical corticosteroid (TCS) or a calcineurin inhibitor.
155. The method of claim 150, wherein the patient, following
administration of the pharmaceutical composition, exhibits an
improvement in one or more AD-associated parameters.
156. The method of claim 155, wherein the improvement in the one or
more AD-associated parameters is selected from the group consisting
of: (a) a decrease from baseline in Investigator's Global
Assessment (IGA) score of at least 25%; (b) a decrease from
baseline in Body Surface Area Involvement of Atopic Dermatitis
(BSA) score of at least 35%; (c) a decrease from baseline in Eczema
Area and Severity Index (EASI) score of at least 45%; (d) a
decrease from baseline in SCORAD score of at least 30%; (e) a
decrease from baseline in 5-D Pruritus Scale of at least 15%; and
(f) a decrease from baseline in Pruritus Numeric Rating Scale (NRS)
score of at least 25%.
157. The method of claim 156, wherein the improvement in an
AD-associated parameter is a decrease from baseline in IGA of at
least 25% on day 22 through at least day 85 after administration of
the pharmaceutical composition.
158. The method of claim 156, wherein the improvement in an
AD-associated parameter is a decrease from baseline in BSA score of
at least 40% on day 29 through at least day 85 after administration
of the pharmaceutical composition.
159. The method of claim 156, wherein the improvement in an
AD-associated parameter is a decrease from baseline in EASI score
of at least 50% on day 29 through at least day 85 after
administration of the pharmaceutical composition.
160. The method of claim 156, wherein the improvement in an
AD-associated parameter is a decrease from baseline in SCORAD score
of at least 30% on day 29 through at least day 85 after
administration of the pharmaceutical composition.
161. The method of claim 156, wherein the improvement in an
AD-associated parameter is a decrease from baseline in 5-D Pruritus
Scale of at least 15% on day 15 through at least day 85 after
administration of the pharmaceutical composition.
162. The method of claim 156, wherein the improvement in an
AD-associated parameter is a decrease from baseline in NRS score of
at least 25% at the end of week 2 through at least the end of week
10 after administration of the pharmaceutical composition.
163. The method of claim 150, wherein the pharmaceutical
composition comprises about 50 mg to about 600 mg of the antibody
or antigen-binding fragment thereof.
164. The method of claim 163, wherein the pharmaceutical
composition comprises about 75 mg to about 300 mg of the antibody
or antigen-binding fragment thereof.
165. The method of claim 150, wherein the pharmaceutical
composition is administered to the patient subcutaneously or
intravenously.
166. The method of claim 150, wherein a second therapeutic agent is
administered to the patient before, after or concurrent with the
pharmaceutical composition.
167. The method of claim 166, wherein the second therapeutic agent
is selected from the group consisting of a TCS and calcineurin
inhibitor.
168. A method for treating moderate-to-severe AD in a patient, the
method comprising: (a) selecting a patient who exhibits an elevated
level of at least one AD-associated biomarker prior to, or at the
time of treatment; and (b) administering to the patient a
pharmaceutical composition comprising a therapeutically effective
amount of an antibody or antigen-binding fragment thereof that
specifically binds IL-4R.
169. The method of claim 168, wherein the antibody or
antigen-binding fragment thereof comprises heavy and light chain
CDRs in a HCVR/LCVR sequence pair of SEQ ID NOs: 162/164.
170. The method of claim 168, wherein the antibody or
antigen-binding fragment thereof comprises three heavy chain
complementarity determining region (HCDR) sequences comprising SEQ
ID NOs: 148, 150, 152, respectively, and three light chain
complementarity determining region (LCDR) sequences comprising SEQ
ID NOs: 156, 158 and 160, respectively.
171. The method of claim 170, wherein the antibody or
antigen-binding fragment thereof comprises an HCVR having the amino
acid sequence of SEQ ID NO: 162 and an LCVR having the amino acid
sequence of SEQ ID NO: 164.
172. The method of claim 168, wherein the AD-associated biomarker
is IgE.
173. The method of claim 172, wherein the patient is selected on
the basis of exhibiting an IgE level of greater than 1500 kU/L
prior to or at the time of treatment ("baseline").
174. The method of claim 168, wherein the AD-associated biomarker
is Thymus and Activation Regulated Chemokine (TARC).
175. The method of claim 174, wherein the patient is selected on
the basis of exhibiting a TARC level of greater than 1000 pg/mL
prior to or at the time of treatment ("baseline").
176. The method of claim 173, wherein the patient exhibits between
about 5% and 20% decrease in IgE level from baseline at day 36 or
later following the administration of the pharmaceutical
composition.
177. The method of claim 175, wherein the patient exhibits between
about 25% and 70% decrease in TARC level from baseline at day 4 or
later following the administration of the pharmaceutical
composition.
178. The method of claim 168, wherein administration of the
pharmaceutical composition to the patient results in a decrease in
at least one AD-associated biomarker in the patient by day 4, 8,
15, 22, 25, 29 or 36 following administration of the pharmaceutical
composition as compared to the level of the biomarker in the
patient prior to the administration.
179. The method of claim 168, wherein the patient is resistant,
non-responsive or inadequately responsive to either a TCS or a
calcineurin inhibitor.
180. The method of claim 168, wherein the pharmaceutical
composition comprises about 50 mg to about 600 mg of the antibody
or antigen-binding fragment that specifically binds IL-4R.
181. The method of claim 180, wherein the pharmaceutical
composition comprises about 75 mg to about 300 mg of the antibody
or antigen-binding fragment that specifically binds IL-4R.
182. The method of claim 168, wherein the pharmaceutical
composition is administered subcutaneously or intravenously to the
patient.
183. The method of claim 168, wherein a second therapeutic agent is
administered to the subject before, after or concurrent with the
pharmaceutical composition.
184. The method of claim 183, wherein the second therapeutic agent
is selected from the group consisting of a TCS and a calcineurin
inhibitor.
185. A method for improving one or more atopic dermatitis
(AD)-associated parameter(s) in a patient in need thereof, or
reducing the level of at least one AD-associated biomarker in the
patient, the method comprising sequentially administering to a
patient in need thereof a single initial dose of a pharmaceutical
composition comprising an antibody or antigen-binding fragment that
specifically binds IL-4R, followed by one or more secondary doses
of the pharmaceutical composition comprising the antibody or
fragment thereof.
186. The method of claim 185, wherein the antibody or fragment
thereof comprises heavy and light chain CDR sequences in a
HCVR/LCVR sequence pair of SEQ ID NOs: 162/164.
187. The method of claim 185, wherein the antibody or
antigen-binding fragment thereof comprises three heavy chain
complementarity determining region (HCDR) sequences comprising SEQ
ID NOs: 148, 150, 152, respectively, and three light chain
complementarity determining (LCDR) sequences comprising SEQ ID NOs:
156, 158 and 160, respectively.
188. The method of claim 187, wherein the antibody or
antigen-binding fragment thereof comprises an HCVR having the amino
acid sequence of SEQ ID NO: 162 and an LCVR having the amino acid
sequence of SEQ ID NO: 164.
189. The method of claim 185, wherein each secondary dose is
administered 1 to 8 weeks after the immediately preceding dose.
190. The method of claim 189, wherein the one or more secondary
doses of the pharmaceutical composition are administered
weekly.
191. The method of claim 185, wherein at least 4 secondary doses of
the anti-IL-4R antibody are administered to the patient, and
wherein each secondary dose is administered 1 week after the
immediately preceding dose.
192. The method of claim 185, wherein the initial dose and the one
or more secondary doses each comprise 50 mg to 600 mg of the
anti-IL-4R antibody.
193. The method of claim 192, wherein the initial dose and the one
or more secondary doses each comprise 75 mg to 300 mg of the
anti-IL-4R antibody.
194. The method of claim 185, wherein the initial dose comprises a
first amount of the antibody or antigen-binding fragment thereof
and the one or more secondary doses each comprise a second amount
of antibody or fragment thereof.
195. The method of claim 194, wherein the first amount of antibody
or antigen-binding fragment thereof is 1.5.times., 2.times.,
2.5.times. or 3.times. the second amount of the antibody or
antigen-binding fragment thereof.
196. The method of claim 185, wherein the pharmaceutical
composition is administered to the patient subcutaneously or
intravenously.
197. The method of claim 185, wherein a second therapeutic agent is
administered to the patient before, after or concurrent with one or
more doses of the pharmaceutical composition.
198. The method of claim 197, wherein the second therapeutic agent
is selected from the group consisting of a TCS and a calcineurin
inhibitor.
199. A method of improving one or more atopic dermatitis
(AD)-associated parameters in a patient in need thereof, the method
comprising: administering a therapeutically effective amount of an
anti-IL-4R antibody or antigen-binding fragment thereof
concomitantly with a topical corticosteroid (TCS) to the patient,
wherein the improvement in the one or more AD-associated parameters
is selected from the group consisting of: (a) a decrease from
baseline in Investigator's Global Assessment (IGA) score of at
least 45%; (b) a decrease from baseline in Pruritus Numeric Rating
Scale (NRS) score of at least 60%; (c) a decrease from baseline in
Eczema Area and Severity Index (EASI) score of at least 65%; and
(d) a decrease from baseline in SCORAD score of at least 50%.
200. The method of claim 199, wherein the anti-IL-4R antibody or
fragment thereof comprises heavy and light chain CDR sequences from
the HCVR/LCVR sequence pair of SEQ ID NOs: 162/164.
201. The method of claim 199, wherein the anti-IL-4R antibody or
antigen-binding fragment thereof comprises three heavy chain
complementarity determining region (HCDR) sequences comprising SEQ
ID NOs: 148, 150, 152, respectively, and three light chain
complementarity determining (LCDR) sequences comprising SEQ ID NOs:
156, 158 and 160, respectively.
202. The method of claim 199, wherein the anti-IL-4R antibody or
antigen-binding fragment thereof comprises an HCVR having the amino
acid sequence of SEQ ID NO: 162 and an LCVR having the amino acid
sequence of SEQ ID NO: 164.
203. The method of claim 199, wherein the patient has
moderate-to-severe AD.
204. The method of claim 199, wherein the TCS is selected from the
group consisting of a group I TCS, group II TCS and group III
TCS.
205. The method of claim 199, wherein the TCS is selected from the
group consisting of methylprednisolone aceponate, mometasone
furoate, fluticasone propionate, betamethasone valerate and
hydrocortisone butyrate.
206. The method of claim 199, wherein the improvement in the one or
more AD-associated parameters is a decrease from baseline in IGA of
at least 50% on day 29 after administration of the antibody or
antigen-binding fragment thereof.
207. The method of claim 199, wherein the improvement in the one or
more AD-associated parameters is a decrease from baseline in NRS of
at least 65% on day 29 after administration of the antibody or
antigen-binding fragment thereof.
208. The method of claim 199, wherein the improvement in the one or
more AD-associated parameters is a decrease from baseline in EASI
of at least 70% on day 29 after administration of the antibody or
antigen-binding fragment thereof.
209. The method of claim 199, wherein the improvement in the one or
more AD-associated parameters is a decrease from baseline in SCORAD
of at least 60% on day 29 after administration of the antibody or
antigen-binding fragment thereof.
210. A method to reduce dependence on a topical corticosteroid
(TCS) for controlling at least one symptom in a patient with
moderate-to-severe AD, the method comprising: selecting a patient
with moderate-to-severe AD; administering a therapeutically
effective amount of an anti-IL-4R antibody or antigen-binding
fragment thereof concomitantly with a TCS to the patient; and
gradually reducing the amount of the TCS, while maintaining the
dose of the anti-IL-4R antibody or antigen-binding fragment
thereof.
211. The method of claim 210, wherein the antibody or fragment
thereof comprises heavy and light chain CDR sequences from the
HCVR/LCVR sequence pair of SEQ ID NOs: 162/164.
212. The method of claim 210, wherein the dosage of TCS is reduced
by about 20% to about 50% over 4 weeks in a patient treated with
the anti-IL-4R antibody as compared to a patient not treated with
anti-IL-4R antibody.
213. The method of claim 210, wherein the antibody or
antigen-binding fragment thereof is administered at a dose of about
50-600 mg.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. provisional application Nos. 61/697,972, filed
on Sep. 7, 2012; 61/738,715, filed on Dec. 18, 2012; 61/748,588,
filed on Jan. 3, 2013; 61/764,624, filed on Feb. 14, 2013;
61/768,229, filed on Feb. 22, 2013; 61/770,091, filed on Feb. 27,
2013; 61/782,420, filed on Mar. 14, 2013; and 61/816,191, filed on
Apr. 26, 2013, and under 35 U.S.C. .sctn.119(b) of French
application No. 1356759, filed on Jul. 10, 2013, the disclosures of
each herein incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to the treatment and/or
prevention of atopic dermatitis and related conditions. More
specifically, the invention relates to the administration of
interleukin-4 receptor (IL-4R) antagonists to treat or prevent
atopic dermatitis in a patient in need thereof.
BACKGROUND
[0003] Atopic dermatitis (AD) is a chronic/relapsing inflammatory
skin disease characterized by intense pruritus (e.g., severe itch)
and by scaly and dry eczematous lesions. AD is often associated
with other atopic disorders such as allergic rhinitis and asthma.
Severe disease can be extremely disabling due to major
psychological problems, significant sleep loss, and impaired
quality of life, leading to high socioeconomic costs.
[0004] The pathophysiology of AD is influenced by a complex
interplay between Immunoglobulin E (IgE)-mediated sensitization,
the immune system, and environmental factors. The primary skin
defect may be an immunological disturbance that causes IgE-mediated
sensitization, with epithelial-barrier dysfunction that is the
consequence of both genetic mutations and local inflammation. AD
often begins in childhood before age 5 and may persist into
adulthood.
[0005] Typical treatments for AD include topical lotions and
moisturizers, topical corticosteroid ointments, creams or
injections. Most treatment options, however, offer only temporary,
incomplete, symptom relief. Moreover, many patients with
moderate-to-severe AD become resistant to treatment by topical
corticosteroids or by calcineurin inhibitors. Thus, a need exists
in the art for novel targeted therapies for the treatment and/or
prevention of AD.
BRIEF SUMMARY OF THE INVENTION
[0006] According to certain aspects of the present invention,
methods are provided for treating, preventing and/or reducing the
severity of symptoms of atopic dermatitis (AD), including
moderate-to-severe AD. Certain embodiments of the invention pertain
to methods for treating, ameliorating or preventing
moderate-to-severe AD in a patient who is resistant to treatment by
a topical corticosteroid or a calcineurin inhibitor. In some
embodiments, the present invention discloses methods of treating
patients with moderate-to-severe AD that is uncontrolled despite
treatment with a topical corticosteroid or a calcineurin inhibitor.
The methods of the present invention comprise administering to a
subject or a patient in need thereof a pharmaceutical composition
comprising a therapeutically effective amount of an interleukin-4
receptor (IL-4R) antagonist. According to certain embodiments of
the present invention, the IL-4R antagonist is an antibody or
antigen-binding fragment thereof that specifically binds IL-4R.
Exemplary anti-IL-4R antibodies that can be used in the context of
the methods of the present invention are described elsewhere
herein, including working Example 1. In certain embodiments, the
IL-4R antagonist is an anti-IL-4R antibody having the binding
characteristics of the reference antibody referred to herein as
"mAb1" (e.g., an antibody or antigen-binding fragment thereof
comprising the complementarity determining regions of mAb1). In one
embodiment, the antibody or antigen-binding fragment thereof that
binds IL-4R comprises complementarity determining regions (CDRs) in
a heavy chain variable region (HCVR)/light chain variable region
(LCVR) sequence pair of SEQ ID NOs: 162/164.
[0007] Some embodiments of the invention are directed to methods
for treating, reducing, ameliorating or preventing pruritus in a
patient, comprising administration of a pharmaceutical composition
comprising a therapeutically effective amount of an IL-4R
antagonist. In one embodiment, the patient suffers from
moderate-to-severe AD. In some embodiments, the patient suffering
from AD is resistant to treatment by either a topical
corticosteroid or a calcineurin inhibitor.
[0008] In certain embodiments, the present invention includes
methods to treat moderate-to-severe AD in a patient, the methods
comprising administering a pharmaceutical composition comprising a
therapeutically effective amount of an antibody or antigen-binding
fragment thereof that binds IL-4R, and determining an improvement
in an AD-associated parameter. The improvement can be determined or
assayed or quantitated by methods well-known in the art.
AD-associated parameters and improvements therein are discussed
elsewhere herein, including e.g., in working Example 7.
[0009] According to certain exemplary embodiments, the present
invention provides methods for improving one or more AD-associated
parameter(s) in a subject in need thereof. Improvements in
AD-associated parameters include, e.g., a decrease in
Investigator's Global Assessment (IGA) score; a decrease in Body
Surface Area Involvement of Atopic Dermatitis (BSA) score; a
decrease in Eczema Area and Severity Index (EASI) score; a decrease
in SCORAD score; a decrease in 5-D Pruritus Scale; and/or a
decrease in Pruritus Numeric Rating Scale (NRS) score. In exemplary
embodiments, the improvement in an AD-associated parameter is
selected from the group consisting of: (i) a decrease from baseline
in IGA score of at least 25%; (ii) a decrease from baseline in BSA
score of at least 35%; (iii) a decrease from baseline in EASI score
of at least 45%; (iv) a decrease from baseline in SCORAD score of
at least 30%; (v) a decrease from baseline in 5-D Pruritus scale of
at least 15%; (vi) a decrease from baseline in Pruritus NRS score
of at least 25%; and (vii) percent responders with .gtoreq.50%
improvement in EASI (EASI50).
[0010] In some embodiments, the improvement in an AD-associated
parameter comprises a decrease from baseline in IGA of at least 25%
on day 22 through at least day 85 after administration of a
pharmaceutical composition comprising an antibody or
antigen-binding fragment thereof that binds IL-4R. In some
embodiments, the improvement in an AD-associated parameter
comprises a decrease from baseline in BSA score of at least 40% on
day 29 through at least day 85 following administration of the
pharmaceutical composition. In some embodiments, the improvement in
an AD-associated parameter comprises a decrease from baseline in
EASI score of at least 50% on day 29 through at least day 85 after
administration of the pharmaceutical composition. In certain
embodiments, the improvement in an AD-associated parameter
comprises a decrease from baseline in EASI score of at least 50% on
day 29 in at least 70% of subjects administered with the
pharmaceutical composition. In some embodiments, the improvement in
an AD-associated parameter comprises a decrease from baseline in
SCORAD score of at least 30% on day 29 through at least day 85
following administration of the pharmaceutical composition. In some
embodiments, the improvement in an AD-associated parameter
comprises a decrease from baseline in 5-D pruritus Scale of at
least 15% on day 15 through at least day 85 after administration of
the pharmaceutical composition. In some embodiments, the
improvement in an AD-associated parameter comprises a decrease from
baseline in NRS score of at least 25% at the end of week 2 through
at least the end of week 10 after administration of the
pharmaceutical composition.
[0011] In some embodiments, the improvement in an AD-associated
parameter comprises a decrease from baseline in IGA of at least 45%
on day 85 through at least day 197 after administration of a
pharmaceutical composition comprising a therapeutically effective
amount of an IL-4R antagonist. In some embodiments, the improvement
in an AD-associated parameter comprises a decrease from baseline in
BSA score of at least 50% on day 85 through at least day 197 after
administration of a pharmaceutical composition comprising a
therapeutically effective amount of an IL-4R antagonist. In some
embodiments, the improvement in an AD-associated parameter
comprises a decrease from baseline in EASI score of at least 60% on
day 85 through at least day 197 after administration of a
pharmaceutical composition comprising a therapeutically effective
amount of an IL-4R antagonist. In some embodiments, the improvement
in an AD-associated parameter comprises a decrease from baseline in
SCORAD score of at least 45% on day 85 through at least day 197
after administration of a pharmaceutical composition comprising a
therapeutically effective amount of an IL-4R antagonist. In some
embodiments, the improvement in an AD-associated parameter
comprises a decrease from baseline in 5-D Pruritus scale of at
least 30% on day 85 through at least day 197 after administration
of a pharmaceutical composition comprising a therapeutically
effective amount of an IL-4R antagonist. In some embodiments, the
improvement in an AD-associated parameter comprises a decrease from
baseline in NRS score of at least 50% on day 85 through at least
day 197 after administration of a pharmaceutical composition
comprising a therapeutically effective amount of an IL-4R
antagonist.
[0012] According to other exemplary embodiments, the present
invention provides methods for treating AD in a subject, the
methods comprising: (a) selecting a subject who exhibits an
elevated level of at least one AD-associated biomarker; and (b)
administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of an IL-4R
antagonist. In certain embodiments, the IL-4R antagonist is an
antibody or antigen-binding fragment thereof that binds IL-4R.
Exemplary AD-associated biomarkers that can be evaluated and/or
measured in the context of the present invention include, but are
not limited to, thymus and activation-regulated chemokine (TARC;
also known as CCL17), immunoglobulin E (IgE), eotaxin-3, lactate
dehydrogenase (LDH), eosinophils, antigen-specific IgE (e.g.,
Phadiatop.TM. test), and periostin. In some embodiments, the
methods of the present invention comprise determining the level of
an AD-associated biomarker in a patient in need thereof, selecting
a patient with an elevated level of the AD-associated biomarker,
and administering a therapeutically effective amount of an antibody
or antigen-binding fragment thereof that specifically binds IL-4R.
In some embodiments, the patient is selected by acquiring
information about the level of an AD-associated biomarker in a
patient. In some embodiments, the level of an AD-associated
biomarker is determined by an assay or test known in the art or as
disclosed elsewhere herein. In one embodiment, the patient is
selected on the basis of exhibiting an IgE level greater than about
1500 kU/L prior to or at the time of treatment. In one embodiment,
the patient is selected on the basis of exhibiting a TARC level of
greater than about 1000 pg/mL prior to or at the time of treatment.
According to a related aspect of the present invention, methods for
treating AD are provided which comprise administering to a subject
a pharmaceutical composition comprising a therapeutically effective
amount of an IL-4R antagonist, wherein administration of the
pharmaceutical composition to the subject results in a decrease in
at least one AD-associated biomarker by day 4, 8, 15, 22, 25, 29,
36 or later in the subject following administration. In certain
embodiments, the patient exhibits between 5% and 20% decrease in
IgE level from the baseline at day 36 or later following
administration. In certain embodiments, the patient exhibits
between 25% and 70% decrease in TARC level from baseline at day 4
or later following administration.
[0013] The present invention also provides methods for decreasing
the level of one or more AD-associated biomarker(s) in a subject,
or improving one or more AD-associated parameter(s) in a subject,
wherein the methods comprise sequentially administering to a
subject in need thereof a single initial dose of a pharmaceutical
composition comprising an IL-4R antagonist, followed by one or more
secondary doses of the pharmaceutical composition comprising the
IL-4R antagonist.
[0014] According to certain embodiments, the present invention
provides methods for decreasing the level of one or more
AD-associated biomarker(s) in a subject, or improving one or more
AD-associated parameter(s) in a subject, wherein the methods
comprise administering to the subject about 50 mg to about 600 mg
of a pharmaceutical composition comprising an antibody or
antigen-binding fragment thereof that specifically binds IL-4R. In
certain embodiments, the initial dose and the one or more secondary
doses each comprise about 75 mg to about 300 mg of the antibody or
antigen-binding fragment thereof. According to this aspect of the
invention, the pharmaceutical composition may be administered to
the subject at a dosing frequency of, e.g., once a week. In some
embodiments, each secondary dose is administered 1 to 8 weeks after
the immediately preceding dose. In certain embodiments, at least 4
doses of the antibody or antigen-binding fragment thereof are
administered. In one embodiment, each secondary dose is
administered 1 week after the immediately preceding dose. In
certain embodiments, the initial dose comprises a first amount of
the antibody or antigen-binding fragment thereof and the one or
more secondary doses each comprise a second amount of the antibody
or antigen-binding fragment thereof. In some embodiments, the first
amount of antibody or fragment thereof is 1.5.times., 2.times.,
2.5.times., 3.times., 3.5.times., 4.times., or 5.times. the second
amount of the antibody or antigen-binding fragment thereof. In some
embodiments, the pharmaceutical composition is administered
subcutaneously or intravenously.
[0015] In some embodiments, the present invention provides methods
for treating moderate-to-severe AD comprising concomitant
administration of an IL-4R antagonist and a topical corticosteroid
(TCS). In some embodiments, the methods further comprise assaying
for an improvement in an AD-associated parameter. In certain
embodiments, the invention provides for methods for improving one
or more AD-associated parameters, the methods comprising
concomitantly administering an IL-4R antagonist and a TCS, wherein
an improvement in an AD-associated parameter is selected from the
group consisting of: (i) a decrease from baseline in IGA score of
at least 45%; (ii) a decrease from baseline in BSA score of at
least 40%; (iii) a decrease from baseline in EASI score of at least
65%; (iv) a decrease from baseline in SCORAD score of at least 50%;
(v) a decrease from baseline in 5-D Pruritus scale of at least 25%;
and (vi) a decrease from baseline in Pruritus NRS score of at least
60%. In some embodiments, the improvement in an AD-associated
parameter is a decrease from baseline in IGA of at least 50% on day
29 after administration of the antibody or antigen-binding fragment
thereof that binds IL-4R. In some embodiments, the improvement in
an AD-associated parameter is a decrease from baseline in NRS of at
least 65% on day 29 after administration. In some embodiments, the
improvement in an AD-associated parameter is a decrease from
baseline in EASI of at least 70% on day 29 after administration. In
some embodiments, the improvement in an AD-associated parameter is
a decrease from baseline in SCORAD of at least 60% on day 29 after
administration.
[0016] In certain embodiments, the TCS is selected from the group
consisting of a group I TCS, a group II TCS and a group III TCS. In
some embodiments, the TCS is selected from the group consisting of
methylprednisolone aceponate, mometasone furoate, fluticasone
propionate, betamethasone valerate and hydrocortisone butyrate.
[0017] In related embodiments, the invention provides for methods
to reduce the dependence on TCS in a patient with
moderate-to-severe AD comprising concomitant administration of an
IL-4R antagonist and a TCS, wherein the dosage of the TCS is
reduced by 50% as compared to subjects without the administration
of the IL-4R antagonist. In one embodiment, the invention provides
methods to reduce the dosage of a TCS in treatment of
moderate-to-severe AD, comprising administration of an IL-4R
antagonist concomitantly with a reduced dosage of the TCS. The
dosage of the TCS may be reduced by more than, for example, 10%,
20%, 30%, 40%, or 50%. In one embodiment, the dosage of the TCS may
be reduced by more than, for example, 10%, 20%, 30%, 40%, or 50% as
compared to the dosage used by the subject before treatment with
the IL-4R antagonist.
[0018] The present invention also includes an IL-4R antagonist as
disclosed herein for use in treating or preventing AD, for
improving an AD-associated parameter, for decreasing the level of
at least one AD-associated biomarker, and/or for treating any of
the other indications or conditions disclosed herein.
[0019] In certain embodiments, the IL-4R antagonist of the present
methods is an antibody or antigen-binding fragment that
specifically binds IL-4R and that comprises heavy and light chain
CDR sequences from a HCVR/LCVR sequence pair selected from the
group consisting of SEQ ID NOs: 2/10, 18/20, 22/24, 26/34, 42/44,
46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116,
118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168,
170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226,
234/236, 238/240, 242/250, 258/260 and 262/264. In one embodiment,
the antibody or antigen-binding fragment that specifically binds
IL-4R comprises heavy and light chain CDR sequences from the
HCVR/LCVR sequence pair of SEQ ID NOs: 162/164. In one embodiment,
the antibody or antigen-binding fragment that specifically binds
IL-4R comprises three heavy chain complementarity determining
region (HCDR) sequences comprising SEQ ID NOs: 148, 150, 152,
respectively, and three light chain complementarity determining
(LCDR) sequences comprising SEQ ID NOs: 156, 158 and 160,
respectively.
[0020] In some embodiments, the pharmaceutical composition is
administered subcutaneously or intravenously to the patient. In
some embodiments, the pharmaceutical composition comprises about 50
mg to about 600 mg of the antibody or antigen-binding fragment
thereof that binds IL-4R. In further embodiments, the
pharmaceutical composition comprises about 75 mg, about 100 mg,
about 150 mg, about 200 mg, about 250 mg or about 300 mg of the
antibody or fragment thereof that binds IL-4R.
[0021] In certain embodiments, the pharmaceutical composition is
administered to the patient before, after or concurrent with a
second therapeutic agent. In some embodiments, the second
therapeutic agent is a topical corticosteroid (TCS) or a
calcineurin inhibitor.
[0022] In another aspect, the invention provides monitoring the
effectiveness of treatment of moderate-to-severe AD in a subject
with an IL-4R antagonist, the method comprising: (a) determining
the expression level of an AD-associated biomarker, such as TARC or
serum IgE in a biological sample acquired from the subject before
treatment with the IL-4R antagonist; (b) determining the expression
level of one or both of TARC and serum IgE in a biological sample
acquired from the subject after treatment with the IL-4R
antagonist; (c) comparing the level determined in step (a) with the
level in step (b); and (d) concluding that the treatment is
effective when the level determined in step (b) is lower than the
level determined in step (a), or concluding that the treatment is
not effective when the level determined in step (b) is the same or
higher than the level determined in step (a). In one embodiment,
the level in step (b) is determined 1 week, 2 weeks, 3 weeks, 4
weeks, or 5 weeks after determining the level in step (a). In one
embodiment, the biomarker is TARC, and if TARC levels decrease
following administration of the IL-4R antagonist, then treatment
with the IL-4R antagonist is determined to be effective. In one
embodiment, the IL-4R antagonist is an anti-IL-4R antibody or
antigen-binding fragment thereof and comprises heavy and light
chain CDR sequences from a HCVR/LCVR sequence pair selected from
the group consisting of SEQ ID NOs: 2/10, 18/20, 22/24, 26/34,
42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106,
114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164,
166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216,
218/226, 234/236, 238/240, 242/250, 258/260 and 262/264.
[0023] The expression level of the biomarker can be determined, for
example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer
after administration of the IL-4R antagonist, and compared to the
expression level prior to administration of the antagonist. The
dose or the dosing regimen of the IL-4R antagonist (e.g., an
anti-IL4R antibody) can be adjusted following the determination.
For example, if the expression of the biomarker fails to decrease
within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer
following administration of the antagonist, then treatment with the
antagonist can be stopped, or the dose of the antagonist can be
increased. If expression of the biomarker decreases following
administration of the antagonist, the dosage of the antagonist can
be maintained or decreased, such as to identify a minimal effective
dose. In some embodiments, treatment is maintained at the minimal
effective dose.
[0024] In another aspect, the invention provides methods for
monitoring a subject's response to treatment with an IL-4R
antagonist, wherein the subject has moderate-to-severe AD, the
method comprising: (a) acquiring information regarding the
expression level of one or both of TARC and IgE in a biological
sample from the subject following administration of the IL-4R
antagonist to the subject; and (b) providing an indication that the
treatment should be continued if the expression level of TARC or
IgE has decreased as compared to the level before treatment with
the IL-4R antagonist. In one embodiment the biomarker is TARC, and
if TARC levels are determined to decrease following administration
of the antagonist, then an indication is provided to continue
treatment with the IL-4R antagonist.
[0025] The invention also includes an IL-4R antagonist as disclosed
herein for use in the manufacture of a medicament for the treatment
and/or prevention of atopic dermatitis (AD) (e.g., moderate to
severe eosinophilic AD, extrinsic AD, intrinsic AD, etc.) or for
treating any of the other indications or conditions disclosed
herein.
[0026] The invention also includes an IL-4R antagonist as disclosed
herein for use in the treatment and/or prevention of AD (e.g.,
moderate to severe eosinophilic AD, etc.) or for treating and/or
prevention of any of the other indications or conditions disclosed
herein.
[0027] In one embodiment, the IL-4R antagonist is an anti-IL-4R
antibody or antigen-binding fragment thereof.
[0028] The invention includes a pharmaceutical composition
comprising an anti-IL4R antibody antagonist or an antigen binding
fragment thereof for use in the treatment and/or prevention of AD
and related conditions.
[0029] The invention also includes a pharmaceutical composition
comprising an anti-IL4R antibody antagonist or an antigen binding
fragment thereof for use in improving one or more AD-associated
parameters in a subject in need thereof.
[0030] In addition, the invention includes a pharmaceutical
composition comprising an anti-IL4R antibody antagonist or an
antigen binding fragment thereof for use in reducing the level of
one or more AD-associated biomarkers in a subject in need
thereof.
[0031] The invention includes a pharmaceutical composition
comprising an anti-IL-4R antibody antagonist or an antigen binding
fragment thereof for use in the treatment of AD in a patient having
an elevated level of a biomarker selected from the group consisting
of thymus and activation-regulated chemokine (TARC), IgE,
eotaxin-3, lactate dehydrogenase (LDH), and periostin.
[0032] The invention further includes a pharmaceutical composition
comprising an anti-IL-4R antibody antagonist or an antigen binding
fragment thereof for use in the treatment of AD in a subject
wherein the treatment results in a decrease in an AD-associated
biomarker in the subject by day 4, 8, 15, 22, 25, 29 or 36
following treatment as compared to the level of biomarker in the
subject prior to treatment. In certain embodiments, the
AD-associated biomarker is one or both of TARC and IgE.
[0033] The invention further includes a pharmaceutical composition
comprising an anti-IL-4R antibody antagonist or an antigen binding
fragment thereof for use in improving an AD-associated parameter,
or for reducing the level of an AD-associated biomarker in a
subject in need thereof, wherein the pharmaceutical composition is
sequentially administered to the subject as a single initial dose
followed by one or more secondary doses.
[0034] In one embodiment, the one or more secondary doses are
administered weekly.
[0035] In some embodiments, the pharmaceutical composition
comprises 75 mg to 600 mg of the anti-IL-4R antibody or
antigen-binding fragment thereof. In one embodiment, the
pharmaceutical composition comprises 300 mg of the anti-IL-4R
antibody or fragment thereof.
[0036] In another aspect, the invention provides methods of
monitoring whether a therapeutic dose of an interleukin-4 receptor
(IL-4R) antagonist administered to a human subject is safe, said
method comprising: acquiring information regarding the safety of
the antagonist following administration to a human, wherein the
information includes the occurrence of one or more events selected
from the group consisting of an anaphylactic reaction or acute
allergic reaction requiring immediate treatment, severe injection
site reaction lasting longer than 24 hours, severe infection, any
parasitic infection, alanine aminotransferase (ALT) increase
.gtoreq.2 Upper Limit Normal Range (ULN), QTc>=500 ms,
pregnancy, overdose, and herpes simplex type II viral infection;
determining that the one or more said events has occurred,
determining that said therapeutic dose is not safe, and, optionally
advising that the therapeutic dose be discontinued or lowered.
[0037] In a related aspect, the invention provides methods of
monitoring whether a therapeutic dose of an interleukin-4 receptor
(IL-4R) antagonist administered to a human subject is safe, said
method comprising: acquiring information regarding the safety of
the antagonist following administration to a human, wherein the
information includes the occurrence of one or more events selected
from the group consisting of: anaphylactic reaction or acute
allergic reaction requiring immediate treatment, severe injection
site reaction lasting longer than 24 hours, severe infection, any
parasitic infection, alanine aminotransferase (ALT) increase
.gtoreq.2 Upper Limit Normal Range (ULN), QTc>=500 ms,
pregnancy, overdose, and herpes simplex type II viral infection;
determining that the one or more of said events has not occurred;
and determining that said therapeutic dose is safe.
[0038] In one embodiment, the infection is upper respiratory tract
injection, pharyngitis, or sinusitis. In one embodiment, the
injection site reaction is erythema, pain, nodule, hematoma or
pruritus. In one embodiment, the pain is greater than 2 mm VAS,
e.g., 3 mm to 30 mm VAS. In one embodiment, the erythema diameter
is .gtoreq.9 mM.
[0039] In one embodiment, the safe therapeutic dose is equal to or
less than 500 mg. In one embodiment, the safe therapeutic dose is
selected from the group consisting of 75 mg, 150 mg, and 300
mg.
[0040] In another aspect, the invention provides methods of
quantifying or monitoring an amount of anti-drug antibodies in
blood serum of a human subject following administration of drug
wherein the drug is an interleukin-4 receptor (IL-4R) antagonist,
said method comprising: (a) obtaining a sample of said blood serum
from a human subject who was administered a dose of said IL-4R
antagonist; and (b) determining the amount of anti-drug antibodies
in said serum sample.
[0041] In another aspect, the invention provides methods of
comparing an interleukin-4 receptor (IL-4R) antagonist manufactured
by a first process and proposed equivalent second process, said
method comprising: acquiring information regarding the safety of
the antagonist following administration of the antagonist
manufactured by the first process to a first human, and following
administration of the antagonist manufactured by the second process
to a second human, wherein the information includes: one or more
events selected from the group consisting of an anaphylactic
reaction or acute allergic reaction requiring immediate treatment,
severe injection site reaction lasting longer than 24 hours, severe
infection, any parasitic infection, alanine aminotransferase (ALT)
increase .gtoreq.2 Upper Limit Normal Range (ULN), QTc>=500 ms,
pregnancy, overdose, and herpes simplex type II viral infection;
and wherein if the information is not significantly different for
the antagonist manufactured by the first process and the antagonist
manufactured by the second process, then the two processes are
determined to be acceptable for manufacturing equivalent
antagonists; and wherein if the information is determined to be
significantly different for the antagonist manufactured by the
first process and the antagonist manufactured by the second
process, then the two processes are determined to be unacceptable
for manufacturing equivalent antagonists.
[0042] In a related aspect, the invention provides methods of
comparing an interleukin-4 receptor (IL-4R) antagonist manufactured
by a first process and proposed equivalent second process, said
method comprising: acquiring information regarding a therapeutic
dose of the antagonist following administration of the dose of the
antagonist manufactured by the first process to a first human, and
following administration of the dose of the antagonist manufactured
by the second process to a second human, wherein the information
includes one or more of: (a) area under the plasma concentration
versus time curve calculated using the trapezoidal method from time
zero to real time (AUC.sub.last) from about 4 mgh/ml to about 20
mgh/ml; (b) maximum plasma concentration observed (C.sub.max) from
about 15 ug/ml to about 42 ug/ml; (c) first time to reach a maximum
plasma concentration (t.sub.max) from about 40 hr to about 280 hr;
(d) area under the plasma concentration versus time curve
extrapolated to infinity (AUC) from about 5,000,000 ng/h*mL to
about 25,000,000 ng/h*mL and (e) time to reach terminal half-life
of (t.sub.1/2.sup.z) from about 50 h to about 200 h, wherein if the
information is not significantly different for the antagonist
manufactured by the first process and the antagonist manufactured
by the second process, then the two processes are determined to be
acceptable for manufacturing equivalent antagonists; and wherein if
the information is determined to be significantly different for the
antagonist manufactured by the first process and the antagonist
manufactured by the second process, then the two processes are
determined to be unacceptable for manufacturing equivalent
antagonists.
[0043] In another aspect, the invention provides a therapeutic
dosage form of a pharmaceutical composition comprising an
interleukin-4 receptor (IL-4R) antagonist, wherein administration
of the dose form to a human provides one or more of: (a) an area
under the plasma concentration versus time curve calculated using
the trapezoidal method from time zero to real time (AUC.sub.last)
from about 4 mgh/ml to about 20 mgh/ml; (b) a maximum plasma
concentration observed (C.sub.max) from about 15 ug/ml to about 42
ug/ml; (c) a first time to reach a maximum plasma concentration
(t.sub.max) from about 40 hr to about 280 hr; (d) an area under the
plasma concentration versus time curve extrapolated to infinity
(AUC) from about 5,000,000 ng/h*mL to about 25,000,000 ng/h*mL and
(e) a time to reach terminal half-life of (t.sub.1/2.sup.z) from
about 50 h to about 200 h.
[0044] In one embodiment, the safe therapeutic dose is equal to or
less than 500 mg. In one embodiment, the safe therapeutic dose is
selected from the group consisting of 75 mg, 150 mg, and 300
mg.
[0045] Certain aspects of the invention are related to methods and
compositions that are useful in vaccine applications. The present
invention provides methods for enhancing or potentiating the immune
response against an antigen in a subject. In some embodiments, the
methods for enhancing or potentiating the immune response against
an antigen in a subject comprise administering a pharmaceutical
composition comprising the antigen and an IL-4R antagonist. Some
embodiments are related to methods comprising (a) administering a
vaccine composition comprising the antigen to the subject; and (b)
administering an IL-4R antagonist prior to, concurrent with, and/or
subsequent to administration of the vaccine composition to the
subject. The present invention also provides for pharmaceutical
compositions to enhance or potentiate an immune response against an
antigen in a subject, the compositions comprising: (a) the antigen;
and (b) an IL-4R antagonist. In one exemplary embodiment, the IL-4R
antagonist is an anti-IL-4R antibody (as exemplified in Example 1
herein). In certain embodiments, the IL-4R antagonist is an
anti-IL-4R antibody having the binding characteristics of the
reference antibody referred to herein as "mAb1" (e.g., an antibody
or antigen-binding fragment thereof comprising the complementarity
determining regions of mAb1).
[0046] Other embodiments of the present invention will become
apparent from a review of the ensuing detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0047] FIG. 1 shows a Cartesian plot of mean (SD) serum functional
mAb1 concentration-time profiles following a single subcutaneous
dose.
[0048] FIG. 2 shows a diagrammatic representation of the injection
procedure and pain assessments as described in Example 5.
[0049] FIG. 3 shows the IGA score responder rate (score of 0 or
1)--last observation carried forward (LOCF) for the study in
Example 6.
[0050] FIG. 4 shows the mean IGA score change from baseline--LOCF
for the study in Example 6.
[0051] FIG. 5 shows the mean IGA score percent change from
baseline--LOCF for the study in Example 6.
[0052] FIG. 6 shows mean EASI score change from baseline--LOCF for
the study in Example 6.
[0053] FIG. 7 shows mean EASI score percent change from
baseline--LOCF for the study in Example 6.
[0054] FIG. 8 shows EASI50 responder rate--LOCF for the study in
Example 6.
[0055] FIG. 9 shows mean BSA change from baseline--LOCF for the
study in Example 6.
[0056] FIG. 10 shows mean BSA percent change from baseline--LOCF
for the study in Example 6.
[0057] FIG. 11 shows mean 5-D change from baseline--LOCF for the
study in Example 6.
[0058] FIG. 12 shows mean 5-D percent change from baseline--LOCF
for the study in Example 6.
[0059] FIG. 13 shows mean NRS change from baseline--LOCF for the
study in Example 6.
[0060] FIG. 14 shows mean NRS percent change from baseline--LOCF
for the study in Example 6.
[0061] FIG. 15 shows percent change from baseline in BSA in
patients administered 75 mg, 150 mg or 300 mg of anti-IL-4R
antibody vs. placebo for the study in Example 8.
[0062] FIG. 16 shows percent change from baseline in IGA in
patients administered 75 mg, 150 mg or 300 mg of anti-IL-4R
antibody vs. placebo for the study in Example 8.
[0063] FIG. 17 shows percent change from baseline in EASI in
patients administered 75 mg, 150 mg or 300 mg of anti-IL-4R
antibody vs. placebo for the study in Example 8.
[0064] FIG. 18 shows percent change from baseline in Pruritus NRS
in patients administered 75 mg, 150 mg or 300 mg of anti-IL-4R
antibody vs. placebo for the study in Example 8.
[0065] FIG. 19 shows EASI response time course in patients with
moderate-to-severe AD to 300 mg anti-IL-4R antibody for the study
in Example 8.
[0066] FIG. 20 shows the percent responders in the EASI score
administered with 75 mg, 150 mg or 300 mg anti-IL-4R antibody vs.
placebo for the study in Example 8.
[0067] FIG. 21 shows EASI responses at week 4 (day 29) to
anti-IL-4R antibody administered at 75 mg, 150 mg or 300 mg doses
vs. placebo for the study in Example 8.
[0068] FIG. 22 shows proportion of patients achieving IGA.ltoreq.1
for the study in Example 8.
[0069] FIG. 23 shows mean EASI score percent change from baseline
to the last observation carried forward (LOCF) for the study in
Example 10.
[0070] FIG. 24 shows IGA score responder rate 9 score of 0 or 1) up
to LOCF for the study in Example 10.
[0071] FIG. 25 shows IGA score responder rate (reduction in score
of 2 or more) up to LOCF for the study in Example 10.
[0072] FIG. 26 shows EASI score responder rate (50% score reduction
from baseline) up to LOCF for the study in Example 10.
[0073] FIG. 27 shows mean EASI score change from baseline up to
LOCF for the study in Example 10.
[0074] FIG. 28 shows mean IGA score change from baseline up to LOCF
for the study in Example 10.
[0075] FIG. 29 shows mean IGA score percent change from baseline up
to LOCF for the study in Example 10.
[0076] FIG. 30 shows mean BSA change from baseline up to LOCF for
the study in Example 10.
[0077] FIG. 31 shows mean SCORAD score change from baseline up to
LOCF for the study in Example 10.
[0078] FIG. 32 shows mean NRS score change from baseline up to LOCF
for the study in Example 10.
[0079] FIG. 33 shows mean 5-D Pruritus score change from baseline
up to LOCF for the study in Example 10.
[0080] FIG. 34 shows mean EASI score percent change from
baseline--censored LOCF, for the study in Example 11.
[0081] FIG. 35 shows mean EASI score change from baseline--censored
LOCF, for the study in Example 11.
[0082] FIG. 36 shows EASI50 responder rate--censored LOCF, for the
study in Example 11.
[0083] FIG. 37 shows a Kaplan-Meier plot of Time to first
EASI50--censored LOCF, for the study in Example 11.
[0084] FIG. 38 shows mean IGA score percent change from
baseline--censored LOCF, for the study in Example 11.
[0085] FIG. 39 shows mean IGA score change from baseline--censored
LOCF, for the study in Example 11.
[0086] FIG. 40 shows IGA score responder rate (score of 0 or
1)--censored LOCF, for the study in Example 11.
[0087] FIG. 41 shows a Kaplan-Meier plot of time to first
IGA.ltoreq.1--censored LOCF, for the study in Example 11.
[0088] FIG. 42 shows proportion of patients with IGA.ltoreq.1 at
each visit who remained relapse-free--censored LOCF, for the study
in Example 11.
[0089] FIG. 43 shows proportion of patients with reduction from
baseline in IGA.gtoreq.2 at each visit--censored LOCF, for the
study in Example 11.
[0090] FIG. 44 shows mean SCORAD score percent change from
baseline--censored LOCF, for the study in Example 11.
[0091] FIG. 45 shows mean SCORAD score change from
baseline--censored LOCF, for the study in Example 11.
[0092] FIG. 46 shows mean pruritus NRS percent change from
baseline--censored LOCF, for the study in Example 11.
[0093] FIG. 47 shows mean pruritus NRS change from
baseline--censored LOCF, for the study in Example 11.
[0094] FIG. 48 shows serum IgE levels at baseline (A) and median
percentage change in response to various doses of mAb1 or placebo
(B) for the study in Example 12.
[0095] FIG. 49 shows serum TARC levels at baseline (A) and mean
percentage change in response to various doses of mAb1 or placebo
(B) for the study in Example 12.
[0096] FIG. 50 shows change in TARC levels for pooled mAb1 group
compared to placebo for the study in Example 12.
[0097] FIG. 51 shows the distribution of baseline levels of (A)
TARC, (B) total serum IgE, and (C) lactate dehydrogenase (LDH) in
patients in the study in section B of Example 12.
[0098] FIG. 52 shows the median percent change in IgE from baseline
for the study in section B of Example 12.
[0099] FIG. 53 shows the median percent change in LDH from baseline
for the study in section B of Example 12.
[0100] FIG. 54 shows the median percent change in TARC from
baseline for the study in section B of Example 12.
[0101] FIG. 55 shows the median percent change in IgE from baseline
for the study in section C of Example 12.
[0102] FIG. 56 shows the median percent change in TARC from
baseline for the study in section C of Example 12.
DETAILED DESCRIPTION
[0103] 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.
[0104] 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.). 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 the named disorder or condition.
[0105] The present invention includes methods which comprise
administering to a subject in need thereof a therapeutic
composition comprising an IL-4R antagonist. As used herein, the
expression "a subject in need thereof" means a human or non-human
animal that exhibits one or more symptoms or indicia of atopic
dermatitis, and/or who has been diagnosed with atopic dermatitis.
In certain embodiments, the methods of the present invention may be
used to treat patients that show elevated levels of one or more
AD-associated biomarkers (described elsewhere herein). For example,
the methods of the present invention comprise administering an
IL-4R antagonist to patients with elevated levels of IgE or TARC or
periostin. In some embodiments, the methods herein may be used to
treat AD in children who are .ltoreq.1 year old. For example, the
present methods may be used to treat infants who are less than 1
month, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7
months, 8 months, 9 months, 10 months, 11 months or less than 12
months old. In other embodiments, the present methods may be used
to treat children and/or adolescents who are .ltoreq.18 years old.
For example, the present methods may be used to treat children or
adolescents less than 17 years, 16 years, 15 years, 14 years, 13
years, 12 years, 11 years, 10 years, 9 years, 8 years, 7 years, 6
years, 5 years, 4 years, 3 years, or less than 2 years old.
[0106] In the context of the present invention, "a subject in need
thereof" may include, e.g., subjects who, prior to treatment,
exhibit (or have exhibited) one or more AD-associated parameters
such as, e.g., elevated IGA, BSA, EASI, SCORAD, 5D-Pruritus, and/or
NRS score, and/or an elevated level of one or more AD-associated
biomarker such as, e.g., IgE and/or TARC (as described elsewhere
herein). In certain embodiments, "a subject in need thereof" may
include a subset of population which is more susceptible to AD or
may show an elevated level of an AD-associated biomarker. For
example, "a subject in need thereof" may include a subset of
population defined by a race or an ethnicity present in the
population.
[0107] "Atopic dermatitis" (AD), as used herein, means an
inflammatory skin disease characterized by intense pruritus (e.g.,
severe itch) and by scaly and dry eczematous lesions. The term
"atopic dermatitis" includes, but is not limited to, AD caused by
or associated with epidermal barrier dysfunction, allergy (e.g.,
allergy to certain foods, pollen, mold, dust mite, animals, etc.),
radiation exposure, and/or asthma. The present invention
encompasses methods to treat patients with mild, moderate-to-severe
or severe AD. As used herein, "moderate-to-severe AD", is
characterized by intensely pruritic, widespread skin lesions that
are often complicated by persistent bacterial, viral or fungal
infections. Moderate-to-severe AD also includes chronic AD in
patients. In many cases, the chronic lesions include thickened
plaques of skin, lichenification and fibrous papules. Patients
affected by moderate-to-severe AD also, in general, have more than
20% of the body's skin affected, or 10% of skin area in addition to
involvement of the eyes, hands and body folds. Moderate-to-severe
AD is also considered to be present in patients who require
frequent treatment with topical corticosteroids. A patient may also
be said to have moderate-to-severe AD when the patient is resistant
or refractory to treatment by either a topical corticosteroid or a
calcineurin inhibitor or any other commonly used therapeutic agent
known in the art.
[0108] The present invention includes methods to treat both the
extrinsic and the intrinsic forms of AD. The extrinsic form of AD
associated with IgE-mediated sensitization and increased levels of
Th2 cytokines involves 70% to 80% of patients with AD. The
intrinsic form without IgE-mediated sensitization involves 20% to
30% of patients with AD; these patients have lower levels of IL-4
and IL-13 than extrinsic AD.
[0109] The present invention includes methods to treat AD in
patients resistant, non-responsive or inadequately responsive to
treatment with a topical corticosteroid (TCS) or a calcineurin
inhibitor. The term "resistant, non-responsive or inadequately
responsive to a TCS or a calcineurin inhibitor", as used herein,
refers to subjects or patients with AD who have been treated with a
TCS or a calcineurin inhibitor and wherein the TCS/calcineurin
inhibitor does not have a therapeutic effect. In some embodiments,
the term refers to reduced patient compliance and/or toxicity and
side effects and/or ineffectiveness of the administered
TCS/calcineurin inhibitor to reduce, ameliorate or decrease the
symptoms of AD. In some embodiments, the term refers to patients
suffering from moderate-to-severe AD who are refractory to
treatment by a TCS/calcineurin inhibitor. In some embodiments, the
term refers to patients with AD which is uncontrolled despite
treatment with a TCS and/or calcineurin inhibitor. In some
embodiments, the patients who are "resistant, non-responsive or
inadequately responsive to a TCS or a calcineurin inhibitor" may
show no improvement in one or more AD-associated parameters.
Examples of AD-associated parameters are described elsewhere
herein. For example, treatment with a TCS/calcineurin inhibitor may
result in no decrease in pruritus or EASI score or BSA score. In
some embodiments, the present invention includes methods to treat
moderate-to-severe AD in patients who have been treated earlier
with a TCS/calcineurin inhibitor for .gtoreq.1 month and do not
show a decrease in one or more AD-associated parameters. For
example, the present methods may be used to treat a patient with
chronic AD who has been on a stable regimen of a TCS/calcineurin
inhibitor and has a BSA score of .gtoreq.10% or an IGA
score.gtoreq.3.
[0110] In alternate embodiments, the term "subject in need thereof"
includes patients with moderate-to-severe AD who have been
administered one or more TCS for more than 6 months, more than 1
year, more than 2 years, more than about 5 years, more than about 7
years, or more than about 10 years. The patients may desire to
minimize or avoid the adverse side effects of the TCS. The present
invention includes methods for long-term safer and more effective
management of moderate-to-severe AD in a patient, the methods
comprising administering an IL-4R antagonist concomitantly with a
TCS wherein the dosage is adjusted to minimize or prevent adverse
side effects of the TCS. In certain embodiments, the present
invention includes methods to reduce dependence on TCS in a patient
with moderate-to-severe AD; the methods comprising administering a
therapeutically effective amount of an IL-4R antagonist
concomitantly with a potent TCS wherein the amount of TCS used by
the patient is reduced by about 50% as compared to a patient not
administered the IL-4R antagonist. In certain embodiments, the
present invention includes methods to reduce dependence on TCS in a
patient with moderate-to-severe AD, the methods comprising
administering a therapeutically effective amount of an IL-4R
antagonist concomitantly with a potent TCS wherein the amount of
TCS used by the patient is reduced by about 50% as compared to the
amount used by the patient before treatment with the IL-4R
antagonist. In certain embodiments, the administration of an IL-4R
antagonist and a TCS results in additive or synergistic activity in
treating AD as compared to monotherapy.
[0111] The term "TCS", as used herein includes group I, group II,
group III and group IV topical corticosteroids. According to the
Anatomical Therapeutic Classification System of World Health
Organization, the corticosteroids are classified as weak (group I),
moderately potent (Group II) and potent (Group III) and very potent
(Group IV), based on their activity as compared to hydrocortisone.
Group IV TCS (very potent) are up to 600 times as potent as
hydrocortisone and include clobetasol propionate and halcinonide.
Group III TCS (potent) are 50 to 100 times as potent as
hydrocortisone and include, but are not limited to, betamethasone
valerate, betamethasone dipropionate, diflucortolone valerate,
hydrocortisone-17-butyrate, mometasone furoate, and
methylprednisolone aceponate. Group II TCS (moderately potent) are
2 to 25 times as potent as hydrocortisone and include, but are not
limited to, clobetasone butyrate, and triamcinolone acetonide.
Group I TCS (mild) includes hydrocortisone.
[0112] 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 publications mentioned herein are incorporated herein by
reference to describe in their entirety.
Methods for Improving Atopic Dermatitis (AD)-Associated
Parameters
[0113] The present invention includes methods for improving one or
more atopic dermatitis (AD)-associated parameters in a subject in
need thereof, wherein the methods comprise administering a
pharmaceutical composition comprising an interleukin-4 receptor
(IL-4R) antagonist to the subject.
[0114] Examples of "AD-associated parameters" include: (a)
Investigators Global Assessment (IGA); (b) Body Surface Area
Involvement of Atopic Dermatitis (BSA); (c) Eczema Area and
Severity Index (EASI); (d) SCORAD; (e) 5-D Pruritus Scale; and (f)
Pruritus Numeric Rating Scale (NRS). An "improvement in an
AD-associated parameter" means a decrease from baseline of one or
more of IGA, BSA, EASI, SCORAD, 5-D Pruritus Scale, or NRS. As used
herein, the term "baseline," with regard to an AD-associated
parameter, means the numerical value of the AD-associated parameter
for a subject prior to or at the time of administration of a
pharmaceutical composition of the present invention.
[0115] To determine whether an AD-associated parameter has
"improved," the parameter is quantified at baseline and at one or
more time points after administration of the pharmaceutical
composition of the present invention. For example, an AD-associated
parameter may be measured at day 1, day 2, day 3, day 4, day 5, day
6, day 7, day 8, day 9, day 10, day 11, day 12, day 14, day 15, day
22, day 25, day 29, day 36, day 43, day 50, day 57, day 64, day 71,
day 85; or at the end of week 1, week 2, week 3, week 4, week 5,
week 6, week 7, week 8, week 9, week 10, week 11, week 12, week 13,
week 14, week 15, week 16, week 17, week 18, week 19, week 20, week
21, week 22, week 23, week 24, or longer, after the initial
treatment with a pharmaceutical composition of the present
invention. The difference between the value of the parameter at a
particular time point following initiation of treatment and the
value of the parameter at baseline is used to establish whether
there has been an "improvement" (e.g., a decrease) in the AD
associated parameter.
[0116] Investigator's Global Assessment (IGA).
[0117] The IGA is an assessment scale used in clinical settings to
determine the severity of AD and clinical response to treatment
based on a 6-point scale ranging from 0 (clear) to 5 (very severe).
According to certain embodiments of the present invention,
administration of an IL-4R antagonist to a patient results in a
decrease in IGA score. For example, the present invention includes
therapeutic methods which result in a decrease from baseline in IGA
score of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, or more at day 4, 8, 15, 22, 25, 29,
36, 43, 50, 57, 64, 71, 85 or later following administration of the
IL-4R antagonist (e.g., following subcutaneous administration of
about 75 mg, 150 mg, or 300 mg of an anti-IL-4R antibody or
antigen-binding fragment thereof). In certain exemplary embodiments
of the present invention, administration of an IL-4R antagonist to
a subject results in a decrease from baseline in IGA of at least
25%. In one embodiment of the present invention, administration of
an IL-4R antagonist to a subject results in a decrease from
baseline in IGA of at least 25% by day 15 after administration. In
certain embodiments of the present invention, administration of an
IL-4R antagonist to a subject results in a decrease from baseline
in IGA of at least 35% by day 22 after administration. In other
embodiments, administration of an IL-4R antagonist to a subject
results in a decrease from baseline in IGA of at least 40% or at
least 45% through day 85 upon treatment.
[0118] Body Surface Area Involvement of Atopic Dermatitis
(BSA).
[0119] BSA is assessed for each major section of the body (head,
trunk, arms and legs) and is reported as a percentage of all major
body sections combined. According to certain embodiments of the
present invention, administration of an IL-4R antagonist to a
patient results in a decrease in BSA score. For example, the
present invention includes therapeutic methods which result in a
decrease from baseline in BSA score of at least about 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more
at day 4, 8, 15, 22, 25, 29, 36, 43, 50, 57, 64, 71, 85 or later
following administration of the IL-4R antagonist (e.g., following
subcutaneous administration of about 75 mg, 150 mg, or 300 mg of an
anti-IL-4R antibody or antigen-binding fragment thereof). In
certain exemplary embodiments of the present invention,
administration of an IL-4R antagonist to a subject results in a
decrease from baseline in BSA score of at least 35% after
administration. In one embodiment of the present invention,
administration of an IL-4R antagonist to a subject results in a
decrease from baseline in BSA score of at least 35% by day 29 after
administration. In one embodiment of the present invention,
administration of an IL-4R antagonist to a subject results in a
decrease from baseline in BSA score of at least 40% by day 29 after
administration. In some embodiments, administration of an IL-4R
antagonist to a subject results in a decrease from baseline in BSA
score of at least 40% or at least 50% through day 85 upon
treatment.
[0120] Eczema Area and Severity Index (EASI).
[0121] The EASI is a validated measure used in clinical settings to
assess the severity and extent of AD. (Hanifin et al. 2001, Exp.
Dermatol. 10:11-18). Four AD disease characteristics are assessed
for severity by a physician or other qualified medical professional
on a scale of 0 (absent) through 3 (severe). In addition, the area
of AD involvement is assessed as a percentage by body area of head,
trunk, arms and legs and converted to a score of 0 to 6. According
to certain embodiments of the present invention, administration of
an IL-4R antagonist to a patient results in a decrease in EASI
score. For example, the present invention includes therapeutic
methods which result in a decrease from baseline in EASI score of
at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75% or more at day 4, 8, 15, 22, 25, 29, 36, 43, 50,
57, 64, 71, 85 or later following administration of the IL-4R
antagonist (e.g., following subcutaneous administration of about 75
mg, 150 mg, or 300 mg of an anti-IL-4R antibody or antigen-binding
fragment thereof). In certain exemplary embodiments of the present
invention, administration of an IL-4R antagonist to a subject
results in a decrease from baseline in EASI score of at least 45%.
In one embodiment of the present invention, administration of an
IL-4R antagonist to a subject results in a decrease from baseline
in EASI score of at least 45% by day 15 after administration. In
one embodiment of the present invention, administration of an IL-4R
antagonist to a subject results in a decrease from baseline in EASI
score of at least 50% by day 29 after administration. In some
embodiments, administration of an IL-4R antagonist to a subject
results in a decrease from baseline in EASI score of at least 55%
or at least 60% through day 85 upon treatment.
[0122] SCORAD.
[0123] SCORing Atopic Dermatitis (SCORAD) is a clinical assessment
of the severity (e.g., extent or intensity) of atopic dermatitis
developed by the European Task Force on Atopic Dermatitis
(Consensus Report of the European Task Force on Atopic Dermatitis,
1993, Dermatology (Basel) 186(1):23-31). The extent of AD is
assessed as a percentage of each defined body area and reported as
the sum of all areas, with a maximum score of 100% (assigned as "A"
in the overall SCORAD calculation). The severity of 6 specific
symptoms of AD is assessed using the following scale: none (0),
mild (1), moderate (2), or severe (3) (for a maximum of 18 total
points, assigned as "B" in the overall SCORAD calculation).
Subjective assessment of itch and sleeplessness is recorded for
each symptom by the patient or relative on a visual analogue scale
(VAS), where 0 is no itch (or sleeplessness) and 10 is the worst
imaginable itch (or sleeplessness), with a maximum possible score
of 20. This parameter is assigned as "C" in the overall SCORAD
calculation. The SCORAD is calculated as: A/5+7B/2+C. According to
certain embodiments of the present invention, administration of an
IL-4R antagonist to a patient results in a decrease in SCORAD
score. For example, the present invention includes therapeutic
methods which result in a decrease from baseline in SCORAD of at
least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75% or more at day 4, 8, 15, 22, 25, 29, 36, 43, 50, 57,
64, 71, 85 or later following administration of the IL-4R
antagonist (e.g., following subcutaneous administration of about 75
mg, 150 mg, or 300 mg of an anti-IL-4R antibody or antigen-binding
fragment thereof). In certain exemplary embodiments of the present
invention, administration of an IL-4R antagonist to a subject
results in a decrease from baseline in SCORAD score of at least
30%. In one embodiment of the present invention, administration of
an IL-4R antagonist to a subject results in a decrease from
baseline in SCORAD score of at least 30% by day 29 after
administration. In one embodiment of the present invention,
administration of an IL-4R antagonist to a subject results in a
decrease from baseline in SCORAD score of at least 35% by day 29
after administration. In some embodiments, administration of an
IL-4R antagonist to a subject results in a decrease from baseline
in SCORAD score of at least 40% or at least 45% through day 85 upon
treatment.
[0124] 5-D Pruritus Scale.
[0125] The 5-D Pruritus Scale is a 1-page, 5-question tool used in
clinical settings to assess 5 dimensions of background itch:
degree, duration, direction, disability, and distribution. (Elman
and Hynan, 2010, Brit. J. Dermatol. 162:587-593). Each question
corresponds to 1 of the 5 dimensions of itch; patients rate their
symptoms as "present" or on a 1 to 5 scale, with 5 being the most
affected. According to certain embodiments of the present
invention, administration of an IL-4R antagonist to a patient
results in a decrease in 5-D Pruritus Scale. For example, the
present invention includes therapeutic methods which result in a
decrease from baseline in 5-D Pruritus Scale of at least about 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or
more at day 4, 8, 15, 22, 25, 29, 36, 43, 50, 57, 64, 71, 85 or
later following administration of the IL-4R antagonist (e.g.,
following subcutaneous administration of about 75 mg, 150 mg, or
300 mg of an anti-IL-4R antibody or antigen-binding fragment
thereof). In certain exemplary embodiments of the present
invention, administration of an IL-4R antagonist to a subject
results in a decrease from baseline in 5-D Pruritus Scale of at
least 15%. In one embodiment of the present invention,
administration of an IL-4R antagonist to a subject results in a
decrease from baseline in 5-D Pruritus Scale of at least 15% by day
15 after administration. In one embodiment of the present
invention, administration of an IL-4R antagonist to a subject
results in a decrease from baseline in 5-D Pruritus Scale of at
least 20% by day 15 after administration. In some embodiments,
administration of an IL-4R antagonist to a subject results in a
decrease from baseline in 5-D Pruritus Scale of at least 25% or at
least 30% through day 85 upon treatment.
[0126] Pruritus Numeric Rating Scale (NRS).
[0127] The Pruritus NRS is a single-question assessment tool that
is used to assess a subject's worst itch, on a scale of 1 to 10, as
a result of AD in the previous 12 hours. According to certain
embodiments of the present invention, administration of an IL-4R
antagonist to a patient results in a decrease in NRS score. For
example, the present invention includes therapeutic methods which
result in a decrease from baseline in NRS score of at least about
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75% or more at the end of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, or later following administration of the IL-4R antagonist
(e.g., following subcutaneous administration of about 75 mg, 150
mg, or 300 mg of an anti-IL-4R antibody or antigen-binding fragment
thereof). In certain exemplary embodiments of the present
invention, administration of an IL-4R antagonist to a subject
results in a decrease from baseline in NRS score of at least 25%.
In one embodiment of the present invention, administration of an
IL-4R antagonist to a subject results in a decrease from baseline
in NRS score of at least 25% by the end of week 2 after
administration. In one embodiment of the present invention,
administration of an IL-4R antagonist to a subject results in a
decrease from baseline in NRS score of at least 30% by the end of
week 2 after administration. In some embodiments, administration of
an IL-4R antagonist to a subject results in a decrease from
baseline in NRS score of at least 45% or at least 50% through day
85 upon treatment.
[0128] Global Individual Signs Score (GISS).
[0129] Individual components of the AD lesions (erythema,
infiltration/population, excoriations, and lichenification) is rate
globally (i.e., each assessed for the whole body, not by anatomical
region) on a 4-point scale (from 0=none to 3=severe) using the EASI
severity grading criteria.
[0130] Pruritus Categorical Scale.
[0131] The Pruritus categorical scale is a 4-point scale used to
assess symptoms that has been used in clinical studies of AD and
has less of a "middling" effect (Kaufmann 2006). The scale is rated
as follows: 0: absence of pruritus; 1: mild, pruritus (occasional
slight itching/scratching); 2: moderate pruritus (constant or
intermittent itching/scratching that does not disturb sleep) and 3:
severe pruritus (bothersome itching/scratching that disturbs
sleep).
[0132] Patient Oriented Eczema Measure (POEM).
[0133] The POEM is a 7-item, validated questionnaire used in
clinical practice and clinical trials to assess disease symptoms in
children and adults (Charman 2004). The format is a response to 7
items (dryness, itching, flaking, cracking, sleep loss, bleeding,
and weeping) with a scoring system of 0 to 28; a high score is
indicative of a poor QOL.
[0134] Dermatology Life Quality Index (DLQI).
[0135] The DLQI is a 10-item, validated questionnaire used in
clinical practice and clinical trials to assess the impact of AD
disease symptoms and treatment on QOL (Badia 1999). The format is a
simple response to 10 items, which assess QOL over the past week,
with an overall scoring system of 0 to 30; a high score is
indicative of a poor QOL.
[0136] Itchy QOL.
[0137] Itchy QOL is a validated pruritus-specific instrument that
addresses the symptom, emotional, and functional impact of
pruritus. There is an overall score as well as subscale scores to
address the 3 types of impact. This is a reliable, valid, and
responsive questionnaire (Desai 2008).
[0138] EQ-5D.
[0139] The EQ-5D is a standardized measure of health status
developed by the EuroQOI Group in order to provide a simple,
generic measure of health for clinical and economic appraisal. The
EQ-5D as a measure of health related QOL, defines health in terms
of 5 dimensions: mobility, self-care, usual activities,
pain/discomfort, and anxiety/depression. Each dimension has 3
ordinal levels of severity: "no problem" (1), "some problems" (2),
"severe problems" (3). Overall health state is defined as a 5-digit
number. Health states defined by the 5-dimensional classification
can be converted into corresponding index scores that quantify
health status, where 0 represents "death" and 1 represents "perfect
health."
[0140] HADS.
[0141] The HADS is a general Likert scale used to detect states of
anxiety and depression (Bjelland 2002). The 14 items on the
questionnaire include 7 that are related to anxiety and 7 that are
related to depression. Each item on the questionnaire is scored; a
person can score between 0 and 21 for either anxiety or
depression.
[0142] Patient Global Assessment of Disease Status and Treatment
Effect.
[0143] Patients rate their overall wellbeing based on a 5-point
Likert scale from poor to excellent. Patients are asked:
"Considering all the ways in which your eczema affects you,
indicate how well you are doing". Response choices are: "Poor";
"Fair"; "Good"; "Very Good"; "Excellent".
[0144] For treatment effect, patients rate their satisfaction with
the study treatment based on a 5-point Likert scale from poor to
excellent. Patients are asked: "How would you rate the way your
eczema responded to the study medication?" Response choices are:
"Poor"; "Fair"; "Good"; "Very Good"; "Excellent".
[0145] Methods for Long-Term Management of Atopic Dermatitis
[0146] The present invention includes methods for long term
management of moderate-to-severe AD in a patient. In certain
embodiments, the methods comprise administering an IL-4R antagonist
concomitantly with a conventional therapeutic agent such as a
topical corticosteroid (TCS). In further embodiments, the IL-4R
antagonist may be an anti-IL-4R antibody as described herein.
[0147] The term "conventional therapeutic agent", as used herein,
refers to therapeutic agents and drugs commonly or routinely used
to treat AD in patients. Conventional therapeutic agents include
systemic as well as topical therapeutics. For example, the most
commonly or frequently prescribed drugs are the topical
corticosteroids (TCS). Other examples of such agents include, but
are not limited to, topical calcineurin inhibitors,
anti-histamines, oral immunosuppressants, and glucocorticoids,
systemic immunosuppressants such as methotrexate, cyclosporine, and
azathioprine. Conventional therapeutic agents are used to relieve
the symptoms of AD; however have numerous and considerable adverse
side effects including diabetes, hypertension, osteoporosis,
myelosuppression, nephrotoxicity, hepatotoxicity, leucopenia, an
increased risk of microbial infections. Topical agents such as
corticosteroids and calcineurin inhibitors are not recommended for
long-term application due to the risk of irreversible skin atrophy,
dyspigmentation, acneiform eruptions and risks associated with
systemic absorption including skin malignancies and lymphomas. Also
repetitive application of any topical therapies over a long period
of time can erode patient compliance.
[0148] The term "long-term management of AD", as used herein,
refers to treatment or containment of one or more symptoms or
disease conditions of AD over a long period of time, typically more
than about 2 years, more than about 5 years, more than about 10
years, or more than about 20 years. Long-term management of AD
includes methods of treatment or methods to improve one or more
AD-associated parameters over a period of more than 6 months, more
than 1 year, more than 2 years, or more than about 5 years, the
methods comprising administering an anti-IL-4R antibody in
combination with a conventional therapeutic agent such as TCS. The
administration regimen and dosage of the IL-4R antibody and the TCS
is adjusted or varied such that one or more AD-associated
parameters is significantly improved as well as the toxicity due to
the conventional agent is prevented or minimized. In some
embodiments, the IL-4R antibody may be administered in higher
loading doses for significant improvement in an AD-associated
parameter followed by lower regular doses to sustain or maintain
the improvement. The concomitantly administered TCS may be
administered at a reduced dose, typically reduced by about 20%,
about 30%, about 40%, about 50% or about 60% as compared to a
patient not treated with the IL-4R antibody. The administration
regimens and dosage amounts are described elsewhere herein. In some
embodiments, the present invention includes methods to reduce
dependence on TCS in a patient with moderate-to-severe AD.
[0149] In certain embodiments, the present invention includes
methods to treat patients who have AD for more than 1 year, more
than about 5 years, more than about 10 years, or more than about 15
years, the methods comprising administering a therapeutically
effective amount of an IL-4R antagonist in combination with a
conventional therapeutic agent such as TCS.
[0150] In another aspect, the present invention includes methods
for a safer and/or more effective therapy in the long-term
management of moderate-to-severe AD in patients. The term "safer
and/or more effective therapy", as used herein, refers to methods
of treatment comprising administering an IL-4R antagonist in
combination with a conventional therapeutic agent such as TCS such
that one or more AD-associated parameters is significantly improved
as well as the side effects and toxicity due to the conventional
agent is minimized or prevented. In certain embodiments, the
improvement in an AD-associated parameter is selected from the
group consisting of: (a) a decrease from baseline in Investigator's
Global Assessment (IGA) score of at least 50%; (b) a decrease from
baseline in Pruritus Numeric Rating Scale (NRS) score of at least
65%; (c) a decrease from baseline in Eczema Area and Severity Index
(EASI) score of at least 70%; and (d) a decrease from baseline in
SCORAD score of at least 60%. In some embodiments, the dosage of
the conventional agent is reduced or lowered to minimize the
adverse side effects. In some embodiments, the methods of treatment
as described herein may reduce or eliminate the risk of rebound
after steroid reduction or discontinuation.
[0151] The present invention includes methods for more effective
and safer therapy in long-term management of AD in patients
including in children or young adults who may be more susceptible
or sensitive to a conventional therapeutic agent.
[0152] In another aspect of the invention, methods for reducing or
eliminating an AD patient's dependence on conventional therapeutics
such as TCS during the treatment of moderate-to-severe AD are
provided. In embodiments of the invention, the methods comprise:
selecting a patient with moderate-to-severe AD that is uncontrolled
or partially controlled with a background therapy; administering to
the patient a defined dose of an IL-4R antagonist, preferably an
anti-IL-4R antibody, for an initial treatment period while
maintaining the patient's background therapy for the initial
treatment period; and gradually reducing the dosage of one or more
components of the background therapy over a subsequent period of
treatment, while continuing to administer the IL-4R antagonist. The
term "background therapy" as used herein, refers to standard or
conventional therapeutic agents known in the art which are used for
treating AD (described elsewhere herein). In certain embodiments,
the background therapy comprises a TCS, or a topical calcineurin
inhibitor. In one embodiment, the background therapy is a potent
Group III TCS such as mometasone furoate or methylprednisolone
aceponate. In some embodiments, the dosage of the conventional
therapeutic such as TCS is eliminated or completely withdrawn upon
the initial treatment period. For example, a TCS is administered in
an initial treatment period and completely stopped or withdrawn in
the subsequent treatment period. In certain embodiments, the TCS is
reduced by about 10%, about 20%, about 30%, about 40%, about 50%,
or more as compared to the dose during the initial treatment
period.
[0153] In one example of a treatment regimen for a patient with
moderate-to-severe AD wherein an IL-4R antagonist is administered
to a patient with moderate-to-severe AD, during an initial
treatment period (also called the "stable phase"), a conventional
therapeutic such as a TCS administered to the patient as background
therapy. During a subsequent treatment period (also called
"withdrawal phase"), the administration of the TCS is gradually
reduced by about 5-60% as compared to the initial treatment period.
In one embodiment, the TCS is stopped, i.e., the TCS is gradually
reduced over the subsequent treatment period until it is withdrawn
or eliminated.
[0154] In a related aspect of the invention, methods of treating AD
comprising an add-on therapy to background therapy with systematic
background therapy withdrawal are provided. In certain embodiments
of the invention, an IL-4R antagonist is administered as an add-on
therapy to an AD patient who is on background therapy for a certain
period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months,
5 months, 12 months, 18 months, 24 months, or longer) (also called
the "stable phase"). In some embodiments, the background therapy
comprises a TCS. The stable phase is followed by a background
therapy withdrawal phase, wherein one or more components comprising
the background therapy are withdrawn, or reduced or eliminated,
while the add-on therapy continues. In some embodiments, the
background therapy may be reduced by about 5%, about 10%, about
20%, about 30%, about 40%, about 50% or by more during the
withdrawal phase. The withdrawal phase may last 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10
weeks, 11 weeks, 12 weeks, or more.
Atopic Dermatitis-Associated Biomarkers
[0155] The present invention also includes methods involving the
use, quantification, and analysis of AD-associated biomarkers. As
used herein, the term "AD-associated biomarker" means any
biological response, cell type, parameter, protein, polypeptide,
enzyme, enzyme activity, metabolite, nucleic acid, carbohydrate, or
other biomolecule which is present or detectable in an AD patient
at a level or amount that is different from (e.g., greater than or
less than) the level or amount of the marker present or detectable
in a non-AD patient. In some embodiments, the term "AD-associated
biomarker" includes a biomarker associated with Type 2 helper
T-cell (Th2)-driven inflammation. Exemplary AD-associated
biomarkers include, but are not limited to, e.g., thymus and
activation-regulated chemokine (TARC; also known as CCL17),
immunoglobulin E (IgE), eotaxin-3 (also known as CCL26), lactate
dehydrogenase (LDH), eosinophils, antigen-specific IgE (e.g.,
Phadiatop.TM. test), and periostin. The term "AD-associated
biomarker" also includes a gene or gene probe known in the art
which is differentially expressed in a subject with AD as compared
to a subject without AD. For example, genes which are significantly
up-regulated in a subject with AD include, but are not limited to,
T-helper 2 (Th2)-associated chemokines such as CCL13, CCL17, CCL18
and CCL26, markers of epidermal proliferation such as K16, Ki67,
and T-cell and dendritic cell antigens CD2, CD1b, and CD1c (Tintle
et al 2011; J. Allergy Clin. Immunol. 128: 583-593). Alternatively,
"AD-associated biomarker" also includes genes which are down
regulated due to AD such as terminal differentiation proteins
(e.g., loricrin, filaggrin and involucrin) (Tintle et al 2011; J.
Allergy Clin. Immunol. 128: 583-593). Certain embodiments of the
invention pertain to use of these biomarkers for monitoring disease
reversal with the administration of the IL-4R antagonist. Methods
for detecting and/or quantifying such AD-associated biomarkers are
known in the art; kits for measuring such AD-associated biomarkers
are available from various commercial sources; and various
commercial diagnostic laboratories offer services which provide
measurements of such biomarkers as well.
[0156] According to certain aspects of the invention, methods for
treating AD are provided which comprise: (a) selecting a subject
who exhibits a level of at least one AD-associated biomarker prior
to or at the time of treatment which signifies the disease state;
and (b) administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of an IL-4R
antagonist. In certain embodiments, the patient is selected by
determining if the level of an AD-associated biomarker is elevated.
The level of an AD-associated biomarker is determined or quantified
by acquiring a sample from the patient for a biomarker assay known
in the art. In certain other embodiments, a patient is selected by
acquiring information relating to an elevated level of an
AD-associated biomarker from the patient. In certain embodiments of
this aspect of the invention, the subject is selected on the basis
of an elevated level of IgE or TARC or periostin.
[0157] For purposes of the present invention, a normal IgE level in
healthy subjects is less than about 114 kU/L (e.g., as measured
using the ImmunoCAP.RTM. assay [Phadia, Inc. Portage, Mich.]).
Thus, the present invention involves methods comprising selecting a
subject who exhibits a serum IgE level greater than about 114 kU/L,
greater than about 150 kU/L, greater than about 500 kU/L, greater
than about 1000 kU/L, greater than about 1500 kU/L, greater than
about 2000 kU/L, greater than about 2500 kU/L, greater than about
3000 kU/L, greater than about 3500 kU/L, greater than about 4000
kU/L, greater than about 4500 kU/L, or greater than about 5000
kU/L, and administering to the subject a pharmaceutical composition
comprising a therapeutically effective amount of an IL-4R
antagonist.
[0158] TARC levels in healthy subjects are in the range of 106 ng/L
to 431 ng/L, with a mean of about 239 ng/L. (An exemplary assay
system for measuring TARC level is the TARC quantitative ELISA kit
offered as Cat. No. DDN00 by R&D Systems, Minneapolis, Minn.)
Thus, the present invention involves methods comprising selecting a
subject who exhibits a serum TARC level greater than about 431
ng/L, greater than about 500 ng/L, greater than about 1000 ng/L,
greater than about 1500 ng/L, greater than about 2000 ng/L, greater
than about 2500 ng/L, greater than about 3000 ng/L, greater than
about 3500 ng/L, greater than about 4000 ng/L, greater than about
4500 ng/L, or greater than about 5000 ng/L, and administering to
the subject a pharmaceutical composition comprising a
therapeutically effective amount of an IL-4R antagonist.
[0159] Another AD-associated biomarker is antigen-specific IgE.
Phadiatop.TM. is a commercially available variant of serum specific
or antigen-specific IgE assay test that was introduced for the
screening of allergic sensitization (Merrett et al 1987, Allergy
17: 409-416). The test provides for simultaneous testing for serum
specific IgE to a mixture of relevant allergens causing common
inhalant allergies. The test gives a qualitative result, either
positive or negative depending upon a fluorescence response
obtained. When a patient sample gives a fluorescence response
higher than or equal to the reference, a positive test result is
indicated. A patient sample with a lower fluorescence response
indicates a negative test result. The present invention includes
methods comprising selecting a subject who exhibits a positive test
result and administering to the subject a therapeutically effective
amount of an IL-4R antagonist.
[0160] Periostin is an extracellular matrix protein involved in the
Th2-mediated inflammatory processes. Periostin levels are found to
be up regulated in patients with AD (Masuoka et al 2012 J Clin
Invest. 122(7):2590-2600. doi:10.1172/JCI58978). The present
invention includes methods comprising administering an IL-4R
antagonist to treat patients with elevated levels of periostin.
[0161] Lactate dehydrogenase (LDH) is used as a marker of tissue
damage and is found to be elevated in patients with AD (Kou et al
2012; Arch. Dermatol. Res. 304: 305-312). The present invention
includes methods comprising administering an IL-4R antagonist to
treat patients with elevated levels of LDH.
[0162] According to other aspects of the invention, methods for
treating AD are provided which comprise administering to a subject
a pharmaceutical composition comprising a therapeutically effective
amount of an IL-4R antagonist, wherein administration of the
pharmaceutical composition to the subject results in a decrease in
at least one AD-associated biomarker (e.g., IgE, TARC, eosinophils,
eotaxin-3, antigen-specific IgE, LDH, etc.) at a time after
administration of the pharmaceutical composition, as compared to
the level of the biomarker in the subject prior to the
administration.
[0163] As will be appreciated by a person of ordinary skill in the
art, an increase or decrease in an AD-associated biomarker can be
determined by comparing (i) the level of the biomarker measured in
a subject at a defined time point after administration of the
pharmaceutical composition comprising an IL-4R antagonist to (ii)
the level of the biomarker measured in the patient prior to the
administration of the pharmaceutical composition comprising an
IL-4R antagonist (i.e., the "baseline measurement"). The defined
time point at which the biomarker is measured can be, e.g., at
about 4 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days, 20 days, 35
days, 40 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75
days, 80 days, 85 days, or more after administration of the of the
pharmaceutical composition comprising an IL-4R antagonist.
[0164] According to certain particular embodiments of the present
invention, a subject may exhibit a decrease in the level of one or
more of TARC and/or IgE following administration of a
pharmaceutical composition comprising an IL-4R antagonist (e.g., an
anti-IL-4R antibody). For example, at about day 4, day 8, day 15,
day 22, day 25, day 29, day 36, day 43, day 50, day 57, day 64, day
71 or day 85, following administration of a first, second, third or
fourth dose of a pharmaceutical composition comprising about 75,
150 or 300 mg of an anti-hIL-4R antibody (e.g., mAb1), the subject,
according to the present invention, may exhibit a decrease in TARC
of about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more from baseline
(wherein "baseline" is defined as the level of TARC in the subject
just prior to the first administration). Similarly, at about day 4,
day 8, day 15, day 22, day 25, day 29, day 36, day 43, day 50, day
57, day 64, day 71 or day 85, following administration of a first,
second, third or fourth dose of a pharmaceutical composition
comprising about 75, 150 or 300 mg of an anti-hIL-4R antibody
(e.g., mAb1), the subject, according to the present invention, may
exhibit a decrease in IgE of about 1%, 2%, 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95% or more from baseline (wherein "baseline" is defined as the
level of IgE in the subject just prior to the first
administration).
[0165] The present invention also includes methods for determining
whether a subject is a suitable subject for whom administration of
a pharmaceutical composition comprising an IL-4R antagonist would
be beneficial. For example, if an individual, prior to receiving a
pharmaceutical composition comprising an IL-4R antagonist, exhibits
a level of an AD-associated biomarker which signifies the disease
state, the individual is therefore identified as a suitable patient
for whom administration of a pharmaceutical composition of the
invention (a composition comprising an anti-IL-4R antibody) would
be beneficial. In a related embodiment, the present invention
includes methods for treating suitable subjects, wherein a suitable
subject may be more susceptible to AD, for example, due to race or
ethnicity. For example, the present invention includes methods
comprising administering an IL-4R antagonist to African-American
subjects who may be more susceptible to AD. Such a subject
population may have an elevated level of an AD-associated
biomarker.
[0166] According to certain exemplary embodiments, an individual
may be identified as a good candidate for anti-IL-4R therapy if the
individual exhibits one or more of the following: (i) an IgE level
greater than about 114 kU/L, greater than about 150 kU/L, greater
than about 500 kU/L, greater than about 1000 kU/L, greater than
about 1500 kU/L, greater than about 2000 kU/L, greater than about
2500 kU/L, greater than about 3000 kU/L, greater than about 3500
kU/L, greater than about 4000 kU/L, greater than about 4500 kU/L,
or greater than about 5000 kU/L; or (ii) a TARC level greater than
about 431 ng/L, greater than about 500 ng/L, greater than about
1000 ng/L, greater than about 1500 ng/L, greater than about 2000
ng/L, greater than about 2500 ng/L, greater than about 3000 ng/L,
greater than about 3500 ng/L, greater than about 4000 ng/L, greater
than about 4500 ng/L, or greater than about 5000 ng/L; or (iii) a
positive Phadiatop.TM. test. Additional criteria, such as other
clinical indicators of AD (e.g., an elevated IGA, BSA, EASI,
SCORAD, 5-D Pruritus, and/or NRS score indicative of AD), may be
used in combination with any of the foregoing AD-associated
biomarkers to identify an individual as a suitable candidate for
anti-IL-4R therapy as described elsewhere herein.
Interleukin-4 Receptor Antagonists
[0167] As disclosed in detail above, the present invention includes
methods which comprise administering to a subject in need thereof a
therapeutic composition comprising an interleukin-4 receptor
(IL-4R) antagonist. As used herein, an "IL-4R antagonist" is any
agent which binds to or interacts with IL-4R and inhibits the
normal biological signaling function of IL-4R when IL-4R is
expressed on a cell in vitro or in vivo. Non-limiting examples of
categories of IL-4R antagonists include small molecule IL-4R
antagonists, anti-IL-4R aptamers, peptide-based IL-4R antagonists
(e.g., "peptibody" molecules), and antibodies or antigen-binding
fragments of antibodies that specifically bind human IL-4R.
[0168] The terms "IL-4R," "hIL-4R," and the like, as used herein,
are intended to refer to the alpha chain of the human cytokine
receptor that specifically binds interleukin-4 (IL-4), IL-4R.alpha.
(SEQ ID NO:274). Unless specifically designated as being from a
non-human species, the term "IL-4R", as used herein, shall be
understood to mean the human interleukin-4 receptor alpha
chain.
[0169] The term "antibody," as used herein, is intended to refer to
immunoglobulin molecules comprising four polypeptide chains, two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, as well as multimers thereof (e.g., IgM). Each
heavy chain comprises a heavy chain variable region (abbreviated
herein as HCVR or V.sub.H) and a heavy chain constant region. The
heavy chain constant region comprises three domains, C.sub.H1,
C.sub.H2 and C.sub.H3. Each light chain comprises a light chain
variable region (abbreviated herein as LCVR or V.sub.L) and a light
chain constant region. The light chain constant region comprises
one domain (C.sub.L1). The V.sub.H and V.sub.L regions can be
further subdivided into regions of hypervariability, termed
complementarity determining regions (CDRs), 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 different
embodiments of the invention, the FRs of the anti-IL-4R antibody
(or antigen-binding portion 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.
[0170] The term "antibody," as used herein, also includes
antigen-binding fragments of full antibody molecules. The terms
"antigen-binding portion" of an antibody, "antigen-binding
fragment" of an antibody, and the like, as used herein, include any
naturally occurring, enzymatically obtainable, synthetic, or
genetically engineered polypeptide or glycoprotein that
specifically binds an antigen to form a complex. Antigen-binding
fragments of an antibody may be derived, e.g., from full antibody
molecules using any suitable standard techniques such as
proteolytic digestion or recombinant genetic engineering techniques
involving the manipulation and expression of DNA encoding antibody
variable and optionally constant domains. Such DNA is known and/or
is readily available from, e.g., commercial sources, DNA libraries
(including, e.g., phage-antibody libraries), or can be synthesized.
The DNA may be sequenced and manipulated chemically or by using
molecular biology techniques, for example, to arrange one or more
variable and/or constant domains into a suitable configuration, or
to introduce codons, create cysteine residues, modify, add or
delete amino acids, etc.
[0171] Non-limiting examples of antigen-binding fragments include:
(i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv)
Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb
fragments; and (vii) minimal recognition units consisting of the
amino acid residues that mimic the hypervariable region of an
antibody (e.g., an isolated complementarity determining region
(CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4
peptide. Other engineered molecules, such as domain-specific
antibodies, single domain antibodies, domain-deleted antibodies,
chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies,
tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies,
bivalent nanobodies, etc.), small modular immunopharmaceuticals
(SMIPs), and shark variable IgNAR domains, are also encompassed
within the expression "antigen-binding fragment," as used
herein.
[0172] An antigen-binding fragment of an antibody will typically
comprise at least one variable domain. The variable domain may be
of any size or amino acid composition and will generally comprise
at least one CDR which is adjacent to or in frame with one or more
framework sequences. In antigen-binding fragments having a V.sub.H
domain associated with a V.sub.L domain, the V.sub.H and V.sub.L
domains may be situated relative to one another in any suitable
arrangement. For example, the variable region may be dimeric and
contain V.sub.H-V.sub.H, V.sub.H-V.sub.L or V.sub.L-V.sub.L dimers.
Alternatively, the antigen-binding fragment of an antibody may
contain a monomeric V.sub.H or V.sub.L domain.
[0173] In certain embodiments, an antigen-binding fragment of an
antibody may contain at least one variable domain covalently linked
to at least one constant domain. Non-limiting, exemplary
configurations of variable and constant domains that may be found
within an antigen-binding fragment of an antibody of the present
invention include: (i) V.sub.H-C.sub.H1; (ii) V.sub.H-C.sub.H2;
(iii) V.sub.H-C.sub.H3; (iv) V.sub.H-C.sub.H1-C.sub.H2; (v)
V.sub.H-C.sub.H1-C.sub.H2-C.sub.H3; (vi) V.sub.H-C.sub.H2-C.sub.H3;
(vii) V.sub.H-C.sub.L; (viii) V.sub.L-C.sub.H1; (ix)
V.sub.L-C.sub.H2; (x) V.sub.L-C.sub.H3; (xi)
V.sub.L-C.sub.H1-C.sub.H2; (xii)
V.sub.L-C.sub.H1-C.sub.H2-C.sub.H3; (xiii)
V.sub.L-C.sub.H2-C.sub.H3; and (xiv) V.sub.L-C.sub.L. In any
configuration of variable and constant domains, including any of
the exemplary configurations listed above, the variable and
constant domains may be either directly linked to one another or
may be linked by a full or partial hinge or linker region. A hinge
region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or
more) amino acids which result in a flexible or semi-flexible
linkage between adjacent variable and/or constant domains in a
single polypeptide molecule. Moreover, an antigen-binding fragment
of an antibody of the present invention may comprise a homo-dimer
or hetero-dimer (or other multimer) of any of the variable and
constant domain configurations listed above in non-covalent
association with one another and/or with one or more monomeric
V.sub.H or V.sub.L domain (e.g., by disulfide bond(s)).
[0174] As with full antibody molecules, antigen-binding fragments
may be monospecific or multispecific (e.g., bispecific). A
multispecific antigen-binding fragment of an antibody will
typically comprise at least two different variable domains, wherein
each variable domain is capable of specifically binding to a
separate antigen or to a different epitope on the same antigen. Any
multispecific antibody format may be adapted for use in the context
of an antigen-binding fragment of an antibody of the present
invention using routine techniques available in the art.
[0175] The constant region of an antibody is important in the
ability of an antibody to fix complement and mediate cell-dependent
cytotoxicity. Thus, the isotype of an antibody may be selected on
the basis of whether it is desirable for the antibody to mediate
cytotoxicity.
[0176] The term "human antibody," as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may nonetheless 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 antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0177] 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.
[0178] Human antibodies can exist in two forms that are associated
with hinge heterogeneity. In one form, an immunoglobulin molecule
comprises a stable four chain construct of approximately 150-160
kDa in which the dimers are held together by an interchain heavy
chain disulfide bond. In a second form, the dimers are not linked
via inter-chain disulfide bonds and a molecule of about 75-80 kDa
is formed composed of a covalently coupled light and heavy chain
(half-antibody). These forms have been extremely difficult to
separate, even after affinity purification.
[0179] The frequency of appearance of the second form in various
intact IgG isotypes is due to, but not limited to, structural
differences associated with the hinge region isotype of the
antibody. A single amino acid substitution in the hinge region of
the human IgG4 hinge can significantly reduce the appearance of the
second form (Angal et al. (1993) Molecular Immunology 30:105) to
levels typically observed using a human IgG1 hinge. The instant
invention encompasses antibodies having one or more mutations in
the hinge, C.sub.H2 or C.sub.H3 region which may be desirable, for
example, in production, to improve the yield of the desired
antibody form.
[0180] An "isolated antibody," as used herein, means an antibody
that has been identified and separated and/or recovered from at
least one component of its natural environment. For example, an
antibody that has been separated or removed from at least one
component of an organism, or from a tissue or cell in which the
antibody naturally exists or is naturally produced, is an "isolated
antibody" for purposes of the present invention. An isolated
antibody also includes an antibody in situ within a recombinant
cell. Isolated antibodies are antibodies that have been subjected
to at least one purification or isolation step. According to
certain embodiments, an isolated antibody may be substantially free
of other cellular material and/or chemicals.
[0181] 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" IL-4R, as used in
the context of the present invention, includes antibodies that bind
IL-4R 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. An isolated antibody that specifically
binds human IL-4R may, however, have cross-reactivity to other
antigens, such as IL-4R molecules from other (non-human)
species.
[0182] The anti-IL-4R antibodies useful for the methods of the
present invention 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 from which the
antibodies were derived. 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 methods involving the use
of 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. The
use of antibodies and antigen-binding fragments obtained in this
general manner are encompassed within the present invention.
[0183] The present invention also includes methods involving the
use of anti-IL-4R 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 the use of anti-IL-4R 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.
[0184] The term "surface plasmon resonance," as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
interactions by detection of alterations in protein concentrations
within a biosensor matrix, for example using the BIAcore.TM. system
(Biacore Life Sciences division of GE Healthcare, Piscataway,
N.J.).
[0185] The term "K.sub.D," as used herein, is intended to refer to
the equilibrium dissociation constant of a particular
antibody-antigen interaction.
[0186] The term "epitope" refers to an antigenic determinant that
interacts with a specific antigen binding site in the variable
region of an antibody molecule known as a paratope. A single
antigen may have more than one epitope. Thus, different antibodies
may bind to different areas on an antigen and may have different
biological effects. Epitopes may be either conformational or
linear. A conformational epitope is produced by spatially
juxtaposed amino acids from different segments of the linear
polypeptide chain. A linear epitope is one produced by adjacent
amino acid residues in a polypeptide chain. In certain
circumstance, an epitope may include moieties of saccharides,
phosphoryl groups, or sulfonyl groups on the antigen.
Preparation of Human Antibodies
[0187] 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 human IL-4R.
[0188] Using VELOCIMMUNE.TM. technology (see, for example, U.S.
Pat. No. 6,596,541, Regeneron Pharmaceuticals) or any other known
method for generating monoclonal antibodies, high affinity chimeric
antibodies to IL-4R 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.
[0189] 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.
[0190] Initially, high affinity chimeric antibodies are isolated
having a human variable region and a mouse constant region. The
antibodies are characterized and selected for desirable
characteristics, including affinity, selectivity, epitope, etc,
using standard procedures known to those skilled in the art. 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.
[0191] In general, the antibodies that can be used in the methods
of the present invention possess high affinities, as described
above, 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.
[0192] Specific examples of human antibodies or antigen-binding
fragments of antibodies that specifically bind IL-4R which can be
used in the context of the methods of the present invention include
any antibody or antigen-binding fragment which comprises the three
heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy
chain variable region (HCVR) having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 2, 18, 22, 26, 42, 46, 50,
66, 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166,
170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258 and 262. The
antibody or antigen-binding fragment may comprise the three light
chain CDRs (LCVR1, LCVR2, LCVR3) contained within a light chain
variable region (LCVR) having an amino acid sequence selected from
the group consisting of SEQ ID NOs: 10, 20, 24, 34, 44, 48, 58, 68,
72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178,
188, 192, 202, 212, 216, 226, 236, 240, 250, 260 and 264. Methods
and techniques for identifying CDRs within HCVR and LCVR amino acid
sequences are well known in the art and can be used to identify
CDRs within the specified HCVR and/or LCVR amino acid sequences
disclosed herein. Exemplary conventions that can be used to
identify the boundaries of CDRs include, e.g., the Kabat
definition, the Chothia definition, and the AbM definition. In
general terms, the Kabat definition is based on sequence
variability, the Chothia definition is based on the location of the
structural loop regions, and the AbM definition is a compromise
between the Kabat and Chothia approaches. See, e.g., Kabat,
"Sequences of Proteins of Immunological Interest," National
Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J.
Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.
Sci. USA 86:9268-9272 (1989). Public databases are also available
for identifying CDR sequences within an antibody.
[0193] In certain embodiments of the present invention, the
antibody or antigen-binding fragment thereof comprises the six CDRs
(HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy and
light chain variable region amino acid sequence pairs (HCVR/LCVR)
selected from the group consisting of SEQ ID NOs: 2/10, 18/20,
22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92,
94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144,
146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202,
210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260 and
262/264.
[0194] In certain embodiments of the present invention, the
antibody or antigen-binding fragment thereof comprises six CDRs
(HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3) having the amino acid
sequences selected from the group consisting of SEQ ID NOs:
4/6/8/12/14/16; 28/30/32/36/38/40; 52/54/56/60/62/64;
76/78/80/84/86/88; 100/102/104/108/110/112;
124/126/128/132/134/136; 148/150/152/156/158/160;
172/174/176/180/182/184; 196/198/200/204/206/208;
220/222/224/228/230/232; and 244/246/248/252/254/256.
[0195] In certain embodiments of the present invention, the
antibody or antigen-binding fragment thereof comprises HCVR/LCVR
amino acid sequence pairs selected from the group consisting of SEQ
ID NOs: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68,
70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130,
138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188,
190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240,
242/250, 258/260 and 262/264.
Pharmaceutical Compositions
[0196] The present invention includes methods which comprise
administering an IL-4R antagonist to a patient, wherein the IL-4R
antagonist is contained within a pharmaceutical composition. The
pharmaceutical compositions of the invention are 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.
[0197] The dose of antibody administered to a patient according to
the methods of the present invention may vary depending upon the
age and the size of the patient, symptoms, conditions, route of
administration, and the like. The dose is typically calculated
according to body weight or body surface area. Depending on the
severity of the condition, the frequency and the duration of the
treatment can be adjusted. Effective dosages and schedules for
administering pharmaceutical compositions comprising anti-IL-4R
antibodies may be determined empirically; for example, patient
progress can be monitored by periodic assessment, and the dose
adjusted accordingly. Moreover, interspecies scaling of dosages can
be performed using well-known methods in the art (e.g., Mordenti et
al., 1991, Pharmaceut. Res. 8:1351). Specific exemplary doses of
anti-IL4R antibodies, and administration regimens involving the
same, that can be used in the context of the present invention are
disclosed elsewhere herein.
[0198] 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.
[0199] 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.
[0200] Numerous reusable pen and autoinjector delivery devices have
applications in the subcutaneous delivery of a pharmaceutical
composition of the present invention. Examples include, but are not
limited to AUTOPEN.TM. (Owen Mumford, Inc., Woodstock, UK),
DISETRONIC.TM. pen (Disetronic Medical Systems, Bergdorf,
Switzerland), HUMALOG MIX 75/25.TM. pen, HUMALOG.TM. pen, HUMALIN
70/30.TM. pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN.TM.
I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN
JUNIOR.TM. (Novo Nordisk, Copenhagen, Denmark), BD.TM. pen (Becton
Dickinson, Franklin Lakes, N.J.), OPTIPEN.TM., OPTIPEN PRO.TM.,
OPTIPEN STARLET.TM., and OPTICLIK.TM. (sanofi-aventis, Frankfurt,
Germany), to name only a few. Examples of disposable pen delivery
devices having applications in subcutaneous delivery of a
pharmaceutical composition of the present invention include, but
are not limited to the SOLOSTAR.TM. pen (sanofi-aventis), the
FLEXPEN.TM. (Novo Nordisk), and the KWIKPEN.TM. (Eli Lilly), the
SURECLICK.TM. Autoinjector (Amgen, Thousand Oaks, Calif.), the
PENLET.TM. (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey,
L.P.), and the HUMIRA.TM. Pen (Abbott Labs, Abbott Park Ill.), to
name only a few.
[0201] In certain situations, the pharmaceutical composition can be
delivered in a controlled release system. In one embodiment, a pump
may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref.
Biomed. Eng. 14:201). 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.
[0202] 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 can be filled in an appropriate
ampoule.
[0203] 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.
[0204] 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
[0205] The amount of IL-4R antagonist (e.g., anti-IL-4R 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 IL-4R antagonist that results in one or more of: (a) an
improvement in one or more AD-associated parameters (as defined
elsewhere herein); and/or (b) a detectable improvement in one or
more symptoms or indicia of atopic dermatitis. A "therapeutically
effective amount" also includes an amount of IL-4R antagonist that
inhibits, prevents, lessens, or delays the progression of AD in a
subject.
[0206] In the case of an anti-IL-4R 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, 75 mg, 150 mg, or 300
mg of an anti-IL-4R antibody is administered to a subject.
[0207] The amount of IL-4R antagonist 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 IL-4R antagonist may be administered to a patient at a
dose of about 0.0001 to about 10 mg/kg of patient body weight.
Combination Therapies
[0208] The methods of the present invention, according to certain
embodiments, comprise administering to the subject one or more
additional therapeutic agents in combination with the IL-4R
antagonist. As used herein, the expression "in combination with"
means that the additional therapeutic agents are administered
before, after, or concurrent with the pharmaceutical composition
comprising the IL-4R antagonist. The term "in combination with"
also includes sequential or concomitant administration of IL-4R
antagonist and a second therapeutic agent.
[0209] For example, when administered "before" the pharmaceutical
composition comprising the IL-4R antagonist, the additional
therapeutic agent may be administered about 72 hours, about 60
hours, about 48 hours, about 36 hours, about 24 hours, about 12
hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours,
about 2 hours, about 1 hour, about 30 minutes, about 15 minutes or
about 10 minutes prior to the administration of the pharmaceutical
composition comprising the IL-4R antagonist. When administered
"after" the pharmaceutical composition comprising the IL-4R
antagonist, the additional therapeutic agent may be administered
about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour,
about 2 hours, about 4 hours, about 6 hours, about 8 hours, about
10 hours, about 12 hours, about 24 hours, about 36 hours, about 48
hours, about 60 hours or about 72 hours after the administration of
the pharmaceutical composition comprising the IL-4R antagonist.
Administration "concurrent" or with the pharmaceutical composition
comprising the IL-4R antagonist means that the additional
therapeutic agent is administered to the subject in a separate
dosage form within less than 5 minutes (before, after, or at the
same time) of administration of the pharmaceutical composition
comprising the IL-4R antagonist, or administered to the subject as
a single combined dosage formulation comprising both the additional
therapeutic agent and the IL-4R antagonist.
[0210] The additional therapeutic agent may be, e.g., another IL-4R
antagonist, an IL-1 antagonist (including, e.g., an IL-1 antagonist
as set forth in U.S. Pat. No. 6,927,044), an IL-6 antagonist, an
IL-6R antagonist (including, e.g., an anti-IL-6R antibody as set
forth in U.S. Pat. No. 7,582,298), an IL-13 antagonist, a TNF
antagonist, an IL-8 antagonist, an IL-9 antagonist, an IL-17
antagonist, an IL-5 antagonist, an IgE antagonist, a CD48
antagonist, an IL-31 antagonist (including, e.g., as set forth in
U.S. Pat. No. 7,531,637), a thymic stromal lymphopoietin (TSLP)
antagonist (including, e.g., as set forth in US 2011/027468),
interferon-gamma (IFN.gamma.) antibiotics, topical corticosteroids,
tacrolimus, pimecrolimus, cyclosporine, azathioprine, methotrexate,
cromolyn sodium, proteinase inhibitors, or combinations thereof. In
certain embodiments, the pharmaceutical composition comprising an
anti-IL4R antagonist is administered to a subject in conjunction
with a non-pharmaceutical therapy such as ultraviolet (UV) light
therapy.
[0211] The methods of the invention comprise administering an IL-4R
antagonist in combination with a second therapeutic agent for
additive or synergistic activity to treat AD. In one embodiment,
the invention includes methods to treat moderate-to-severe AD.
Certain embodiments of the invention include methods to treat
moderate-to-severe AD by administering an IL-4R antagonist
concomitantly with a TCS. The TCS may be a potent TCS such as a
Group III TCS. Examples of Group II TCS include methylprednisolone
aceponate, mometasone furoate, fluticasone propionate and
betamethasone valerate. In some embodiments, the TCS may be a
moderate TCS such as Group II TCS or a weak TCS such as Group I
TCS.
Administration Regimens
[0212] The present invention includes methods comprising
administering to a subject a pharmaceutical composition comprising
an IL-4R antagonist at a dosing frequency of about four times a
week, twice a week, once a week, once every two weeks, once every
three weeks, once every four weeks, once every five weeks, once
every six weeks, once every eight weeks, once every twelve weeks,
or less frequently so long as a therapeutic response is achieved.
In certain embodiments involving the administration of a
pharmaceutical composition comprising an anti-IL-4R antibody, once
a week dosing at an amount of about 75 mg, 150 mg, or 300 mg, can
be employed.
[0213] According to certain embodiments of the present invention,
multiple doses of an IL-4R antagonist may be administered to a
subject over a defined time course. The methods according to this
aspect of the invention comprise sequentially administering to a
subject multiple doses of an IL-4R antagonist. As used herein,
"sequentially administering" means that each dose of IL-4R
antagonist is administered to the subject at a different point in
time, e.g., on different days separated by a predetermined interval
(e.g., hours, days, weeks or months). The present invention
includes methods which comprise sequentially administering to the
patient a single initial dose of an IL-4R antagonist, followed by
one or more secondary doses of the IL-4R antagonist, and optionally
followed by one or more tertiary doses of the IL-4R antagonist.
[0214] The terms "initial dose," "secondary doses," and "tertiary
doses," refer to the temporal sequence of administration of the
IL-4R antagonist. Thus, the "initial dose" is the dose which is
administered at the beginning of the treatment regimen (also
referred to as the "baseline dose"); the "secondary doses" are the
doses which are administered after the initial dose; and the
"tertiary doses" are the doses which are administered after the
secondary doses. The initial, secondary, and tertiary doses may all
contain the same amount of IL-4R antagonist, but generally may
differ from one another in terms of frequency of administration. In
certain embodiments, however, the amount of IL-4R antagonist
contained in the initial, secondary and/or tertiary doses varies
from one another (e.g., adjusted up or down as appropriate) during
the course of treatment. In certain embodiments, one or more (e.g.,
1, 2, 3, 4, or 5) doses are administered at the beginning of the
treatment regimen as "loading doses" followed by subsequent doses
that are administered on a less frequent basis (e.g., "maintenance
doses"). For example, an IL-4R antagonist may be administered to a
patient with AD at a loading dose of about 300 mg or about 600 mg
followed by one or more maintenance doses of about 75 mg to about
300 mg. In one embodiment, the initial dose and the one or more
secondary doses each include 50 mg to 600 mg of the IL-4R
antagonist, e.g., 100 mg to 400 mg of the IL-4R antagonist, e.g.,
100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg or 500 mg of the
IL-4R antagonist. In some embodiments, the initial dose and the one
or more secondary doses each contain the same amount of the IL-4R
antagonist. In other embodiments, the initial dose comprises a
first amount of the IL-4R antagonist, and the one or more secondary
doses each comprise a second amount of the IL-4R antagonist. For
example, the first amount of the IL-4R antagonist can be
1.5.times., 2.times., 2.5.times., 3.times., 3.5.times., 4.times. or
5.times. or more than the second amount of the IL-4R
antagonist.
[0215] In one exemplary embodiment of the present invention, each
secondary and/or tertiary dose is administered 1 to 14 (e.g., 1,
11/2, 2, 21/2, 3, 31/2, 4, 41/2, 5, 51/2, 6, 61/2, 7, 71/2, 8,
81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2, 14,
141/2, or more) weeks after the immediately preceding dose. The
phrase "the immediately preceding dose," as used herein, means, in
a sequence of multiple administrations, the dose of IL-4R
antagonist which is administered to a patient prior to the
administration of the very next dose in the sequence with no
intervening doses.
[0216] The methods according to this aspect of the invention may
comprise administering to a patient any number of secondary and/or
tertiary doses of an IL-4R antagonist. For example, in certain
embodiments, only a single secondary dose is administered to the
patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7,
8, or more) secondary doses are administered to the patient.
Likewise, in certain embodiments, only a single tertiary dose is
administered to the patient. In other embodiments, two or more
(e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are
administered to the patient.
[0217] In embodiments involving multiple secondary doses, each
secondary dose may be administered at the same frequency as the
other secondary doses. For example, each secondary dose may be
administered to the patient 1 to 2 weeks after the immediately
preceding dose. Similarly, in embodiments involving multiple
tertiary doses, each tertiary dose may be administered at the same
frequency as the other tertiary doses. For example, each tertiary
dose may be administered to the patient 2 to 4 weeks after the
immediately preceding dose. Alternatively, the frequency at which
the secondary and/or tertiary doses are administered to a patient
can vary over the course of the treatment regimen. The frequency of
administration may also be adjusted during the course of treatment
by a physician depending on the needs of the individual patient
following clinical examination.
[0218] The present invention includes methods comprising sequential
administration of an IL-4R antagonist and a second therapeutic
agent, to a patient to treat AD. In some embodiments, the present
methods comprise administering one or more doses of an IL-4R
antagonist followed by one or more doses of a second therapeutic
agent. For example, one or more doses of about 75 mg to about 300
mg of the IL-4R antagonist may be administered after which one or
more doses of a second therapeutic agent (e.g., a topical
corticosteroid or a calcineurin inhibitor or any other therapeutic
agent, as described elsewhere herein) may be administered to treat,
alleviate, reduce or ameliorate one or more symptoms of AD. In some
embodiments, the IL-4R antagonist is administered at one or more
doses resulting in an improvement in one or more AD-associated
parameters followed by the administration of a second therapeutic
agent to prevent recurrence of at least one symptom of AD.
Alternative embodiments of the invention pertain to concomitant
administration of an IL-4R antagonist and a second therapeutic
agent. For example, one or more doses of an IL-4R antagonist are
administered and a second therapeutic agent is administered at a
separate dosage at a similar or different frequency relative to the
IL-4R antagonist. In some embodiments, the second therapeutic agent
is administered before, after or concurrently with the IL-4R
antagonist.
IL-4R Antagonists as Vaccine Adjuvants
[0219] The present invention also includes compositions and methods
that are useful in vaccine applications. For example, an IL-4R
antagonist (e.g., an anti-IL-4R antibody disclosed herein) may be
administered to a subject in conjunction with a vaccine to improve
or potentiate the immune response (including humoral and cellular
immune responses) elicited by the vaccine, i.e., as a vaccine
adjuvant. In certain embodiments, an IL-4R antagonist is
administered just prior to, concurrent with, and/or subsequent to
administration of a vaccine composition to a subject. For example,
the present invention includes methods of eliciting or enhancing an
immune response to an antigen in a subject by first administering
to the subject a pharmaceutical composition comprising an IL-4R
antagonist, followed by administering to the subject a vaccine
composition comprising the antigen (by itself or in combination
with the IL-4R antagonist), and optionally administering additional
doses of the IL-4R antagonist for a period of time following
administration of the vaccine antigen to the subject.
[0220] The IL-4R antagonists of the present invention may be
administered as adjuvants with any type of vaccine including, e.g.,
live vaccines, live/attenuated vaccines, killed vaccines, subunit
vaccines, DNA vaccines, and cancer immunotherapeutic vaccines. The
vaccines that may be used in connection with the IL-4R antagonists
of the invention include vaccines against bacterial pathogens,
viruses, parasites, and other infectious agents. Non-limiting
examples of infectious agents and diseases against which the
vaccine compositions and methods of the invention may be targeted
include, e.g., HIV, HCV, RSV, Neisseria meningitides,
streptococcus, tuberculosis, malaria, smallpox, diphtheria,
pertussis, tetanus, polio, measles, rubella, mumps, influenza,
Anthrax, SARS, Ebola virus, Hanta virus, Dengue virus, etc.
[0221] The present invention also includes pharmaceutical
compositions comprising an IL-4R antagonist and one or more vaccine
antigen. The pharmaceutical compositions according to this aspect
of the invention may comprise one or more additional immune
potentiators such as MPL, MDP, CpG oligonucleotides, lipopeptides,
saponins, dsRNA, small molecule immune potentiators, etc.
[0222] Besides IL-4R antagonists, other inhibitors of the
IL-4/IL-13 signaling pathway (e.g., anti-IL-4 antibodies,
anti-IL-13 antibodies, bispecific anti-IL-4/anti-IL-13 antibodies,
etc.) may be used in the context of vaccine methods and
compositions as disclosed herein.
EXAMPLES
[0223] 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
Generation of Human Antibodies to Human IL-4R
[0224] Human anti-hIL-4R antibodies were generated as described in
U.S. Pat. No. 7,608,693. Table 1 sets forth the sequence
identifiers for the heavy and light chain variable region amino
acid sequence pairs, and CDR amino acid sequences, of selected
anti-IL-4R antibodies and their corresponding antibody
designations.
TABLE-US-00001 TABLE 1 Antibody SEQ ID NOs: Designation HCVR HCDR1
HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H095-a 2 4 6 8 10 12 14 16
H1H095-b 18 4 6 8 20 12 14 16 H1H095-c 22 4 6 8 24 12 14 16
H1H097-a 26 28 30 32 34 36 38 40 H1H097-b 42 28 30 32 44 36 38 40
H1H097-c 46 28 30 32 48 36 38 40 H1H093-a 50 52 54 56 58 60 62 64
H1H093-b 66 52 54 56 68 60 62 64 H1H093-c 70 52 54 56 72 60 62 64
H1H093-d 74 76 78 80 82 84 86 88 H1H093-e 90 76 78 80 92 84 86 88
H1H093-f 94 76 78 80 96 84 86 88 H1H094-a 98 100 102 104 106 108
110 112 H1H094-b 114 100 102 104 116 108 110 112 H1H094-c 118 100
102 104 120 108 110 112 H1H096-a 122 124 126 128 130 132 134 136
H1H096-b 138 124 126 128 140 132 134 136 H1H096-c 142 124 126 128
144 132 134 136 H1H098-a 146 148 150 152 154 156 158 160 H1H098-b
162 148 150 152 164 156 158 160 H1H098-c 166 148 150 152 168 156
158 160 H1H099-a 170 172 174 176 178 180 182 184 H1H099-b 186 172
174 176 188 180 182 184 H1H099-c 190 172 174 176 192 180 182 184
H4H083-a 194 196 198 200 202 204 206 208 H4H083-b 210 196 198 200
212 204 206 208 H4H083-c 214 196 198 200 216 204 206 208 H4H121-a
218 220 222 224 226 228 230 232 H4H121-b 234 220 222 224 236 228
230 232 H4H121-c 238 220 222 224 240 228 230 232 H4H118-a 242 244
246 248 250 252 254 256 H4H118-b 258 244 246 248 260 252 254 256
H4H118-c 262 244 246 248 264 252 254 256
[0225] The exemplary IL-4R antagonist used in the following
Examples is the human anti-IL-4R antibody designated in Table 1 as
H1H098-b (also referred to herein as "mAb1").
Example 2
Single Ascending Dose Clinical Trial of Intravenously and
Subcutaneously Administered Anti-IL-4R Antibody (mAb1) in Healthy
Subjects
A. Study Design
[0226] This study was a randomized, double-blind,
placebo-controlled, sequential, single ascending-dose study of
intravenous (IV) and subcutaneous (SC) administered mAb1 in healthy
subjects. The main purpose of this study was to evaluate the safety
and tolerability of intravenously and subcutaneously administered
mAb1 in healthy subjects.
[0227] Screening occurred from day -21 to day -3. On day 1
(baseline), subjects were randomized to receive either IV or SC
study drug (mAb1 or placebo) infused over a 2-hour period. Subjects
returned on days 4, 8, 11, 15, 22, 29, 43, 57 and 85 (end-of-study)
for safety assessments and blood sampling for clinical laboratory
testing.
[0228] Forty-eight total subjects participated in the study. Four
sequential ascending dose cohorts (1.0, 3.0, 8.0, and 12.0 mg/kg)
were planned for IV dosing and 2 sequential ascending dose cohorts
(150 and 300 mg) were planned for SC dosing. Each dose cohort
consisted of 8 subjects (if there was no cohort expansion): 6
randomized to receive mAb1 and 2 randomized to receive placebo. In
order to optimize safety, the first 3 subjects in IV cohort 1 (1.0
mg/kg) were dosed at least 24 hours apart and the remaining 5
subjects were dosed 5 to 7 days later. In subsequent IV cohorts, 3
of the 8 subjects were dosed on day 1 and the remaining 5 subjects
were dosed 5 to 7 days later. All 8 subjects in SC dose cohort 1
(150 mg) were dosed on the same day, and all 8 subjects in the
subsequent SC cohort (300 mg) were dosed on the same day. The SC
cohorts were administered after the IV cohorts were completed.
[0229] Inclusion criteria for the study were as follows: (1) Male
or female 18 to 65 years of age; (2) Weight>50 kg and <120
kg; (3) For women of childbearing potential, a negative serum
pregnancy test at the screening visit (visit 1) and a negative
urine pregnancy test on day -1; (4) Willingness to refrain from the
consumption of more than 2 standard alcoholic drinks in any 24-hour
period during the duration of the study. A standard alcoholic drink
was considered to be the equivalent of 12 ounces of beer, 5 ounces
of wine, or 1.5 ounces of hard liquor; (5) Willingness to refrain
from the consumption of alcohol for 24 hours prior to each study
visit; (6) For men and women of childbearing potential, willingness
to utilize adequate contraception and not become pregnant (or have
their partner[s] become pregnant) during the full duration of the
study. Adequate contraceptive measures include intrauterine device
(IUD); bilateral tubal ligation; vasectomy; condom or diaphragm
plus either contraceptive sponge, foam or jelly; and (7)
Willingness, commitment, and ability to return for all clinic
visits and complete all study-related procedures.
[0230] Exclusion criteria for the study were as follows: (1) Onset
of a new exercise routine or major change to a previous exercise
routine within 4 weeks prior to screening (visit 1). Subjects had
to be willing to maintain a similar level of exercise for the
duration of the study and to refrain from unusually strenuous
exercise for the duration of the trial; (2) Pregnant or
breast-feeding women; (3) Significant concomitant illness or
history of significant illness such as cardiac, renal,
neurological, endocrinological, metabolic or lymphatic disease, or
any other illness or condition that would have adversely affected
the subject's participation in this study; (4) Any clinically
significant abnormalities observed during the screening visit; (5)
Hospitalization for any reason within 60 days of screening (visit
1); (6) Known history of human immunodeficiency virus (HIV),
hepatitis B or hepatitis C, and/or positive hepatitis B surface
antigen, positive hepatitis C antibody or positive HIV serology at
the screening visit; (7) History of or positive drug screen for
drug or alcohol abuse within a year prior to the screening visit;
(8) History of a hypersensitivity to doxycycline or similar
compound; (9) Participation in any clinical research study
evaluating another investigational drug or therapy within 30 days
or at least 5 half-lives (whichever was longer), of the
investigational drug prior to the screening visit; (10) Previous
exposure to any therapeutic or investigational biological agent;
(11) Any medical or psychiatric condition which, in the opinion of
the investigator, would have placed the subject at risk; interfered
with participation in the study or interfered with the
interpretation of study results; (12) Subjects with a positive
QuantiFERON tuberculosis (TB) test; (13) History of a parasitic
infection or recent (within the previous 6 months) travel to a
parasitic endemic area; (14) History of alcohol or substance abuse
within previous 5 years; (15) Positive urine drug screen result at
screening (visit 1) or baseline (visit 2); and/or (16)
Live/attenuated vaccinations within 12 weeks of screening or during
the study.
B. Investigational Treatment
[0231] mAb1 drug product was supplied as a lyophilized powder in a
20 ml glass vial for either IV or SC administration. When delivered
IV, mAb1 drug product was reconstituted in a single use vial with
7.8 ml of sterile water for injection yielding a solution
containing 50 mg/mL of mAb1. The pharmacist or designee withdrew
the required amount of reconstituted mAb1 (dependent upon the
subject's dose and weight) or placebo, and injected it into an
infusion bag with 0.9% saline for IV delivery. The infusion was
given over a 2-hour period.
[0232] When delivered SC, the mAb1 drug product was reconstituted
with 2.3 ml of sterile water for injection, yielding a solution
containing 150 mg/mL of mAb1. The pharmacist or designee
administered the injections in the abdomen; administration to the
extremities was not allowed due to the possibility of different
absorption and bioavailability. If administration of multiple
injections were required on the same day, each injection was
delivered at a different injection site.
[0233] The dose levels of mAb1 tested were: 1.0, 3.0, 8.0, and 12.0
mg/kg for IV administration, and 150 and 300 mg for SC
administration.
[0234] Placebo matching mAb1 was prepared in the same formulation
as mAb1, but without addition of antibody.
C. Results and Conclusions
[0235] mAb1 was generally well-tolerated with a favorable safety
profile. The overall adverse event (AE) profile was characteristic
of a healthy population. Less than 55% of subjects treated with
mAb1 (19/36) experienced 1 or more treatment-emergent adverse event
(TEAE) as compared to less than 59% for the subjects treated with
placebo (7/12). The most frequently reported TEAEs were: Blood
creatine phosphokinase (CPK) Increased, Blood Pressure Increased,
Nasopharyngitis, and Toothache. Most subjects experienced an
intensity of TEAEs as mild or moderate; only 3 subjects reported
TEAEs that were considered severe. Only 1 severe TEAE (Blood CPK
Increased) was considered by the investigator to be related to
treatment. One serious adverse event (SAE) was reported during the
study, which was considered by the investigator to be unrelated to
the study drug. No subjects were withdrawn from the study due to an
AE and no deaths were reported. No other clinically significant
laboratory test results (blood chemistry, hematology, or
urinalysis) were reported during the study. No trends were seen in
mean/median baseline in any laboratory parameter. There were no
significant trends in mean or median changes from baseline in
temperature or pulse throughout the study. No clinically
significant abnormalities were seen on physical examination
results, ECGs or vital signs.
[0236] This study was significant in that the subject population
consisted of a high proportion of Black/African-American subjects
(Table 2).
TABLE-US-00002 TABLE 2 Demographic Characteristics of the Treatment
Groups Intravenous dose Subcutaneous dose Placebo 1 mg/kg 3 mg/kg 8
mg/kg 12 mg/kg 150 mg 300 mg Variable (n = 12) (n = 6) (n = 6) (n =
6) (n = 6) (n = 6) (n = 6) Age, years, mean (SD) 35.8 (10.1) 34.3
(9.4) 37.7 (9.8) 35.7 (12.3) 37.2 (9.7) 35.0 (10.4) 33.3 (8.3) Sex,
n (%) Male 5 (41.7) 5 (83.3) 2 (33.3) 4 (66.7) 5 (83.3) 4 (66.7) 4
(66.7) Female 7 (58.3) 1 (16.7) 4 (66.7) 2 (33.3) 1 (16.7) 2 (33.3)
2 (33.3) Race, n (%) White 4 (33.3) 2 (33.3) 1 (16.7) 2 (33.3) 1
(16.7) 0 1 (16.7) Black/African American 8 (66.7) 4 (66.7) 5 (83.3)
4 (66.7) 5 (83.3) 6 (100) 5 (83.3)
[0237] Although the subjects were healthy volunteers,
African-Americans as a group may be more susceptible to atopic
diseases (Caggana et al 1999; Genet. Med. 1: 267-271), and this
population may therefore be considered appropriate for evaluation
of proof of mechanism based on the exploratory biomarker
analysis.
[0238] With regard to pharmacokinetic (PK) analysis, nonlinear
kinetics were observed. The target-mediated pathway of elimination
appeared saturated at IV doses of 8 and 12 mg/kg, when
concentrations of functional mAb1 were above about 30 mg/L. Low
anti-drug antibody (ADA) titers were observed in 9 subjects. No
sudden and persistent drop in concentrations of functional mAB1 was
observed indicating that the ADAs did not have a major impact on
PK.
Example 3
Clinical Trial of Two Different Drug Products of Anti-IL-4R
Antibody (mAb1) Following Subcutaneous Administration of Anti-IL-4R
Antibody (mAb1) in Healthy Patients
A. Study Design
[0239] This study was a single-center, single-dose, double-blind,
randomized, no placebo-controlled study to assess the safety and
pharmacokinetic profile of subcutaneous administration of two
different anti-IL-4R mAb (mAb1) drug products generated from
different cell lines and manufacturing processes. The drug products
were provided in 150 mg/mL 2 mL doses, and 300 mg (2 mL) were
administered subcutaneously to 30 healthy adults in two parallel
groups (15 subjects per group). Subjects included 30 subjects
represented by 22 males (73.3%) and 8 females (26.7%) aged 19 to 45
years old, with weights ranging from 54.8 to 94.3 kg.
[0240] Serum concentration of mAb1 was used to determine the
following PK parameters: maximum serum concentration (C.sub.max),
area under the [serum concentration versus time] curve from time 0
to the real time corresponding to the last concentration above the
lower limit of quantification (t.sub.last (AUC.sub.last), and area
under the serum concentration versus time curve from time zero
extrapolated to infinity (AUC). Also measured with the time to
reach maximum concentration (t.sub.max) and terminal half-life
(t.sub.1/2z).
B. Criteria for Evaluation and Methods
[0241] Safety was assessed by measuring adverse events, including
treatment-emergent adverse events (TEAEs) up to two months
postdose, clinical laboratory evaluations (biochemistry,
hematology, urinalysis), vital signs, electrocardiograms (ECGs)
with automatic reading, anti-mAb1 antibodies (negative or titer),
and local tolerability assessments (including injection site pain
using a Visual Analog Scale [VAS; 100 mm ungraduated line],
Erythema [diameter in mm at injection site], and edema [diameter in
mm at injection site]).
[0242] Adverse events of special interest (AESI) were AEs (serious
or nonserious) of scientific and medical concern that needed
specific monitoring, documentation, and management as described in
the protocol. The following AEs were defined as AESI:
hypersensitivity/anaphylaxis: anaphylactic reaction or acute
allergic reaction requiring immediate treatment, severe injection
site reaction lasting longer than 24 hours, severe infection, any
parasitic infection, alanine aminotransferase (ALT) increase
.gtoreq.2 ULN, QTc.gtoreq.500 ms, pregnancy, or overdose.
[0243] Blood samples for hematology and biochemistry evaluations
were collected predose on Day -1 and on Days 2 (i.e., 24 hours
postdose) and 57, and biochemistry limited to liver function on
Days 8, 15, 22, 29, 36, 43, and 50.
[0244] Blood samples for the determination of anti-mAb1 antibodies
in serum were collected on Day 1 and on Days 15, 29 and 57.
[0245] Local tolerability assessments were performed at predose on
Day 1 and at 2 minutes, 2 hours, 6 hours and 12 hours postdose and
on Days 2 (i.e., 24 hours postdose), 3, 4 and 8 following mAb1
administration.
[0246] For pharmacokinetic and pharmacogenetic sampling, blood
samples were collected at predose on Day 1 and 12 hours post-dose,
and on Days 2, 3, 4, 8, 11, 15, 22, 29, 36, 43, 50, and 57
following mAb1 administration. Serum concentrations of functional
mAb1 were determined using a validated enzyme-linked immunosorbent
assay (ELISA) with a lower limit of quantification (LLOQ) of 78
ng/mL.
[0247] Samples were collected at baseline (Day 1 predose) for
optional pharmacogenetic analyses.
[0248] Pharmacokinetic parameters of serum functional mAb1 were
summarized by treatment group using descriptive statistics (mean,
geometric mean, median, standard deviation (SD), coefficient of
variation [CV], minimum, and maximum). For log transformed
C.sub.max, AUC.sub.last, and AUC, the test/reference treatment
ratios were assessed using a linear fixed effects model with gender
and treatment as fixed effects, and with weight as covariate.
Estimates and 90% confidence intervals (Cis) for treatment ratios
were provided for C.sub.max, AUC.sub.last, and AUC.
[0249] Evaluation of safety was based on the review of individual
values and descriptive statistics. All AEs were coded using Medical
Dictionary for Regulatory Activities (MedDRA) version 15.0, and
frequencies of treatment emergent adverse events (TEAEs) were
classified and tabulated (counts and percents) by primary system
organ class, preferred term, and treatment group. Potentially
clinically significant abnormalities (PCSAs; definitions according
to version 2.0 dated 14 Sep. 2009) for clinical laboratory data,
vital signs, and ECG values and out of normal range values for
clinical laboratory data were flagged and summarized by treatment
group.
[0250] Anti-mAb1 antibody results were listed as either negative or
with a titer value if positive in the confirmation assay by
treatment group, subject and visit. Data were summarized as number
of subjects (counts and percent) with negative or positive
anti-drug antibody (ADA) response by treatment group.
[0251] Descriptive statistics (mean, SD, minimum, median, and
maximum) of the pain VAS, erythema diameter, and edema diameter
were provided by treatment group for each scheduled time point.
Each of these measurements was further summarized by treatment
group as time-averaged (from study drug administration to Day 8
assessment included) and peak values (using post-dose
assessments).
C. Pharmacokinetic Results
TABLE-US-00003 [0252] TABLE 3 Mean .+-. SD (geometric mean) [CV %]
of serum functional mAb1 PK parameters Serum mAb1 PK Parameters
Drug product A Drug Product B N 15 13 C.sub.max 28900 .+-. 9110
27200 .+-. 9950 (ng/mL) .sup. (27300) [31.6] .sup. (25300) [36.6]
t.sub.max.sup.a 168.00 168.00 (h) (72.00-240.00) (48.00-240.00)
AUC.sub.last 11700000 .+-. 4790000 12000000 .+-. 4300000 (ng h/mL)
(10500000) [41.0].sup. (11200000) [35.7].sup.c AUC 13300000 .+-.
3910000 12500000 .+-. 4770000 (ng h/mL) (12800000) [29.4].sup.b
(11600000) [38.2].sup.c t.sub.1/2.sup.z 137 .+-. 46.3 131 .+-. 47.4
(h) (129) [33.8].sup.b (124) [36.2].sup.c .sup.aMedian(Min-Max),
.sup.bN = 13 since terminal log-linear phase could not be
determined in 2 subjects, .sup.cN = 11 since 2 subjects
discontinued from the study.
TABLE-US-00004 TABLE 4 Point estimates of treatment ratios with 90%
confidence interval Comparison Parameter Estimate 90% CI Drug
Product A vs. C.sub.max 1.10 (0.89 to 1.35) Drug Product B
AUC.sub.last 0.90 (0.71 to 1.16) AUC 1.05 (0.86 to 1.29) Drug
Product A: Group A (test), 150 mg/mL (Drug Product 1) .times. 2.0
mL (300 mg of mAb1); Drug Product B: Group B (reference), 150 mg/mL
(Drug Product 2) .times. 2.0 mL (300 mg of mAb1).
[0253] The estimates are based on the linear fixed effect model
with fixed terms for gender, weight, and treatment.
D. Safety Results
[0254] Twelve out of 15 subjects (80.0%) on Drug Product A (test
drug product) versus 8 out of 15 subjects (53.3%) on Drug Product B
(reference drug product) had TEAEs. The apparent treatment
imbalance appeared in some primary system organ classes only and
appeared to be due to events not related to the IMP as often
another cause was identified. Four SAEs were reported in two
subjects during the study.
[0255] A 23 years old male subject in the Drug Product 1 treatment
group experienced a "herpes simplex type II viral infection" with
symptoms of blurred vision, diaphoresis, fever, and headache
starting 4 days after dosing, followed by a swollen tongue (6 days
after dosing) and cough, chest congestion, and muscle cramps in
both calves (7 days after dosing). During the course of this event,
the subject went to the emergency room on several occasions and
received multiple treatments including Solumedrol.RTM. (3 IV
doses), prednisone (for 9 days), Rocephin.RTM. (1 IV dose),
Zithromax.RTM. (for 5 days). It is noteworthy that the subject had
a tongue barbell piercing placed 3 months before dosing. All
symptoms had resolved 19 days after dosing. Initial Herpes Simplex
Virus (HSV) II Ig G titers performed 10 days after dosing were
negative which converted to positive when re-assessed 7 weeks after
dosing. This SAE was judged as related to the IMP by the
Investigator and the company. More than 4 weeks after dosing, the
subject was diagnosed with "Bell's palsy" on the left side that was
considered by the Investigator to be consequent to the HSV II
infection. This event was treated with prednisone (for 6 days) and
acyclovir (for 10 days). This SAE was deemed to be not related to
the IMP by the Investigator due to the multiple and repeated
steroids administrations in the acute state of the HSV II
infection, which were considered an alternative explanation. The
company considered that a causal relationship to the IMP could not
be excluded. Both events were recovering at the end of the study.
This subject did not develop ADA at any time during the study
[0256] A 22 years old male subject in the Drug Product A treatment
group experienced "elevated ALT" (ALT up to 11.4 ULN) together with
"rhabdomyolysis" (creatine phosphokinase up to 392 ULN) both
discovered by routine laboratory exams 7 weeks after dosing. These
events followed a physical challenge (consisting of swimming,
push-ups, pull-ups, sit-ups and other endurance type exercises;
subject is a life guard) and an injury to his triceps muscle
(NSAE). The subject was admitted to the hospital for hydration.
Liver causes of the increased liver function tests were ruled out,
and the elevated ALT (as well as the aspartate aminotransferase
[AST] elevation, up to 50.5 ULN) was judged as related to the
rhabdomyolysis. Creatinine and glomerular function remained within
normal ranges during the course of the events. Both events resolved
within 3 weeks. These 2 SAEs were deemed to be not related to the
IMP by the Investigator. This subject also developed ADA with
positive titers detected on Day 29 (titer value=120) and at EOSV on
Day 58 (titer value=30).
[0257] Apart from the ALT increase, no other AESI were observed
during the study.
[0258] Four (4) subjects experienced infection during the study.
Apart from the herpes simplex infection described above, cases of
mild upper respiratory tract infection, pharyngitis, and sinusitis
(1 case each) were observed 54, 7 and 1 day after dosing,
respectively. These 3 latter events were observed in subjects
treated with C1P2.
[0259] Fifteen (15) cases of injection site reaction occurred in 12
subjects: erythema (8 cases in 8 subjects, 4 in each group), pain
(3 cases, 2 on C2P1 and 1 on C1P2), nodule (2 cases, 1 in each
group), haematoma (1 case on C1P2), and pruritus (1 case on C2P1).
All were mild and resolved within 24 hours of injection.
[0260] Other TEAEs were not observed in more than 1 subject in each
treatment group, except for 2 cases of pruritus (not at injection
site) in 2 subjects treated with C2P1 and 3 cases of headache (in 1
subject treated with C2P1 and 2 subjects treated with C1P2).
[0261] Apart from the laboratory abnormalities already described,
there were no other laboratory increases above the predefined
thresholds for PCSAs.
[0262] Anti-mAb1 antibodies were positive in 6 out of 27 (22.22%)
subjects who completed the study (no subject who did not complete
the study had any detected ADA). Among the 6 subjects with positive
ADA titers, 4 were treated with C2P1 and 2 were treated with C1P2.
No association was observed between ADA development and TEAEs.
E. Specific Local Tolerability Assessments
[0263] On the pain VAS, the mean peak values were 4.4 and 4.2 mm
(on the 100 mm scale) in the C2P1 and C1P2 treatment groups,
respectively, with a median value at 2.0 mm in both groups. Five
(5) out of 15 subjects in each group had "no pain" (peak value at 0
mm). The highest measurements were 17 and 18 mm in the C2P1 and
C1P2 treatment groups, respectively, and were generally observed 2
minutes after dosing (range between 2 minutes and 12 hours
postdose).
[0264] The mean peak values for erythema diameters measured were
12.5 and 10.9 mm in the C2P1 and C1P2 treatment groups,
respectively. Nine (9) out of 15 subjects in each group had no
erythema at any time. The maximum values observed were 40 mm in
both groups, and were all observed 2 minutes after dosing, except
for one subject whose maximum (3 mm) was observed 48 hours
post-dose.
[0265] The mean peak values for edema diameters measured were 1.1
and 0 mm in the C2P1 and C1P2 treatment groups, respectively.
Thirteen (13) out of 15 subjects and 15 out of 15 subjects in the
C2P1 and C1P2 treatment groups, respectively, had no edema at any
time. The maximum values were 15 and 1 mm in 2 subjects in the C2P1
treatment group, and were observed 2 hours post-dose.
F. Conclusions
[0266] After a single subcutaneous dose of 300 mg of mAb1 to
healthy subjects, serum functional mAb1 exposure was similar in the
two test drug products. The geometric mean treatment ratios
(DP1/DP2) with 90% CIs were 1.10 (0.89 to 1.35) for C.sub.max, 0.90
(0.71 to 1.16) for AUC.sub.last, and 1.05 (0.86 to 1.29) for
AUC.
[0267] mAb1 was generally well-tolerated. One subject administered
with DP1 experienced a serious adverse event of "herpes simplex
type II viral infection" followed by "Bell's Palsy".
[0268] There were no clinically important local tolerability issues
and no apparent differences in local tolerability parameters (ie,
pain, erythema, and edema) between treatment groups.
[0269] The most common TEAE was erythema at injection site (8 out
of 30 subjects) and was observed with the same incidence in both
treatment groups (4 out of 15 subjects [26.7%] in each group).
[0270] In conclusion, after a single 300 mg SC administration of
mAb1 in healthy subjects, there was no clinically important
difference identified in the PK profiles, safety, and local
tolerability of the two different drug products.
Example 4
Clinical Trial of Safety, Tolerability and Pharmacokinetics of
Ascending Single Subcutaneous Dose of Anti-IL-4R Antibody in
Healthy Japanese Adult Male Subjects
A. Study Design
[0271] This study was a randomized, double-blind,
placebo-controlled study of ascending, single subcutaneous doses of
an anti-IL-4R antibody (mAb1) in healthy Japanese adult male
subjects. The primary objective was to assess the safety and
tolerability of mAb1 after ascending single subcutaneous doses in
healthy Japanese male subjects. The secondary objectives were to
assess the pharmacokinetics, the immunogenicity and exploratory
pharmacodynamics of ascending single subcutaneous doses of mAb1 in
healthy Japanese male subjects.
[0272] mAb1 was derived from cell line 2 and supplied in liquid
formulation of either 75 mg/mL or 150 mg/mL concentration in vials.
Single ascending doses of 75, 150, 300, and 600 mg of mAb1 were
administered subcutaneously on day 1 (1 injection for 75 mg and for
150 mg; 2 injections for 300 mg; and 4 injections for 600 mg).
Duration of observation was for approximately 11 weeks (including a
screening period of 2 to 21 days prior to dosing, 5 days in the
clinic [day -1 to day 4 with 1 treatment day], and outpatient
follow-up visits up to 57 days after dosing) for each subject.
B. Criteria for evaluation
[0273] Safety: Adverse events (AEs), physical examination, clinical
laboratory evaluations (hematology, biochemistry, urinalysis),
vital signs (supine and standing blood pressure and heart rate,
body temperature), 12-lead electrocardiograms (ECGs), and anti-mAb1
antibodies
[0274] Pharmacokinetics: The following mAb1 serum functional
pharmacokinetics parameters were calculated with non-compartmental
analysis--maximum observed serum concentration (C.sub.max), time to
reach maximum serum concentration (t.sub.max), dose normalized
C.sub.max (C.sub.max/Dose), area under the serum concentration
versus time curve from time zero to the real time corresponding to
the last concentration above the lower limit of quantification t
(AUC.sub.last), dose normalized AUC.sub.last (AUC.sub.last/Dose),
area under the serum concentration versus time curve from time zero
extrapolated to infinity (AUC), apparent volume of distribution at
steady state (V.sub.ss/F), apparent total body clearance (CL/F),
mean residence time (MRT), and terminal half-life (t.sub.1/2z)
[0275] Pharmacodynamics (PD): pharmacodynamics effects of mAb1 on
total IgE and TARC
[0276] Blood samples for PK evaluation were collected at predose
(Day 1) and days 1, 2, 4, 8, 11, 15, 18, 22, 25, 29, 36, 43, 50,
and 57 (.+-.1 day for days 15 to 25; .+-.2 days for days 29 to 57)
following mAb1 administration. Serum concentrations of mAb1 were
determined using a validated ELISA with a lower limit of
quantification (LLOQ) of 78 ng/mL (0.078 mg/mL). Blood samples for
PD evaluation were collected prior to dosing at Day -1 and on day
1, then on days 8, 15, 22, 29, 43, and 57 (.+-.1 day for days 15 to
25; .+-.2 days for days 29 to 57) following mAb1 administration.
Serum screens for total IgE and TARC were determined using a
validated method.
C. Statistical Methods
[0277] Evaluation of safety was based on the review of individual
values and descriptive statistics. All adverse events were coded
using MedDRA version 15.1, and frequencies of treatment-emergent
adverse events (TEAEs) were classified and tabulated (counts and
percentages) by primary system organ class, preferred term, and
treatment group. Potentially clinically significant abnormalities
for clinical laboratory data, vital signs, and ECG data and out of
normal range values for clinical laboratory data were flagged and
summarized by treatment group. In addition, raw data and changes
from baseline for vital signs, ECGs, and limited laboratory
parameters were summarized in descriptive statistics.
[0278] Pharmacokinetic parameters of serum functional mAb1 were
summarized for each dose group using descriptive statistics (mean,
geometric mean, standard error of the can [SEM], median, standard
deviation [SD], and coefficient of variation [CV], minimum and
maximum). Dose proportionality was assessed using a power model for
C.sub.max, AUC.sub.last, and AUC. The dose effect on t.sub.1/2z was
assessed with a linear fixed effect model. The distribution of
t.sub.max values was represented by histogram plots. mAb1 PD
biomarkers (total IgE and TARC: CCL17) were summarized for each
dose group using descriptive statistics.
D. Safety Results
[0279] mAb1 administration of a single subcutaneous dose of up to
600 mg was well tolerated in healthy Japanese adult male subjects
with a median weight of 65.1 kg. No serious TEAEs or premature
discontinuations were reported during the study. During the 57-day
period of observation after dosing, a total of 3 TEAEs were
reported among the 32 study subjects as follows: 1 out of 8
subjects in the placebo group (influenza), 1 out of 6 subjects in
the 150 mg group (influenza) and 1 out of 6 subjects in the 600 mg
group (orthostatic hypotension).
[0280] There were no local cutaneous reactions or discomfort at the
site on injection at volumes up to 2.0 mL.times.4 sites (600
mg).
[0281] Anti-mAb1 antibodies (ADAs) were positive in 5 out of 32
subjects with low titer levels (1 in 75 mg group, 2 in 150 mg
group, 1 in 300 mg group, and 1 in 600 mg group). ADAs were
undetectable at baseline and in the placebo group in all subjects.
No ADA positive subject experienced any TEAE.
[0282] Very few PCSAs in hematology and biochemistry values were
identified in the mAb1 treatment group without any dose-incidence
relationship. In particular, there were no changes in liver enzymes
observed. There were few PCSAs for vital signs or ECG, with no dose
relationship. No subjects experienced a prolonged QTcB (>450 ms)
and no changes from baseline over 60 ms were observed during the
study.
E. Pharmacokinetic Results
[0283] Mean (SD) serum functional mAb1 concentration-time profiles
following single subcutaneous doses are shown in FIG. 1.
Pharmacokinetic parameters for serum functional mAb1 are summarized
for all subjects treated with mAb1 in Table 5.
TABLE-US-00005 TABLE 5 Mean .+-. SD (geometric mean) [CV %] of
serum mAb1 PK parameters PK parameter mAb1 75 mg mAb1 150 mg mAb1
300 mg mAb1 600 mg N 6 6 6 6 Cmax (mg/L) 5.33 .+-. 1.50 .sup. 10.4
.+-. 2.95 38.3 .+-. 15.3 70.1 .+-. 24.1 (5.09) [28.2] (10.1) [28.2]
(36.1) [40.1] (66.8) [34.4] t.sub.max.sup.a (day) 7.01 7.01 7.01
7.01 (3.00-7.03) (3.00-7.03) (6.99-10.00) (3.00-7.02)
t.sub.last.sup.a (day) 17.02 24.03 42.00 52.51 (14.01-21.05)
(21.01-24.04) (35.00-42.02) (42.00-56.02) AUC.sub.last 59.2 .+-.
20.8 .sup. 150 .+-. 41.3 700 .+-. 234 1780 .+-. 699 (mg day/L)
(55.2) [35.2] (146) [27.5] (667) [33.5] (1680) [39.3] AUC 72.4 .+-.
10.6.sup.b 155 .+-. 41.6 709 .+-. 231 1870 .+-. 852 (mg day/L)
(71.9) [14.6] (151) [26.8] (677) [32.6] (1740) [45.5] V.sub.ss/F
(L) 7.96 .+-. 0.673.sup.b 10.7 .+-. 3.04 6.67 .+-. 2.34 6.60 .+-.
1.78 (7.94) [8.5] (10.3) [28.4] (6.32) [35.1] (6.41) [26.9] CL/F
(L/day) 1.05 .+-. 0.144.sup.b 1.02 .+-. 0.238 0.464 .+-. 0.155
0.366 .+-. 0.126 (1.04) [13.7] (0.993) [23.4] (0.443) [33.5]
(0.344) [34.4] t.sub.1/2z (day) 2.77 .+-. 0.567.sup.b 3.18 .+-.
0.805 5.13 .+-. 1.42 8.77 .+-. 5.18 (2.72) [20.5] (3.11) [25.3]
(4.96) [27.7] (7.66) [59.1] .sup.aMedian (Min-Max); .sup.bN = 4
since terminal log-linear phase could not be determined in 2
subjects
[0284] Median t.sub.max of mAb1 was 7 days at all doses. Mean
terminal elimination half-life (t.sub.1/2z) was dose-dependent
(p<0.01) and ranged from 2.77 days at 75 mg to 8.77 days at 600
mg. An 8-fold increase in dose from 75 mg to 600 mg resulted in
13.1-, 30.4-, and 24.2-fold increase in geometric mean C.sub.max,
AUC.sub.last, and AUC respectively.
F. Pharmacodynamics Results
[0285] Serum IgE and TARC values were highly variable within the
treatment groups. Regarding the serum IgE (percent change from
baseline), no drug-related effect was observed over time at single
administrations of subcutaneous doses at 75 mg and 150 mg. At 300
mg and 600 mg, there was a trend of decreasing serum IgE
post-treatment. A treatment effect was observed on TARC. Single
administrations of subcutaneous doses between 75 mg and 600 mg were
associated with reduced serum TARC levels as compared to placebo. A
more sustained reduction was associated with increasing dose.
G. Conclusions
[0286] mAb1 with single subcutaneous dose up to 600 mg was well
tolerated in healthy Japanese male subjects. No serious TEAEs or
premature discontinuations were reported during the study. A total
of 3 TEAEs were reported among the 32 study subjects. There were no
local cutaneous reactions or discomfort at the site on injection at
volumes up to 2.0 mL.times.4 sites (600 mg). Overall, the reported
TEAEs and laboratory, vital signs and ECG assessments did not
suggest dose-related effects.
[0287] After single doses to healthy Japanese adult males, mAb1 was
absorbed with a median tmax of 7 days and eliminated with a
dose-dependent mean terminal elimination half-life (t.sub.1/2z)
ranging from 2.77 days at 75 mg to 8.77 days at 600 mg. Mean serum
functional mAb1 exposure increased in a greater than dose
proportional manner, with an 8-fold increase in dose from 75 mg to
600 mg resulting in a 13.1-, 30.4-, and 24.2-fold increase in
geometric mean C.sub.max, AUC.sub.last, and AUC, respectively.
[0288] A pharmacodynamic effect was observed. Serum levels of TARC
were reduced post-treatment with mAb1. A more sustained decline was
associated with increasing dose. Low titers of ADA were detected in
5 out of 32 subjects. ADAs were undetectable at baseline and in the
placebo group in all subjects. No ADA positive subject experienced
any TEAE.
Example 5
Clinical Trial to Assess the Effect of Injection Rate on Safety and
Tolerability of mAb1 Administered Subcutaneously to Healthy
Volunteers
A. Overview and Study Design
[0289] This study was conducted to support the development of a
large volume injection device for administering mAb1. The study
assessed comparatively two different injection rates approximating
the corresponding attributes of two different subcutaneous (SC)
delivery devices: A fast injection representing an autoinjector and
a slow injection representing a microinfuser. The primary objective
of the study was to assess the comparative safety and tolerability
of a single 300 mg/2 mL dose of mAb1 administered SC at 2 different
rates to normal healthy volunteers. The secondary objectives of the
study were: to compare the pharmacokinetic (PK) profiles of a
single 300 mg/2 mL dose of mAb1 administered SC at 2 different
rates in two separate cohorts of NHV; and to assess the comparative
immunogenicity of a single 300 mg/2 mL dose of mAb1 administered SC
at 2 different rates in NHV.
[0290] This was an open label, randomized, parallel-group,
single-dose study of the safety, tolerability, PK and
immunogenicity of mAb1 administered SC at 2 different injection
rates. The study was randomized to avoid any potential bias in
assigning subjects to study treatment and to minimize systematic
differences between treatment groups with respect to baseline
variables that could affect the outcome. Injection method and
duration could not be effectively blinded, so the study was
open-label. Thirty-six subjects (18 subjects per treatment group)
were recruited and randomized at 1 site in the US. The sample size
for this study was selected empirically. No formal sample size or
power calculations based on the primary endpoint were used.
However, it was estimated that the enrollment of 18 subjects per
group would provide 80% power to detect the difference of 20 in
pain VAS between 2 groups, assuming the common standard deviation
is 20.8 in pain VAS with a 2-sided test at the 0.05 significance
level.
[0291] Subjects underwent screening between day -14 and day -2. On
day -1, subjects were admitted to the clinic for training and
familiarization with the injection procedure, and were randomized
to either group 1 (fast injection) or group 2 (slow injection):
[0292] Group 1 (Fast injection): subjects received study drug via
manual SC injection administered over 30 seconds. [0293] Group 2
(Slow injection): subjects received study drug via a SC infusion
set connected to a syringe pump programmed to deliver 2 mL in 10
minutes.
[0294] On day -1, all subjects underwent a mock injection, in which
the SC infusion set was briefly attached to the skin. The process
included the insertion of a 27G 6-mm needle, which was left in
place for about 10-15 seconds and then removed. Subjects rated
their pain/discomfort related to each respective step of the mock
injection procedure as follows: [0295] Immediately (within 10
seconds) of needle insertion and needle removal, subjects rated
their pain/discomfort related to each respective step of the
procedure. [0296] Global assessment (GA): 1 minute after needle
removal subjects were asked to provide a GA by recalling and rating
the pain/discomfort experienced during the entire procedure. [0297]
Comparative assessment (CA): approximately 1 minute after needle
removal (immediately post GA), in addition to the visual analog
scale (VAS), subjects provided a CA by relating their global
pain/discomfort to familiar experiences, like a bee sting or a flu
shot.
[0298] On day 1, all subjects received 300 mg mAb1 in 2 mL volume
and completed VAS assessments according to the diagram in FIG.
2.
[0299] Information on the incidence, extent, and severity of
injection site reactions (ISRs), such as erythema, edema,
induration, tenderness, and itching, were monitored for all
subjects (in groups 1 and 2). The extent (largest diameter in mm)
of erythema, edema, and induration, as well as the severity of
erythema and edema, were assessed 1, 2, 4, and 8 hours post
completion of the injection and at follow-up visits through the end
of the study or until the injection site appeared normalized at 2
consecutive assessments, based on all parameters evaluated.
Subjects were also asked to rate any pruritus (itching) and
tenderness (pain on palpation) present using VAS.
[0300] Subjects were discharged from the clinic on day 2. Subjects
returned to the clinic for outpatient follow-up visits on days 4,
8, 11, 15, 22, 29, 36, 43, 50, 57, and 64 (end of study). Day 8,
11, and 15 clinic visits could have occurred within a window of
+/-1 day. Visits from day 22 through day 64 could have occurred
within a window of +/-2 days. The total observation period for each
subject was 9 weeks following day 1 dosing.
B. Analysis Variables and Statistical Methods
[0301] The following demographic and Baseline characteristics
variables were summarized: Age at screening (year), Gender,
Ethnicity, Race, Baseline Weight (kg), Height (m), and BMI (kg/m2),
Pain/discomfort VAS. Primary variables include the following
measurements for safety and tolerability: (i) incidence and
severity of treatment-emergent adverse events (TEAEs) through day
64 (end of study); Incidence, extent, severity and duration of ISRs
through day 64; (ii) overall pain/discomfort associated with the
injection procedure (GA); (iii) individual pain/discomfort
components on needle insertion, while injecting study drug and on
needle removal; and (iv) residual pain/discomfort over time:
present pain/discomfort at 5 minutes, 10 minutes, 15 minutes, 30
minutes, 1, 2, 4, and 8 hours after study administration, and at
subsequent study visits.
[0302] Information on the incidence, extent, and severity of ISRs,
such as erythema, edema, induration, tenderness, and itching, were
monitored for all subjects (in groups 1 and 2). The extent (largest
diameter in mm) of erythema, edema and induration were assessed. In
addition, the severity of erythema and edema were qualitatively
assessed using standard 0-4 dermal tolerability scales (Draize) 1,
2, 4, and 8 hours post completion of the injection, and at
follow-up visits through the end of the study or until the
injection site appeared normalized at 2 consecutive assessments,
based on all parameters evaluated.
[0303] The following scales were used to grade the severity of
erythema and edema:
[0304] Erythema: [0305] 0=no erythema [0306] 1=very slight erythema
(barely perceptible) [0307] 2=well defined erythema [0308]
3=moderate to severe erythema [0309] 4=severe erythema (beet
redness) to slight eschar formation (injuries in depth)
[0310] Edema: [0311] 0=no edema [0312] 1=very slight edema (barely
perceptible) [0313] 2=slight edema (edges well defined) [0314]
3=moderate edema (raised >1 mm) [0315] 4=severe edema (raised
>1 mm and beyond area of exposure)
[0316] The VAS is a continuous rating scale (0-100 mm) used by
patients to rate their pain/discomfort related to study drug
injection. The VAS was anchored by "no pain/discomfort" on the left
and "worst possible pain/discomfort" on the right. The same scale
was used to quantify injection site itching and tenderness, which
were assessed as part of ISR.
[0317] Safety and tolerability of mAb1 were assessed by physical
examination, vital signs, electrocardiograms (ECGs), and clinical
laboratory evaluations. Subjects were asked to monitor and report
all adverse events (AEs) experienced from the time the informed
consent is signed until the end of study visit on day 64. Adverse
events, serious adverse events, and treatment-emergent adverse
events have been defined elsewhere herein.
[0318] Blood samples were collected for PK analyses at every study
visit starting at baseline (day 1, pre-dose and post-dose [at the
end of the injection and 1, 2, 4, 8, and 12 hours post dose]).
Blood samples were collected for the analysis of anti-mAb1 antibody
levels on day 1 (pre-dose), and on days 29 and 64 (end of
study).
[0319] For continuous variables, descriptive statistics included
the following: the number of patients reflected in the calculation
(n), mean, median, standard deviation, minimum, and maximum. For
categorical or ordinal data, frequencies and percentages were
displayed for each category.
C. Results
[0320] Pain on Injection and Residual Pain:
[0321] Both injection modalities--fast and slow--were well
tolerated and associated with relatively low levels of pain on
injection. For both modalities, pain peaked approximately 15-30
seconds after the start of injection. Mean peak pain levels were
below 15 mm on the 0-100 mm VAS. Mean pain scores, including the
global assessment (overall pain recalled at 1 minute after the
injection), as well as residual pain over time, were comparable
between the fast and slow injection; differences observed were not
clinically relevant (i.e., .DELTA.<10 on the 0-100 VAS scale).
More subjects on slow injection reported little to no pain
(VAS<5 mm), compared to those who received the fast injection.
Overall, injection pain profile seemed slightly more favorable for
the slow injection, but did not clearly differentiate the two
injection modalities.
[0322] Injection Site Reactions:
[0323] ISR incidence overall was similar between the two study
groups (89% for the fast injection, versus 94% for the slow
injection). However, incidence of objective ISR findings (erythema
and/or induration) was higher in the in the slow injection group
(83%) compared with the fast injection group (44%), especially for
injection site erythema (61% vs. 11%, respectively). Subjective
ISRs included tenderness and pruritus at the injection site and
their incidence was somewhat higher for the fast injection (72%)
compared with the slow injection (56%), especially for injection
site tenderness (72% vs. 39%, respectively). ISR onset was noted
from 1 hour to several days after the injection. Time to ISR
resolution was also reported from 1 hour to several days after
onset. Overall, the ISR profile appeared somewhat more favorable to
the fast injection group, but did not provide a clear
differentiation between the two injection modalities.
[0324] Adverse Events:
[0325] The number and incidence of treatment emergent adverse
events were higher in the slow-injection group (35 TEAEs reported
in 15 subjects) compared with the fast-injection group (19 TEAEs
reported in 11 subjects). Most TEAEs were ISRs that were reported
as adverse events based on investigator's assessment of clinical
relevance. No TEAE led to discontinuation. There were 3 serious
TEAEs related to a single case which were not related to the study
drug or injection modality. Overall, the adverse event profile
appeared slightly more favorable to the fast-injection group,
mostly on the account of ISRs reported as adverse events.
D. Conclusion
[0326] The study achieved the primary and secondary objectives
stated in the protocol. mAb1 was safe and well tolerated when
administered by either fast or slow injection. The results of the
study did not provide a clear differentiation between the two
injection modalities.
Example 6
Sequential Ascending Repeat-Dose Clinical Trial of Subcutaneously
Administered Anti-IL-4R Antibody (mAb1) in Patients with
Moderate-to-Severe Atopic Dermatitis
A. Study Design
[0327] This study was a phase 1b, randomized, double-blind,
placebo-controlled, sequential ascending, repeat-dose study of mAb1
subcutaneously administered in patients with moderate-to-severe
extrinsic atopic dermatitis (AD). Thirty patients were randomized
into the study (6 in placebo, 8 each in of 75 mg, 150 mg and 300 mg
groups). Twenty-eight patients received all the treatments. The
treatment period was 4 weeks in duration; patients were followed
for 8 weeks after end of the treatment period. Patients were
randomized in a 4:1 ratio to receive mAb1 or placebo in each of 3
ascending dose cohorts (75, 150, or 300 mg mAb1). The primary
objective of the trial was to access the safety and tolerability,
with PK as a secondary objective. Exploratory objectives included
efficacy and biomarker endpoints. The exploratory efficacy
variables included: (i) proportion of patients who achieved an IGA
score of 0 or 1 through week 4 and each study visit; (ii) change
and percent change in BSA, EASI, and 5-D pruritus scale from
baseline to each visit; and (iii) weekly change from baseline in
NRS scale.
B. Efficacy Variables
[0328] The efficacy variables IGA, BSA, EASI, SCORAD, 5-D pruritus
scale, and pruritus NRS rating have been described elsewhere herein
(see, for example, Example 7).
[0329] The IGA, BSA, EASI and SCORAD scores were assessed at every
clinic visit. Patients underwent 5-D pruritus assessment at the
following visits: screening, day 1/baseline (pre-dose), and days
15, 29, 43, 57, 71, and 85 (end of study) or early termination.
Patients used the IVRS to record their Pruritus NRS score twice
daily through the last study visit.
[0330] Baseline for efficacy variable is defined as the last
non-missing value on or before the date of randomization. For the
patient who has no value on or before his/her randomization date
the last non-missing value on or before the date of first dose
injection will be used as baseline.
C. Statistical Methods
[0331] Summary of safety and exploratory efficacy variables was
generated by dose group and overall. The summary of safety and
tolerance were performed based on the safety analysis set (SAF).
The safety analyses were based on the reported adverse events
(AEs), clinical laboratory evaluations, vital signs, and 12-lead
ECG.
[0332] All the categorical variables were analyzed using the
Fisher's exact test with nominal p-value and confidence intervals
reported.
[0333] All continuous variables were analyzed by the ANalysis of
COVAriance (ANCOVA). Unless otherwise specified, assessments of
changes from baseline and construction of confidence intervals for
continuous measures were based on an ANCOVA model which included
treatment as the main factor and baseline value as covariates.
Point estimate and 95% CI of the difference in adjusted mean change
from baseline between two treatment groups were provided. Due to
small sample size of this study, p-values from the tests of the
exploratory efficacy variables were provided for descriptive
purpose. Missing values were imputed by the last observation
carried forward (LOCF).
D. Patient Disposition
[0334] The patients in placebo group were the youngest, and 33% of
patients in placebo group were Hispanic or Latino compared to the
treatment groups where all the patients were non-Hispanic. Table 6
summarizes the demographic characteristics of the patient
population.
TABLE-US-00006 TABLE 6 Summary of Demographic Characteristics
Placebo 150 mg All Doses (N = 6) 75 mg (N = 8) (N = 8) 300 mg (N =
8) (N = 24) Mean age, 23.0 35.8 45.6 46.1 42.5 years (SD) (4.00)
(12.51) (10.78) (11.36) (12.09) Ethnicity, n (%) Hispanic or 2
(33.3%) 0 0 0 0 Latino Non-Hispanic 4 (66.7%) 8 (100%) 8 (100%) 8
(100%) 24 (100%) Race, n (%) American- 0 0 0 1 (12.5%) 1 (4.2%)
Indian/Alaska Native Asian 0 0 0 1 (12.5%) 1 (4.2%) African- 2
(33.3%) 4 (50.0%) 2 (25.0%) 2 (25.0%) 8 (33.3%) American White 4
(66.7%) 4 (50.0%) 5 (62.5%) 4 (50.0%) 13 (54.2%) Other 0 0 1
(12.5%) 0 1 (4.2%) Gender, n (%) Male 3 (50.0%) 6 (75.0%) 5 (62.5%)
2 (25.0%) 13 (54.2%) Female 3 (50.0%) 2 (25.0%) 30 (37.5%) 6
(75.0%) 11 (45.8%) Mean BMI, 26.81 26.41 27.32 31.60 24.45
kg/m.sup.3 (SD) (8.191) (4.489) (5.285) (12.890) (8.399)
[0335] Table 7 summarizes the baseline disease characteristics of
the patient population.
TABLE-US-00007 TABLE 7 Baseline Disease Characteristics Placebo All
Doses (N = 6) 75 mg (N = 8) 150 mg (N = 8) 300 mg (N = 8) (N = 24)
Duration of 17.8 (6.82) 24.5 (16.95) 27.5 (16.34) 33.9 (17.92) 28.6
(16.80) chronic AD, years EASI score 18.1 (7.17) 36.9 (11.75) 25.6
(13.84) 29.8 (6.44) 30.8 (11.65) IGA score 3.2 (0.41) 4.1 (0.35)
3.9 (0.64) 3.8 (0.46) 3.9 (0.50) % BSA of AD 31.6 (24.27) 64.4
(17.03) 46.9 (28.20) 47.0 (20.49) 52.84 (22.99) 5-D pruritus 15.5
(3.83) 21.5 (3.55) 19.3 (2.92) 19.6 (2.97) 20.1 (3.18) scale
Pruritus NRS 5.8 (1.60) 7.0 (1.78) 6.7 (1.62) 6.0 (1.18) 6.6 (1.54)
score
[0336] The mean baseline IGA, EASI, BSA, and pruritus NRS for the
study participants was approximately 3.8, 28.2, 48.5, and 6.4
respectively.
E. Results
[0337] Subcutaneous administration of mAb1 to patients with
moderate-to-severe AD was safe and well tolerated in this study. A
single serious adverse event was recorded for a patient in the 150
mg group, who was diagnosed with exercise-associated CPK increase.
No deaths were reported. 25 of the treated patients or 83% reported
at least one treatment emergent adverse event (TEAE). The most
frequent TEAE from the treatment groups were infections and
infestations (n=7 [29%] vs. 1 [17%] for placebo), and headaches in
patients dosed with mAb1 (n=3 [13%] vs. 1 [17%] for placebo).
[0338] The baseline and exploratory efficacy results obtained from
the study are summarized in FIGS. 3-14. mAb1 administration did not
induce statistically significant improvement in any exploratory
endpoints of AD. This may be due to the small sample size and the
fact that the placebo patients were less severe and younger than
active treatment groups.
Example 7
Clinical Trial of Subcutaneously Administered Anti-IL-4R Antibody
(mAb1) in Patients with Moderate-to-Severe Atopic Dermatitis
A. Study Design
[0339] This study was a 12-week, double-blind, randomized,
placebo-controlled, sequential ascending, repeated-dose study to
assess the safety and pharmacokinetic profile of subcutaneous
administration of the anti-IL-4R mAb, referred to herein as "mAb1,"
in adult patients with moderate-to-severe atopic dermatitis.
Patients with moderate-to-severe AD had an Eczema Area and Severity
Index (EASI).gtoreq.12 and minimum 10% body surface area
involvement. The treatment period was four weeks in duration, with
patients being followed for 8 weeks after the end of the treatment
period. Patients were withdrawn from topical agents (e.g.,
pimecrolimus, tacrolimus, and topical corticosteroids) for at least
1 week prior to baseline. Oral corticosteroids and
immunosuppressives (e.g., cyclosporine, mycophenolate-mofetil,
IFN.gamma.) were also prohibited from .gtoreq.4 weeks prior to
baseline.
[0340] Patients were randomized in a 3:1 ratio to receive mAb1 or
placebo in each of two ascending dose cohorts (150 mg or 300 mg).
The study consisted of a screening period (day -14 through day -3),
a treatment period (day 1 through day 29) (topical steroids were
not allowed), and a follow-up period (day 29 through day 85)
(topical steroids were allowed). During the treatment period,
patients were seen in the clinic at least once weekly for safety,
laboratory, and clinical effect assessments on days 1, 4, 8, 15,
22, 25 and 29 (week 4). Patients received a dose of study drug on
days 1, 8, 15 and 22. Patients were monitored at the study site for
2 hours after each dose of study drug. The end of the treatment
period study visit occurred on day 29 (week 4). During the
follow-up period patients were seen in the clinic for follow-up
assessments at days 36, 43, 50, 57, 64, 71 and 85 (end of study
visit).
B. Efficacy Variables
[0341] The exploratory efficacy variables measured in this study
included: (1) proportion of patients who achieved an investigator's
global assessment (IGA) score of 0 or 1 through week 4 and each
study visit; (2) change and percent change in body surface area
involvement of atopic dermatitis (BSA), eczema area and severity
index (EASI), SCORAD, and 5-D pruritus scale from baseline to each
visit; (3) weekly change from baseline in pruritus numeric rating
scale (NRS); (4) change from baseline in circulating eosinophils,
TARC, eotaxin-3, and total IgE through week 4; (5) change from
baseline in circulating eosinophils, TARC, eotaxin-3, and total IgE
through week 12; and (6) change from baseline in eosinophils, TARC,
eotaxin-3, Phadiatop.TM. results, and total IgE associated with
response through week 4.
[0342] Baseline for efficacy variable is defined as the last
non-missing value on or before the date of randomization. For the
patient who has no value on or before his/her randomization date
the last non-missing value on or before the date of first dose
injection will be used as baseline.
[0343] Investigator's Global Assessment (IGA):
[0344] The IGA is an assessment scale used in clinical studies to
determine severity of AD and clinical response to treatment based
on a 6-point scale ranging from 0 (clear) to 5 (very severe). The
IGA score was assessed at every clinic visit.
[0345] Body Surface Area Involvement of Atopic Dermatitis
(BSA):
[0346] BSA affected by AD was assessed for each major section of
the body (head, trunk, upper extremities, and lower extremities)
and was reported as the total of percentage from each body
sections. Patients were assessed for BSA at the following visits:
screening, day 1/baseline (pre-dose), and days 15, 29, 36, 43, 57,
71, and 85 (end of study) or early termination.
[0347] Eczema Area and Severity Index (EASI):
[0348] The EASI is a validated measure used in clinical practice
and clinical trials to assess the severity and extent of AD
(Hanifin et al 2001, Exp. Dermetol. 10: 11-18). The EASI score
calculation is based upon the Physician's Assessment of Individual
Signs [erythema (E), induration/papulation (I), excoriation (X),
and lichenification (L)], where each sign is scored as 0=Absent,
1=Mild, 2=Moderate, or 3=Severe, and also upon the Area Score
[based on the % (BSA) affected] where 0=0% BSA, 1=1-9% BSA,
2=10-29% BSA, 3=30-49% BSA, 4=50-69% BSA, 5=70-89% BSA, 6=90-100%
BSA.
[0349] For each of major section of the body (head, upper
extremities, trunk and lower extremities), EASI
score=(E+I+X+L).times.Area Score. The total EASI score is the
weighted total of the section EASI using the weights 10%=head,
20%=upper extremities, 30%=trunk, 40%=lower extremities. The
minimum possible EASI score is 0 and the maximum possible EASI
score is 72 where a higher score indicates increased severity of
atopic dermatitis. Achieving an EASI 50 (50% or greater improvement
in EASI score is considered by dermatology investigators to a
clinically significant level of improvement to use as an
endpoint.
[0350] Patients underwent EASI score assessment at the following
visits: screening, day 1/baseline (pre-dose), and days 15, 29, 36,
43, 57, 71, and 85 (end of study) or early termination.
[0351] SCORAD:
[0352] The SCORAD is a validated tool used in clinical research and
clinical practice that was developed to standardize the evaluation
of the extent and severity of AD (Dermatology 1993, 186: 23-31).
The extent of AD is assessed as a percentage of each defined body
area and reported as the sum of all areas, with a maximum score of
100% (assigned as "A" in the overall SCORAD calculation). The
severity of 6 specific symptoms (erythema, oedema/papulation,
excoriations, lichenification, oozing/crusts and dryness) of AD is
assessed using the following scale: none (0), mild (1), moderate
(2), or severe (3) (for a maximum of 18 total points, assigned as
"B" in the overall SCORAD calculation). Subjective assessment of
itch and sleeplessness is recorded for each symptom by the patient
or relative on a visual analogue scale (VAS), where 0 is no itch
(or sleeplessness) and 10 is the worst imaginable itch (or
sleeplessness), with a maximum possible score of 20. This parameter
is assigned as "C" in the overall SCORAD calculation. The SCORAD
score is calculated as N5+7B/2+C. The maximum SCORAD score is
103.
[0353] Patients underwent SCORAD assessment at the following
visits: screening, day 1/baseline (pre-dose), and days 15, 29, 36,
43, 57, 71, and 85 (end of study) or early termination.
[0354] 5-D Pruritus Scale:
[0355] The 5-D Pruritus Scale is a 5-question tool used in clinical
trials to assess 5 dimensions of background itch: degree, duration,
direction, disability, and distribution (Elman et. al. 2010, Brit.
J. Dermatol. 162: 587-593). Patients rate their symptoms over the
preceding 2-week period as "present" or on a 1 to 5 scale, with 5
being the most affected for each question in degree, duration,
direction and disability. Single-item domain scores (duration,
degree and direction) are equal to the value indicated below the
response choice (range 1-5).
[0356] The disability domain includes four items that assess the
impact of itching on daily activities: sleep, leisure/social
activities, housework/errands and work/school. The score for the
disability domain is achieved by taking the highest score on any of
the four items.
[0357] For the distribution domain, the number of affected body
parts is tallied (potential sum 0-16) and the sum is sorted into
five scoring bins: sum of 0-2=score of 1, sum of 3-5=score of 2,
sum of 6-10=score of 3, sum of 11-13=score of 4, and sum of
14-16=score of 5.
[0358] The scores of each of the five domains are achieved
separately and then summed together to obtain a total 5-D score.
5-D scores can potentially range between 5 (no pruritus) and 25
(most severe pruritus).
[0359] Patients underwent 5-D pruritus assessment at the following
visits: screening, day 1/baseline (pre-dose), and days 15, 29, 43,
57, 71, and 85 (end of study) or early termination.
[0360] Pruritus Numeric Rating Scale (NRS):
[0361] The Pruritus NRS is a single-question assessment tool that
was used to assess the patient's worst itch as a result of AD in
the previous 12 hours. Patients call in to the IVRS twice daily
from the evening of the screening visit and be asked the following
question, "on a scale of 0-10, with 0 being `no itch` and 10 being
the `worst itch imaginable`, how would you rate your worst degree
of itch experienced during the previous 12 hours?" Patients are
instructed on using the IVRS to record their Pruritus NRS score at
the screening visit and are queried for compliance at each
following clinic visit. Patients complete the rating scale twice
daily through the last study visit.
[0362] The baseline NRS is defined as the average of the reported
NRSs during right after the screening visit and right before the
baseline visit. For post-baseline NRS, The mean weekly NRS is
calculated as the average of the reported daily NRS within the week
(prorated mean).
C. Safety Assessment
[0363] Safety was assessed throughout the study by monitoring
Adverse Events and Serious Adverse Events.
[0364] An Adverse Event (AE) is any untoward medical occurrence in
a subject or clinical investigation subject administered a
pharmaceutical product. An AE can, therefore, be any unfavorable
and unintended sign (including abnormal laboratory finding),
symptom, or disease temporally associated with the use of a
medicinal product, whether or not considered related to the
medicinal (investigational) product. AEs also include: any
worsening (i.e., any clinically significant change in frequency
and/or intensity) of a pre-existing condition that is temporally
associated with the use of the study drug; abnormal laboratory
findings considered by the Investigator to be clinically
significant; and any untoward medical occurrence.
[0365] A Serious Adverse Event (SAE) is any untoward medical
occurrence that at any dose results in death; is life-threatening;
requires in-patient hospitalization or prolongation of existing
hospitalization; results in persistent or significant
disability/incapacity; is a congenital anomaly/birth defect; or is
an important medical event.
[0366] In addition, laboratory safety variables, vital sign
variables, 12-lead electrocardiography (ECG) variables, and
physical examination variables were measured throughout the
study.
[0367] The clinical laboratory data consists of hematology, blood
chemistry and urinalysis. Blood samples for hematology testing were
collected at every study visit; blood samples for serum chemistry
testing and urine samples for urinalysis were collected to measure
overall patient health at screening, day 1/baseline (pre-dose), day
8, day 15, day 29, day 36, day 57, day 85 (end-of-study) or early
termination if subject is discontinued from the study.
[0368] Vital sign parameters include respiratory rate (bpm), pulse
rate (bpm), systolic and diastolic blood pressure (mmHg) and body
temperature (.degree. C.). Vital signs were collected (pre-dose, on
dosing days) at screening and day 1/baseline, and days 4, 8, 15,
22, 25, 29, 36, and 85 (end of study) or early termination. Vital
signs were taken at 1 and 2 hours post-injection following the
study drug dose on days 1, 8, 15, and 22.
[0369] 12-Lead ECG parameters include: Ventricular HR, PR interval,
QRS interval, corrected QT interval (QTcF=QT/[RR.sup.0.33] and
QTcB=QT/[RR.sup.0.5]) ECG status: normal, abnormal not clinical
significant or abnormal clinical significant. A standard 12-lead
ECG was performed at screening, day 29, and day 85 (end of study)
or early termination.
[0370] A thorough and complete physical examination was performed
at screening, day 29, and day 85 (end of study) or early
termination.
D. Data Analysis
[0371] 1. Analyses of Exploratory Efficacy Variables
[0372] All categorical variables were analyzed using the Fisher's
Exact test with nominal p-value and confidence intervals reported.
All continuous variables were analyzed by the ANalysis of
COVAriance (ANCOVA). Unless otherwise specified, assessments of
changes from baseline and construction of confidence intervals for
continuous measures were based on an ANCOVA model which includes
treatment as the main factor and baseline value as covariates.
Point estimate and 95% CI of the difference in adjusted mean change
from baseline between two treatment groups are provided. Missing
values were imputed by the last observation carried forward (LOCF)
approach. In the event that the model assumptions were not
warranted, the Rank-based analysis of covariates was used.
Correlation analyses were performed using Spearman's correlation
coefficient.
[0373] 2. Analysis of Safety Data
[0374] The safety analysis is based on the reported AEs, clinical
laboratory evaluations, vital signs, and 12-lead ECG. Thresholds
for Potentially Clinically Significant Values (PCSV) in laboratory
variables, vital signs and ECG are defined in SAP. The time
interval to detect any event or abnormality is between the infusion
of study medication and end of study. Data collected outside this
interval are excluded from the calculation of descriptive
statistics and identification of abnormalities for laboratory
evaluations, vital sign and ECG.
E. Results
[0375] As noted above, patients were treated either with 150 mg or
300 mg subcutaneous mAb1 once a week for four weeks, or with
placebo. Except for a higher age at diagnosis in the 300 mg
treatment group, demographic and clinical characteristics were
generally similar among treatments (Table 8). The study population
was primarily male (62.2%), white (94.6%), with a mean age of 43.6
(15.4) years. Of the 37 patients, 31 (83.8%) completed treatments
and 25 (67.6%) completed the whole study. The most frequent reason
for withdrawal was lack of efficacy (4 placebo patients and 1 in
each treatment group). There were no withdrawals due to adverse
events with the administered mAb1.
[0376] The baseline and exploratory efficacy results obtained from
the study are summarized in Tables 9-14.
TABLE-US-00008 TABLE 8 Summary of Baseline Characteristics - all
values represented as Mean (SD) mAb1 All Doses All Subjects Placebo
150 mg 300 mg Combined Combined No. Patients 10 14 13 27 37 Age,
years, mean (SD) 46.0 (16.2) 40.6 (11.7) 45.0 (18.6) 42.7 (15.3)
43.6 (15.4) Gender, n (%) Male 8 (80.0) 7 (50.0) 8 (61.5) 15 (55.6)
23 (62.2) Female 2 (20.0) 7 (50.0) 5 (38.5) 12 (44.4) 14 (37.8)
Race, n (%) White 9 (90.0) 14 (100) 12 (92.3) 26 (96.3) 35 (94.6)
Black/African-American 0 0 1 (7.7) 1 (3.7) 1 (2.7) Other 1 (10.0) 0
0 0 1 (2.7) BMI, kg/m.sup.2 mean (SD) 25.0 (4.6) 24.7 (2.8) 25.3
(3.5) 25.0 (3.1) 25.0 (3.5) Chronic Atopic Dermatitis 5.8 (10.50)
5.9 (11.92) 16.2 (23.79) 10.9 (18.98) 9.5 (17.11) Diagnosis Age BSA
45.5 (26.43) 51.4 (29.98) 49.0 (24.08) 50.2 (26.80) 48.9 (26.42)
EASI Score 25.6 (13.73) 32.6 (18.56) 25.9 (13.38) 29.4 (16.32) 28.3
(15.56) IGA Score 3.9 (0.74) 3.9 (0.73) 3.3 (0.48) 3.6 (0.69) 3.7
(0.70) NRS Score 5.8 (1.92) 5.6 (1.87) 5.4 (1.69) 5.5 (1.75) 5.6
(1.78) SCORAD Score 65.3 (11.09) 66.2 (17.07) 58.6 (13.93) 62.6
(15.81) 63.3 (14.59) Pruritus 5-D Score 17.8 (3.94) 18.9 (3.05)
18.1 (3.99) 18.5 (3.49) 18.3 (3.58) CCL17, ng/mL, mean 7001
(2669.8) 9162.2 (4851.7) 4601.4 (1957.3) 6881.8 (2280.4) 6914.9
(2001.3) (SD)
TABLE-US-00009 TABLE 9 Summary of Percentage and Absolute Change in
IGA Score from Baseline - all values represented as Mean (SD) mAb1
All Doses Placebo 150 mg 300 mg Combined No. Patients 10 14 13 27
Baseline IGA Score 3.9 3.9 3.3 3.6 (0.74) (0.73) (0.48) (0.69) Day
4 IGA Score 3.9 3.9 3.3 3.6 (0.74) (0.73) (0.48) (0.69) % Change
from Baseline to Day 4 0.0 0.0 0.0 0.0 (0.00) (0.00) (0.00) (0.00)
Absolute change from Baseline to Day 4 0.0 0.0 0.0 0.0 (0.00)
(0.00) (0.00) (0.00) Day 8 IGA Score 3.9 3.6 3.2 3.4 (0.60) (0.93)
(0.55) (0.80) % Change from Baseline to Day 8 4.6 -8.0 -4.5 -6.3
(18.22) (13.46) (11.08) (12.27) Absolute change from Baseline to
Day 8 0.1 -0.3 -0.2 -0.2 (0.60) (0.47) (0.38) (0.42) Day 15 IGA
Score 4.0 2.9 2.8 2.8 (0.71) (1.19) (0.83) (1.01) % Change from
Baseline to Day 15 9.3 -27.2 -16.7 -21.9 (29.89) (18.24) (21.25)
(20.13) Absolute change from Baseline to Day 15 0.2 -1.0 -0.5 -0.8
(0.97) (0.71) (0.66) (0.71) Day 22 IGA Score 3.5 2.7 2.2 2.4 (0.55)
(0.89) (0.90) (0.91) Change from Baseline to Day 22 -2.8 -32.8
-34.6 -33.7 (21.52) (11.68) (28.23) (21.49) Absolute change from
Baseline to Day 22 -0.2 -1.3 -1.2 -1.2 (0.75) (0.45) (0.90) (0.71)
Day 25 IGA Score 3.5 2.6 2.0 2.3 (0.55) (1.00) (1.00) (1.02) %
Change from Baseline to Day 25 -1.4 -34.4 -39.7 -37.2 (28.59)
(18.73) (29.30) (24.44) Absolute change from Baseline to Day 25
-0.2 -1.3 -1.3 -1.3 (0.98) (0.78) (0.95) (0.85) Day 29 IGA Score
3.3 2.2 2.1 2.1 (0.52) (0.94) (0.95) (0.93) % Change from Baseline
to Day 29 -8.3 -44.4 -37.8 -41.0 (12.91) (19.86) (26.05) (23.05)
Absolute change from Baseline to Day 29 -0.3 -1.8 -1.2 -1.5 (0.52)
(0.87) (0.83) (0.87) Day 36 IGA Score 3.5 2.0 2.1 2.1 (0.58) (0.74)
(0.35) (0.60) % Change from Baseline to Day 36 -6.3 -48.9 -36.5
-43.9 (12.50) (15.51) (8.84) (14.39) Absolute change from Baseline
to Day 36 -0.3 -1.9 -1.3 -1.7 (0.50) (0.67) (0.46) (0.67) Day 43
IGA Score 3.0 2.2 1.7 2.0 (1.10) (1.03) (0.79) (0.93) % Change from
Baseline to Day 43 -20.0 -45.1 -47.7 -46.4 (29.15) (23.40) (22.39)
(22.44) Absolute change from Baseline to Day 43 -0.8 -1.8 -1.5 -1.7
(1.17) (0.87) (0.69) (0.78) Day 50 IGA Score 3.2 2.3 1.6 2.0 (1.48)
(1.14) (0.67) (0.98) % Change from Baseline to Day 50 -10.0 -42.1
-50.0 -45.9 (37.91) (25.51) (21.08) (23.32) Absolute change from
Baseline to Day 50 -0.4 -1.7 -1.6 -1.7 (1.52) (1.07) (0.67) (0.88)
Day 57 IGA Score 3.5 2.2 1.7 2.0 (0.58) (1.01) (0.79) (0.93) %
Change from Baseline to Day 57 2.1 -42.6 -47.0 -44.6 (23.94)
(24.94) (23.65) (23.93) Absolute change from Baseline to Day 57 0.0
-1.7 -1.5 -1.6 (0.82) (1.03) (0.82) (0.92) Day 64 IGA Score 3.2 2.5
1.7 2.1 (0.45) (1.13) (0.65) (0.97) % Change from Baseline to Day
64 -10.0 -36.8 -47.0 -41.9 (13.69) (27.24) (18.36) (23..26)
Absolute change from Baseline to Day 64 -0.4 -1.5 -1.5 -1.5 (0.55)
(1.04) (0.69) (0.86) Day 71 IGA Score 3.0 2.8 1.6 2.2 (.071) (0.87)
(0.90) (1.07) % Change from Baseline to Day 71 -16.7 -25.6 -52.8
-39.8 (15.59) (31.74) (26.67) (31.72) Absolute change from Baseline
to Day 71 -0.6 -1.2 -1.8 -1.5 (0.55) (1.33) (0.87) (1.12) Day 85
IGA Score 3.3 2.8 2.2 2.5 (0.96) (1.08) (0.94) (1.04) % Change from
Baseline to Day 85 -6.3 -26.5 -33.3 -30.1 (29.95) (35.84) (29.09)
(31.92) Absolute change from Baseline to Day 85 -0.3 -1.2 -1.1 -1.1
(0.96) (1.33) (0.90) (1.10)
TABLE-US-00010 TABLE 10 Summary of Percentage and Absolute Change
in EASI Score from Baseline - all values represented as Mean (SD)
mAb1 All Doses Placebo 150 mg 300 mg Combined No. Patients 10 14 13
27 Baseline EASI Score 25.6 32.6 25.9 29.4 (13.73) (18.56) (13.38)
(16.32) Day 15 EASI Score 31.4 20.6 14.1 17.5 (22.59) (16.87)
(9.62) (14.00) % Change from Baseline to 23.9 -35.6 -47.2 -41.2 Day
15 (79.60) (28.97) (20.13) (25.32) Absolute change from Baseline
5.7 -11.9 -11.8 -11.9 to Day 15 (17.21) (14.03) (7.53) (11.16) Day
29 EASI Score 21.4 12.7 11.7 12.2 (15.36) (13.52) (14.25) (13.62) %
Change from Baseline to -13.1 -61.5 -63.1 -62.3 Day 29 (15.14)
(26.54) (29.00) (27.27) Absolute change from Baseline -2.2 -19.6
-14.2 -16.8 to Day 29 (5.14) (15.59) (8.00) (12.29) Day 36 EASI
Score 13.9 8.7 10.6 9.4 (2.11) (10.94) (11.30) (10.83) % Change
from Baseline to -10.7 -74.7 -58.9 -68.3 Day 36 (19.60) (20.64)
(25.75) (23.54) Absolute change from Baseline -1.9 -23.7 -12.9
-19.4 to Day 36 (3.08) (16.13) (8.44) (14.36) Day 43 EASI Score
13.9 9.5 8.6 9.0 (5.85) (12.71) (9.96) (11.23) % Change from
Baseline to -29.1 -71.3 -66.7 -69.1 Day 43 (27.93) (25.68) (25.72)
(25.22) Absolute change from Baseline -6.3 -22.8 -14.9 -19.0 to Day
43 (5.61) (16.59) (8.88) (13.78) Day 57 EASI Score 14.1 12.1 8.2
10.3 (12.14) (14.93) (10.22) (12.87) % Change from Baseline to
-15.2 -63.3 -65.5 -64.3 Day 57 (78.20) (34.42) (27.26) (30.70)
Absolute change from Baseline -4.7 -21.6 -15.3 -18.7 to Day 57
(15.74) (19.92) (10.72) (16.36) Day 71 EASI Score 13.3 13.7 7.2
10.5 (8.00) (12.01) (8.61) (10.75) % Change from Baseline to -42.3
-56.8 -73.7 -65.3 Day 71 (29.40) (36.08) (21.83) (30.41) Absolute
change from Baseline -12.2 -18.6 -18.9 -18.8 to Day 71 (10.91)
(16.96) (11.33) (14.11) Day 85 EASI Score 10.1 15.9 7.9 11.8 (3.73)
(17.57) (8.31) (13.85) % Change from Baseline to -41.6 -51.2 -68.6
-60.3 Day 85 (33.96) (44.04) (25.00) (35.68) Absolute change from
Baseline -10.5 -18.1 -15.5 -16.7 to Day 85 (11.97) (20.78) (8.12)
(15.20)
TABLE-US-00011 TABLE 11 Summary of Percentage and Absolute Change
in BSA Score from Baseline - all values represented as Mean (SD)
mAb1 All Doses Placebo 150 mg 300 mg Combined No. Patients 10 14 13
27 Baseline BSA Score 45.5 51.4 49.0 50.2 (26.43) (29.98) (24.08)
(26.80) Day 15 BSA Score 42.8 42.3 38.7 40.6 (26.62) (27.46)
(24.88) (25.81) % Change from Baseline to -3.0 -16.6 -26.4 -21.3
Day 15 (14.43) (24.86) (23.61) (24.30) Absolute change from
Baseline -2.4 -9.1 -10.3 -9.7 to Day 15 (6.25) (17.98) (9.10)
(14.15) Day 29 BSA Score 34.3 28.8 36.0 32.6 (25.78) (31.01)
(29.38) (29.76) % Change from Baseline to -2.5 -44.7 -35.6 -40.0
Day 29 (22.46) (41.65) (37.02) (38.76) Absolute change from
Baseline -0.5 -22.0 -13.0 -17.3 to Day 29 (10.41) (30.09) (13.81)
(23.05) Day 36 BSA Score 21.0 25.2 29.5 26.9 (5.48) (25.27) (29.51)
(26.37) % Change from Baseline to 10.1 -51.5 -38.6 -46.4 Day 36
(37.06) (35.29) (41.01) (37.18) Absolute change from Baseline 1.3
-25.7 -11.5 -20.0 to Day 36 (7.23) (27.96) (14.60) (24.12) Day 43
BSA Score 29.0 27.8 28.9 28.3 (19.79) (31.69) (25.09) (28.08) %
Change from Baseline to -4.2 -46.4 -44.4 -45.4 Day 43 (31.32)
(47.42) (32.77) (40.17) Absolute change from Baseline -3.2 -23.1
-16.4 -19.9 to Day 43 (8.35) (32.26) (10.04) (24.04) Day 57 BSA
Score 27.3 31.2 28.3 29.8 (28.19) (31.48) (22.40) (27.15) % Change
from Baseline to -31.6 -40.5 -42.7 -41.5 Day 57 (31.63) (52.06)
(34.66) (44.01) Absolute change from Baseline -11.0 -22.6 -17.0
-20.0 to Day 57 (20.58) (34.40) (13.33) (26.51) Day 71 BSA Score
26.6 30.6 23.2 26.9 (20.63) (24.59) (20.72) (22.56) % Change from
Baseline to -27.9 -38.3 -55.7 -47.0 Day 71 (35.30) (44.29) (26.21)
(36.69) Absolute change from Baseline -12.2 -20.3 -26.2 -23.2 to
Day 71 (23.53) (26.80) (17.24) (22.24) Day 85 BSA Score 20.0 32.2
18.1 24.8 (14.00) (32.71) (14.85) (25.46) % Change from Baseline to
-41.5 -38.5 -62.6 -51.1 Day 85 (6.32) (54.33) (19.64) (41.06)
Absolute change from Baseline -16.3 -22.2 -27.2 -24.8 to Day 85
(15.88) (36.78) (12.77) (26.51)
TABLE-US-00012 TABLE 12 Summary of Percentage and Absolute Change
in SCORAD Score from Baseline - all values represented as Mean (SD)
mAb1 All Doses Placebo 150 mg 300 mg Combined No. Patients 10 14 13
27 Baseline SCORAD score 65.3 66.2 58.6 62.6 (11.09) (17.07)
(13.93) (15.81) Day 15 SCORAD score 68.9 52.5 41.4 47.1 (17.01)
(18.61) (13.78) (17.01) % Change from Baseline to 8.1 -21.0 -29.4
-25.1 Day 15 (22.20) (15.53) (14.80) (15.49) Absolute change from
Baseline 5.0 -13.7 -17.3 -15.4 to Day 15 (13.08) (10.94) (9.27)
(10.14) Day 29 SCORAD score 56.2 38.8 32.9 35.7 (17.51) (17.39)
(20.53) (18.94) % Change from Baseline to -10.1 -38.9 -46.5 -42.9
Day 29 (17.58) (19.00) (26.30) (22.94) Absolute change from
Baseline -5.5 -24.3 -25.8 -25.1 to Day 29 (11.20) (13.06) (15.03)
(13.85) Day 36 SCORAD score 53.8 28.6 34.0 30.8 (8.97) (14.79)
(12.78) (13.93) % Change from Baseline to -8.9 -54.7 -44.6 -50.7
Day 36 (8.74) (17.10) (15.77) (16.93) Absolute change from Baseline
-5.4 -34.5 -27.2 -31.6 to Day 36 (5.33) (14.35) (10.64) (13.20) Day
43 SCORAD score 46.4 31.3 25.9 28.7 (10.88) (17.18) (14.30) (15.75)
% Change from Baseline to -25.3 -50.3 -55.9 -53.0 Day 43 (15.86)
(22.09) (18.41) (20.15) Absolute change from Baseline -16.6 -31.8
-30.8 -31.3 to Day 43 (10.29) (16.14) (10.69) (13.15) Day 57 SCORAD
score 49.3 36.2 27.6 32.3 (22.19) (21.67) (15.77) (19.29) % Change
from Baseline to -19.0 -44.4 -49.9 -46.9 Day 57 (33.04) (27.74)
(24.92) (26.06) Absolute change from Baseline -11.2 -29.0 -29.1
-29.0 to Day 57 (20.24) (19.89) (15.70) (17.71) Day 71 SCORAD score
46.4 41.9 28.6 35.3 (14.80) (17.40) (18.83) (18.98) % Change from
Baseline to -27.5 -34.0 -53.4 -43.7 Day 71 (18.86) (23.47) (23.21)
(24.88) Absolute change from Baseline -17.2 -21.2 -29.9 -25.5 to
Day 71 (11.67) (14.43) (12.18) (13.78) Day 85 SCORAD score 48.8
42.3 31.2 36.5 (21.01) (21.07) (19.86) (20.77) % Change from
Baseline to -19.7 -33.4 -48.4 -41.2 Day 85 (37.22) (26.47) (27.53)
(27.49) Absolute change from Baseline -12.8 -22.3 -25.8 -24.1 to
Day 85 (22.14) (20.81) (12.96) (16.85)
TABLE-US-00013 TABLE 13 Summary of Percentage and Absolute Change
in 5-D Pruritus Scale from Baseline - all values represented as
Mean (SD) mAb1 All Doses Placebo 150 mg 300 mg Combined No.
Patients 10 14 13 27 Baseline 5-D Pruritus Scale 17.8 18.9 18.1
18.5 (3.94) (3.05) (3.99) (3.49) Day 15 5-D Pruritus Scale 17.0
14.3 13.0 13.7 (4.61) (4.68) (5.03) (4.8) % Change from Baseline to
3.6 -24.8 -28.2 -26.5 Day 15 (30.77) (19.73) (19.49) (19.31)
Absolute change from Baseline 0.0 -4.6 -5.1 -4.9 to Day 15 (6.18)
(3.97) (4.15) (3.99) Day 29 5-D Pruritus Scale 16.7 12.2 11.4 11.8
(3.50) (5.02) (5.97) (5.45) % Change from Baseline to 8.1 -35.4
-37.1 -36.3 Day 29 (18.85) (20.28) (26.40) (23.30) Absolute change
from Baseline 1.2 -6.5 -6.7 -6.6 to Day 29 (3.19) (4.25) (5.57)
(4.91) Day 43 5-D Pruritus Scale 14.5 11.8 10.0 11.0 (2.17) (4.43)
(5.44) (4.91) % Change from Baseline to -9.4 -37.1 -42.5 -39.7 Day
43 (23.24) (19.61) (24.75) (21.86) Absolute change from Baseline
-2.3 -7.0 -7.5 -7.2 to Day 43 (5.20) (3.95) (5.26) (4.52) Day 57
5-D Pruritus Scale 14.5 13.4 11.2 12.4 (2.74) (5.45) (5.40) (5.43)
% Change from Baseline to -7.8 -29.6 -36.2 -32.6 Day 57 (28.55)
(25.38) (22.7) (23.90) Absolute change from Baseline -2.0 -5.5 -6.3
-5.9 to Day 57 (5.14) (4.75) (4.71) (4.65) Day 71 5-D Pruritus
Scale 16.4 16.2 12.3 14.3 (2.88) (5.47) (5.66) (5.79) % Change from
Baseline to 8.1 -15.4 -31.6 -23.5 Day 71 (22.85) (21.50) (24.97)
(24.25) Absolute change from Baseline 1.0 -2.7 -5.6 -4.1 to Day 71
(3.24) (3.70) (4.87) (4.48) Day 85 5-D Pruritus Scale 17.5 15.3
14.0 14.6 (3.00) (4.92) (5.83) (5.33) % Change from Baseline to
20.6 -19.1 -21.4 -20.3 Day 85 (34.03) (23.10) (26.23) (24.25)
Absolute change from Baseline 2.5 -3.7 -3.7 -3.7 to Day 85 (5.00)
(4.50) (4.66) (4.48)
TABLE-US-00014 TABLE 14 Summary of Percentage and Absolute Change
in Average Weekly NRS Score from Baseline - all values represented
as Mean (SD) mAb1 All Doses Placebo 150 mg 300 mg Combined No.
Patients 10 14 13 27 Baseline NRS Score 5.8 (1.92) 5.6 (1.87) 5.4
(1.69) 5.5 (1.75) Week 1 NRS Score 5.5 (1.46) 5.1 (1.88) 4.3 (1.59)
4.7 (1.75) % Change from Baseline to Week 1 -2.9 (14.99) -9.1
(15.88) -16.5 (28.61) -12.8 (22.98) Absolute change from Baseline
to Week 1 -0.3 (0.99) -0.5 (0.93) -1.1 (1.76) -0.8 (1.42) Week 2
NRS Score 5.4 (1.42) 4.2 (2.41) 3.5 (1.83) 3.9 (2.13) % Change from
Baseline to Week 2 1.3 (25.45) -26.6 (31.37) -31.9 (31.90) -29.3
(31.11) Absolute change from Baseline to Week 2 -0.1 (1.50) -1.3
(1.67) -1.9 (2.01) -1.6 (1.84) Week 3 NRS Score 5.5 (2.02) 3.6
(1.80) 3.2 (1.35) 3.4 (1.57) % Change from Baseline to Week 3 2.9
(16.38) -35.7 (17.81) -37.2 (30.15) -36.4 (24.23) Absolute change
from Baseline to Week 3 0.2 (0.83) -1.8 (0.92) -2.2 (1.87) -2.0
(1.45) Week 4 NRS Score 4.5 (1.33) 3.7 (2.18) 2.9 (2.18) 3.3 (2.17)
% Change from Baseline to Week 4 -1.1 (28.49) -35.0 (25.50) -45.4
(40.95) -40.2 (33.78) Absolute change from Baseline to Week 4 -0.1
(1.35) -1.8 (1.37) -2.5 (2.18) -2.1 (1.81) Week 5 NRS Score 4.4
(1.68) 3.2 (2.08) 2.8 (1.92) 3.0 (1.96) % Change from Baseline to
Week 5 -5.4 (34.34) -44.6 (26.15) -43.1 (37.67) -43.9 (31.42)
Absolute change from Baseline to Week 5 -0.3 (1.60) -2.3 (1.54)
-2.3 (2.10) -2.3 (1.79) Week 6 NRS Score 3.8 (1.89) 3.2 (1.98) 2.7
(2.08) 3.0 (2.00) % Change from Baseline to Week 6 -19.0 (35.26)
-43.6 (23.27) -49.7 (33.29) -46.3 (27.72) Absolute change from
Baseline to Week 6 -0.9 (1.72) -2.3 (1.37) -2.6 (1.98) -2.4 (1.64)
Week 7 NRS Score 3.8 (2.18) 3.2 (2.52) 2.3 (1.84) 2.7 (2.21) %
Change from Baseline to Week 7 -17.3 (42.69) -45.0 (33.78) -55.2
(35.03) -49.9 (33.99) Absolute change from Baseline to Week 7 -0.8
(2.06) -2.3 (1.90) -3.1 (2.13) -2.7 (2.00) Week 8 NRS Score 3.5
(2.01) 3.2 (2.10) 2.5 (1.93) 2.9 (2.00) % Change from Baseline to
Week 8 -22.1 (38.80) -43.0 (35.08) -50.7 (34.39) -46.7 (34.18)
Absolute change from Baseline to Week 8 -1.0 (1.90) -2.3 (1.84)
-2.8 (2.18) -2.6 (1.98) Week 9 NRS Score 3.7 (1.93) 3.6 (2.12) 2.5
(2.34) 3.1 (2.25) % Change from Baseline to Week 9 -20.7 (34.06)
-34.2 (36.64) -52.9 (39.71) -43.1 (38.47) Absolute change from
Baseline to Week 9 -0.9 (1.65) -1.8 (1.74) -2.8 (2.25) -2.3 (2.02)
Week 10 NRS Score 3.7 (1.86) 4.3 (2.39) 3.1 (2.38) 3.7 (2.40) %
Change from Baseline to Week 10 -19.7 (31.94) -25.1 (32.66) -43.6
(38.72) -34.4 (36.22) Absolute change from Baseline to Week 10 -0.8
(1.54) -1.2 (1.30) -2.3 (2.10) -1.7 (1.80) Week 11 NRS Score 3.9
(2.18) 4.9 (2.45) 2.9 (1.92) 3.9 (2.39) % Change from Baseline to
Week 11 -17.1 (37.62) -10.1 (43.91) -43.4 (37.56) -26.0 (43.46)
Absolute change from Baseline to Week 11 -0.7 (1.77) -0.5 (1.85)
-2.2 (1.93) -1.4 (2.03) Week 12 NRS Score 3.9 (1.88) 4.3 (2.68) 3.4
(2.59) 3.9 (2.60) % Change from Baseline to Week 12 -13.6 (29.53)
-21.1 (39.41) -38.0 (35.65) -29.1 (37.65) Absolute change from
Baseline to Week 12 -0.5 (1.35) -1.1 (2.00) -1.9 (1.93) -1.5
(1.96)
F. Conclusions
[0377] Subcutaneous administration of an anti-IL-4R antibody (mAb1)
to adult patients with moderate-to-severe atopic dermatitis was
generally safe and well tolerated after 4 weekly doses of 150 or
300 mg with an adverse event (AE) rate similar to placebo and no
dose limiting toxicities or serious AEs. The most common AEs with
mAb1 were nasopharyngitis and headache. The mAb1 rapidly (by Day 8)
reduced pruritus and improved skin disease in a dose-dependent
fashion. Administration of mAb1 at 150 and 300 mg resulted in
significant improvement in IGA, EASI, BSA, SCORAD and NRS pruritus
as early as Day 8 through day 85 in both mean and absolute and
percent change, as compared to baseline (see Tables 9-14). In the
300 mg arm at day 29, proportion of patients who achieved an EASI50
response was 71.4% vs 18.8% for placebo (p=0.0025) and NRS pruritus
score decreased by 45.4% vs 18.6% for placebo (p=0.0016). The
effect was sustained through day 85 for EASI50 and day 75 for NRS
pruritus. For the 300 mg treatment group, the difference from
placebo was significant for an additional 6 weeks after end of
treatment period. The mAb significantly improved other clinical
outcomes at day 29, mean % change IGA (p=0.0002), EASI
(p<0.0001), BSA (p=0.0037), and 5D pruritus (p<0.0001). These
improvements were generally observed by Day 8 and persisted after
end of treatment. No rebound phenomena were observed after end of
treatment.
[0378] The results shown in this Example therefore demonstrate that
mAb1 is safe and effective for the treatment of atopic
dermatitis.
Example 8
Treatment of Patients with Moderate-to-Severe Atopic Dermatitis
with Anti-IL-4R Antibody: Analysis of Pooled Phase 1b Studies
[0379] AD efficacy parameters were measured and pooled for analysis
from two separate clinical trials in patients with
moderate-to-severe AD. "Study A" was a 12-week, double-blind,
randomized, placebo-controlled, sequential ascending dose study to
assess the safety and tolerability of administered anti-IL-4R
antibody (mAb1) in patients with atopic dermatitis. The treatment
period was 4 weeks with patients being followed for 8 weeks after
the end of the treatment period. Patients were randomized in a 4:1
ratio to receive mAb1 or placebo in each of the three ascending
dose cohorts (75 mg, 150 mg, or 300 mg). The study consisted of a
screening period (day -14 to day -3), a treatment period (day 1
through day 29), and a follow-up period (day 29 through day 85).
During the treatment period, patients were seen in the clinic once
weekly for safety, laboratory and clinical effect assessments on
days 1, 4, 8, 15, 22, 25 and 29 (week 4). Patients received a dose
of mAb1 or placebo on days 1, 8, 15 and 22. The end of the
treatment period study was on day 29 (week 4). Patients were
monitored at the study site for 6 hours after the injection (of
mAb1 or placebo) on day 1, and for 3 hours after the injection on
days 8, 15 and 22. During the follow-up period, patients were seen
in the clinic for follow-up assessments at days 36, 43, 50, 57, 64,
71, and 85 (end of study visit).
[0380] "Study B" was a 12-week, double-blind, randomized,
placebo-controlled, sequential ascending, repeated-dose study in
patients with moderate-to-severe AD. AD subjects were administered
150 mg or 300 mg of mAb1, or placebo on days 1, 8, 15 and 22 of the
study (four weekly doses) (See Example 3 herein). All
administrations for both studies were subcutaneous.
[0381] The patient inclusion criteria for the studies were: (1)
should be male or female.gtoreq.18 years; (2) have chronic atopic
dermatitis for 3 years; (3) have EASI.gtoreq..sub.12; (4)
IGA.gtoreq.3; (5) .gtoreq.15% BSA of AD involvement (in the US) or
.gtoreq.10% BSA of AD involvement (ex-US); and (6) history of
inadequate response to stable regimen of topical corticosteroids
(TCS) or calcineurin inhibitors.
[0382] The patient exclusion criteria for the study were: (1)
WBC<3.5.times.10.sup.3/.mu.l; (2)
platelets<125.times.10.sup.3/.mu.l; (3)
neutrophils<1.75.times.10.sup.3/.mu.l; (4)
AST/ALT>1.5.times.ULN; (5) positive for hepatitis B or hepatitis
C; and (6) treatment with TCS or calcineurin inhibitors within 1
week of baseline.
[0383] The primary endpoint of the studies was to monitor incidence
of treatment-emergent adverse events (TEAEs) from baseline through
week 12. The exploratory endpoints for efficacy variables were: (i)
% achieving an IGA of 0 or 1 through week 4; (ii) % improvement in
BSA and EASI from baseline; and (iii) change from baseline in NRS
scale.
[0384] The efficacy variables IGA, BSA, EASI, SCORAD, 5-D Pruritus
scale, and Pruritus NRS rating have been described elsewhere herein
(see, e.g., Example 4).
[0385] The IGA, BSA, EASI and SCORAD scores were assessed at every
clinic visit. Patients underwent 5-D pruritus assessment at the
following visits: screening, day 1/baseline (pre-dose), and days
15, 29, 43, 57, 71, and 85 (end of study) or early termination.
Patients used the IVRS to record their Pruritus NRS score twice
daily through the last study visit.
[0386] Baseline for efficacy variable is defined as the last
non-missing value on or before the date of randomization. For the
patient who has no value on or before his/her randomization date
the last non-missing value on or before the date of first dose
injection will be used as baseline.
[0387] The baseline demographics for the patient population are
presented below in Table 15.
TABLE-US-00015 TABLE 15 Baseline Demographics Placebo 150 mg 300 mg
All Doses (N = 16) 75 mg (N = 8) (N = 22) (N = 21) (N = 51) Mean
age, 37.4 (17.16) 35.8 (12.51) 42.5 (11.37) 45.4 (15.92) 42.6
(13.73) years (SD) Race, n (%) Caucasian 13 (81.3%) 4 (50.0%) 19
(86.4%) 16 (76.2%) 39 (76.5%) Non- 3 (18.7%) 4 (50.0%) 3 (13.6%) 5
(23.8%) 12 (23.5%) Caucasian Gender, n (%) Male 11 (68.8%) 6
(75.0%) 12 (54.5%) 10 (47.6%) 28 (54.9%) Female 5 (31.3%) 2 (25.0%)
10 (45.5%) 11 (52.4%) 23 (45.1%) Mean BMI, 25.69 (5.993) 26.41
(4.489) 25.68 (3.991) 27.71 (8.667) 26.63 (6.361) kg/m.sup.3
(SD)
[0388] The mean baseline disease characteristics are given in Table
16.
TABLE-US-00016 TABLE 16 Mean Baseline Disease Characteristics
Placebo 150 mg All Doses (N = 16) 75 mg (N = 8) (N = 22) 300 mg (N
= 21) (N = 51) Duration of 31.8 (18.67) 24.5 (16.95) 32.1 (15.44)
30.7 (16.95) 30.4 (16.19) chronic AD, years EASI score 22.8 (12.02)
36.9 911.75) 30.0 (17.00) 27.4 (11.21) 30.0 (14.19) IGA score 3.6
(0.72) 4.1 (0.35) 3.9 (0.68) 3.5 (0.51) 3.8 (0.62) % BSA of AD 40.3
(25.77) 64.4 917.03) 49.8 (28.75) 48.2 (22.26) 51.4 (24.87) 5-D
pruritus 16.9 (3.94) 21.5 (3.55) 19.0 (2.94) 18.7 (3.64) 19.3
(3.41) scale Pruritus 5.8 (1.75) 7.0 (1.78) 6.0 (1.82) 5.7 (1.51)
6.0 (1.72) NRS score
[0389] The exploratory efficacy results obtained from the pooled
studies are summarized in Tables 17-25 and in FIGS. 15-22.
TABLE-US-00017 TABLE 17 Summary of subjects achieving IGA .ltoreq.
1 at Day 29 and all study visits Number and % All Doses subjects
with Placebo 75 mg 150 mg 300 mg Combined IGA .ltoreq. 1 (N = 16)
(N = 8) (N = 22) (N = 21) (N = 51) Week 4, 1 (6.3%) 0 4 (18.2%) 2
(9.5%) 6 (11.8%) Day 29 Day 4 0 0 0 0 0 Week 1, Day 8 0 0 0 0 0
Week 2, 0 0 0 1 (4.8%) 1 (2.0%) Day 15 Week 3, 0 0 0 2 (9.5%) 2
(3.9%) Day 22 Week 3, 1 (6.3%) 0 1 (4.5%) 4 (19.0%) 5 (9.8%) Day 25
Week 5, 1 0 4 (18.2%) 2 (9.5%) 6 (11.8%) Day 36 Week 6, 2 (12.5%) 0
5 (22.7%) 3 (14.3%) 8 (15.7%) Day 43 Week 7, 2 (12.5%) 0 4 (18.2%)
3 (14.3%) 7 (13.7%) Day 50 Week 8, 2 (12.5%0 0 3 (13.6%) 5 (23.8%)
8 (15.7%) Day 57 Week 9, 1 (6.3%) 0 3 (13.6%) 4 (19.0%) 7 (13.7%)
Day 64 Week 10, 1 (6.3%) 0 1 (4.5%) 5 (23.8%) 6 (11.8%) Day 71 Week
12, 1 (6.3%) 0 0 3 (14.3%) 3 (5.9%) Day 85
TABLE-US-00018 TABLE 18 Summary of Percentage and Absolute Change
in BSA Score from Baseline - all values represented as Mean (SD)
mAb1 All Doses Placebo 75 mg 150 mg 300 mg Combined No. Patients 16
8 22 21 51 Baseline BSA Score 40.3 64.4 49.8 48.2 51.4 (25.77)
(17.03) (28.75) (22.26) (24.87) Day 15 BSA Score 37.6 52.3 40.9
34.4 40.0 (26.61) (12.54) (25.66) (22.66) (23.23) % Change from
Baseline to Day 15 -4.8 -16.8 -13.9 -30.5 -21.4 (14.80) (15.17)
(21.77) (27.09) (24.27) Absolute change from Baseline to Day 15
-1.7 -12.1 -7.0 -13.9 -10.7 (5.37) (11.58) (15.07) (14.73) (14.51)
Day 29 BSA Score 31.1 46.3 31.1 31.5 33.8 (29.69) (12.42) (28.78)
925.33) (25.47) % Change from Baseline to Day 29 -15.3 -26.4 -38.8
-40.3 -37.4 (31.02) (16.41) (37.00) (33.78) (32.88) Absolute change
from Baseline to Day 29 -2.1 -18.1 -18.2 -16.7 -17.5 (10.93)
(13.14) (24.61) (16.05) (19.31) Day 36 BSA Score 25.1 41.2 24.9
26.0 28.0 (26.81) (15.59) (24.15) (22.67) (22.70) % Change from
Baseline to Day 36 -13.3 -33.7 -48.6 -44.2 -44.4 (39.22) (21.53)
(32.13) (34.61) (31.41) Absolute change from Baseline to Day 36
-1.8 -22.4 -24.3 -18.0 -21.6 (10.33) (15.26) (25.07) (17.82)
(20.85) Day 43 BSA Score 29.9 48.4 24.8 26.2 29.1 (27.04) (21.56)
(26.36) (21.03) (24.42) % Change from Baseline to Day 43 -11.0
-29.2 -43.3 -47.2 -42.7 (39.52) (24.87) (42.81) (30.07) (35.05)
Absolute change from Baseline to Day 43 -2.0 -19.0 -22.2 -19.8
-20.7 (10.74) (15.63) (29.35) (14.41) (21.52) Day 57 BSA Score 27.2
57.5 31.2 28.3 33.7 (31.12) (23.40) (28.60) (20.11) (26.24) %
Change from Baseline to Day 57 -33.6 -18.7 -37.4 -41.9 -36.6
(32.95) (23.06) (42.74) (29.38) (35.90) Absolute change from
Baseline to Day 57 -8.3 -12.4 -20.0 -17.6 -18.0 (16.62) (16.36)
(28.38) (13.86) (21.99) Day 71 BSA Score 27.4 58.4 30.7 23.2 31.1
(28.13) (19.79) (24.56) (19.85) (24.32) % Change from Baseline to
Day 71 -29.0 -13.2 -35.7 -52.0 -39.9 (36.38) (11.92) (37.54)
(35.43) (36.13) Absolute change from Baseline to Day 71 -7.5 -8.5
-18.4 -25.2 -20.1 (17.71) (8.10) (23.12) (18.53) (20.14) Day 85 BSA
Score 25.1 58.0 30.7 23.6 30.7 (27.73) (19.52) (28.38) (17.95)
(25.04) % Change from Baseline to Day 85 -33.4 -16.9 -37.8 -49.0
-40.4 (32.68) (16.63) (43.59) (37.34) (39.14) Absolute change from
Baseline to Day 85 -8.4 -11.9 -20.6 -22.7 -20.5 (14.45) (11.45)
(29.67) (15.74) (22.31)
TABLE-US-00019 TABLE 19 Summary of Percentage and Absolute Change
in EASI Score from Baseline - all values represented as Mean (SD)
mAb1 All Doses Placebo 75 mg 150 mg 300 mg Combined No. Patients 16
8 22 21 51 Baseline EASI Score 22.8 36.9 30.0 27.4 30.0 (12.02)
(11.75) (17.00) (11.21) (14.19) Day 15 EASI Score 25.4 26.2 19.8
15.4 19.0 (20.13) (7.72) (15.05) (8.57) (12.06) % Change from
Baseline to Day 15 8.7 -26.9 -31.1 -45.1 -36.3 (66.05) (19.29)
(27.24) (19.90) (24.02) Absolute change from Baseline to Day 15 2.8
-10.7 -9.7 -12.0 -10.8 (14.11) (9.83) (12.02) (6.93) (9.67) Day 29
EASI Score 17.2 17.7 13.1 11.3 13.1 (15.11) (6.05) (11.89) (11.84)
(11.17) % Change from Baseline to Day 29 -25.4 -47.0 -55.0 -64.3
-57.7 (34.98) (21.93) (30.36) (25.83) (27.45) Absolute change from
Baseline to Day 29 -3.6 -19.2 -16.6 -16.1 -16.8 (7.25) (15.11)
(14.58) (7.69) (11.97) Day 36 EASI Score 13.2 16.3 9.4 10.5 11.0
(11.97) (7.74) (10.27) (8.69) (9.42) % Change from Baseline to Day
36 -28.4 -51.5 -69.6 -61.9 -63.6 (41.10) (25.53) (22.46) (22.69)
(23.41) Absolute change from Baseline to Day 36 -3.9 -21.5 -20.5
-16.1 -19.0 (7.94) (17.30) (14.98) (8.23) (13.13) Day 43 EASI Score
12.9 19.8 9.6 9.3 11.1 97.13) (10.41) (11.01) (8.29) (10.32) %
Change from Baseline to Day 43 -33.8 -39.4 -64.2 -66.4 -61.2
(28.94) (31.87) (33.89) (22.39) (29.98) Absolute change from
Baseline to Day 43 -6.2 -17.0 -19.7 -16.8 -18.0 (4.71) (19.33)
(16.63) (7.84) (13.76) Day 57 EASI Score 13.0 27.0 12.2 10.4 13.5
(11.95) (16.46) (12.88) (9.40) (13.11) % Change from Baseline to
Day 57 -28.7 -24.5 -57.3 -61.1 -54.3 (62.63) (47.21) (33.38)
(24.91) (33.99) Absolute change from Baseline to Day 57 -5.4 -11.9
-18.4 -15.8 -16.5 (11.79) (22.95) (17.88) (9.69) (15.81) Day 71
EASI Score 11.8 28.3 13.0 8.5 13.1 (9.22) (13.06) (10.86) (9.21)
(12.06) % Change from Baseline to Day 71 -45.8 -14.5 -54.9 -71.3
-56.8 (31.06) (41.14) (32.01) (24.14) (34.68) Absolute change from
Baseline to Day 71 -9.6 -9.4 -16.9 -19.1 -16.9 (8.23) (22.16)
(15.41) (9.88) (14.32) Day 85 EASI Score 9.8 27.1 14.2 10.5 14.0
(4.87) (11.99) (14.30) (9.26) (12.77) % Change from Baseline to Day
85 -44.8 -28.3 -51.3 -63.0 -53.9 (30.60) (29.69) (37.58) (25.55)
(32.86) Absolute change from Baseline to Day 85 -9.3 -13.4 -16.6
-15.4 -15.7 (8.01) (18.94) (17.67) (7.57) (13.81)
TABLE-US-00020 TABLE 20 Summary of Percentage and Absolute Change
in 5-D Pruritus Scale from Baseline - all values represented as
Mean (SD) mAb1 All Doses Placebo 75 mg 150 mg 300 mg Combined No.
Patients 16 8 22 21 51 Baseline 5-D Pruritus Scale 16.9 21.5 19.0
18.7 19.3 (3.94) (3.55) (2.94) (3.64) (3.41) Day 15 5-D Pruritus
Scale 15.0 14.0 14.0 12.5 13.4 (4.66) (3.55) (4.46) (4.08) (4.15) %
Change from Baseline to Day 15 -5.6 -34.3 -26.6 -32.4 -30.3 (29.83)
(15.43) (19.26) (17.60) (17.95) Absolute change from Baseline to
Day 15 -1.4 -7.5 -5.0 -6.1 -5.9 (5.55) (3.82) (3.97) (3.93) (3.94)
Day 29 5-D Pruritus Scale 14.8 14.1 13.1 11.0 12.3 93.77) (3.31)
(5.03) (4.86) (4.79) % Change from Baseline to Day 29 -3.9 -33.0
-30.8 -40.8 -35.6 (20.07) (17.25) (23.71) (21.83) (22.02) Absolute
change from Baseline to Day 29 -0.8 -7.4 -5.9 -7.7 -6.9 (3.41)
(4.47) (4.84) (4.78) (4.73) Day 43 5-D Pruritus Scale 13.8 16.5
12.1 10.7 12.3 (3.71) (4.54) (4.64) (4.83) (5.04) % Change from
Baseline to Day 43 -10.4 -21.4 -35.0 -40.8 -35.0 (31.60) (25.01)
(22.07) (23.87) (23.86) Absolute change from Baseline to Day 43
-2.3 -5.0 -6.6 -7.6 -6.8 (5.25) (5.66) (4.45) (5.04) (4.90) Day 57
5-D Pruritus Scale 12.3 19.9 13.9 11.6 14.0 (3.35) (3.98) 94.75)
(5.18) (5.46) % Change from Baseline to Day 57 -19.0 -9.0 -27.2
-37.2 -28.1 (25.37) (20.15) (21.28) (21.68) (22.85) Absolute change
from Baseline to Day 57 -3.4 -2.3 -5.1 -6.8 -5.3 (4.43) (4.46)
(4.03) (4.61) (4.49) Day 71 5-D Pruritus Scale 13.5 19.4 15.3 12.9
14.7 (4.03) (3.51) (4.78) (5.61) (5.36) % Change from Baseline to
Day 71 -11.6 -8.3 -18.9 -31.7 -23.3 (25.71) (14.91) (19.50) (24.53)
(22.58) Absolute change from Baseline to Day 71 -2.0 -2.0 -3.4 -5.8
-4.3 (4.12) (3.39) (3.56) (4.70) (4.24) Day 85 5-D Pruritus Scale
14.1 18.6 15.2 14.6 15.3 (4.48) (1.34) (3.99) (5.26) (4.53) %
Change from Baseline to Day 85 -5.4 -10.0 -18.5 -21.9 -19.0 (32.44)
(22.58) (21.29) (23.41) (22.18) Absolute change from Baseline to
Day 85 -1.2 -2.8 -3.7 -4.1 -3.7 (5.09) (4.92) (4.04) (4.52)
(4.27)
TABLE-US-00021 TABLE 21 Summary of Percentage and Absolute Change
in Average Weekly NRS Score from Baseline - all values represented
as Mean (SD) mAb1 All Doses Placebo 75 mg 150 mg 300 mg Combined
No. Patients 10 8 22 21 51 Baseline NRS Score 5.8 7.0 6.0 5.7 6.0
(1.75) (1.78) (1.82) (1.51) (1.72) Week 1 NRS Score 5.1 5.2 5.2 4.3
4.8 (1.73) (2.50) (1.91) (1.52) (1.88) % Change from Baseline to
Week 1 -11.9 -27.3 -12.7 -21.6 -18.8 (23.13) (20.25) (18.26)
(26.03) (22.42) Absolute change from Baseline to Week 1 -0.8 -1.7
-0.8 -1.4 -1.2 (1.40) (1.22) (1.30) (1.59) (1.44) Week 2 NRS Score
4.7 4.0 4.5 3.7 4.1 (2.00) (2.36) (2.38) (1.59) (2.07) % Change
from Baseline to Week 2 -14.8 -44.6 -26.9 -33.3 -32.4 (36.13)
(21.90) (29.96) (26.69) (27.63) Absolute change from Baseline to
Week 2 -1.0 -3.0 -1.5 -2.0 -1.9 (2.16) (1.350 (1.76) (1.71) (1.73)
Week 3 NRS Score 5.0 3.9 4.0 3.3 3.7 (2.29) (2.12) (2.12) (1.30)
(1.81) % Change from Baseline to Week 3 -10.2 -45.6 -35.4 -39.4
-38.8 (33.75) (21.67) (23.84) (25.92) (24.17) Absolute change from
Baseline to Week 3 -0.7 -3.1 -2.0 -2.4 -2.3 (2.01) (1.30) (1.49)
(1.65) (1.55) Week 4 NRS Score 4.1 4.1 3.9 3.1 3.6 (2.03) (1.95)
(2.38) (1.84) (2.10) % Change from Baseline to Week 4 -18.6 -42.3
-36.7 -45.4 -41.3 (40.12) (22.62) (29.33) (32.89) (29.63) Absolute
change from Baseline to Week 4 -1.2 -2.9 -2.1 -2.6 -2.4 (2.29)
(1.38) (1.85) (1.77) (1.74) Week 5 NRS Score 4.2 4.1 3.5 3.0 3.4
(2.29) (2.03) (2.36) (1.80) (2.09) % Change from Baseline to Week 5
-18.9 -41.9 -43.4 -44.2 -43.5 (43.93) (24.53) (30.89) (32.74)
(30.09) Absolute change from Baseline to Week 5 -1.2 -2.9 -2.5 -2.5
-2.6 (2.43) (1.55) (1.97) (1.92) (1.85) Week 6 NRS Score 4.0 4.1
3.7 3.0 3.5 (2.40) (2.22) (2.38) (1.84) (2.14) % Change from
Baseline to Week 6 -24.9 -42.7 -40.0 -46.9 -43.3 (42.63) (24.23)
(30.52) (28.41) (28.31) Absolute change from Baseline to Week 6
-1.4 -2.8 -2.2 -2.6 -2.5 (2.36) (1.44) (1.86) (1.68) (1.71) Week 7
NRS Score 3.4 4.4 3.7 2.8 3.4 (2.59) (2.39) (2.56) (1.78) (2.26) %
Change from Baseline to Week 7 -35.5 -41.3 -40.3 -49.9 -44.5
(42.70) (21.96) (33.56) (30.73) (30.73) Absolute change from
Baseline to Week 7 -1.9 -2.8 -2.2 -2.8 -2.5 (2.33) (1.10) (1.90)
(1.83) (1.77) Week 8 NRS Score 3.5 5.4 3.7 3.0 3.7 (2.61) (2.40)
(2.24) (1.98) (2.24) % Change from Baseline to Week 8 -33.9 -27.8
-38.2 -45.6 -39.8 (38.63) (21.17) (33.09) (32.23) (31.29) Absolute
change from Baseline to Week 8 -1.8 -1.9 -2.2 -2.6 -2.3 (2.19)
(1.19) (1.80) (1.99) (1.80) Week 9 NRS Score 3.6 5.5 4.1 3.0 3.9
(2.26) (2.44) (2.10) (2.27) (2.32) % Change from Baseline to Week 9
-32.8 -26.1 -31.5 -46.2 -36.9 (35.28) (17.08) (32.14) (36.56)
(32.95) Absolute change from Baseline to Week 9 -1.7 -1.7 -1.8 -2.5
-2.1 (2.01) (1.02) (1.59) (2.10) (1.77) Week 10 NRS Score 3.7 5.3
4.6 3.2 4.1 (2.51) (2.33) (2.18) (1.99) (2.21) % Change from
Baseline to Week 10 -30.3 -21.7 -24.6 -43.4 -32.5 (41.78) (24.33)
(28.77) (31.24) (30.36) Absolute change from Baseline to Week -1.6
-1.4 -1.3 -2.4 -1.8 10 (2.31) (1.51) (1.37) (1.70) (1.59) Week 11
NRS Score 2.8 5.8 5.0 3.2 4.4 (2.03) (2.11) (2.19) (1.81) (2.23) %
Change from Baseline to Week 11 -40.2 -13.1 -14.2 -41.2 -25.1
(40.04) (26.33) (36.88) (31.87) (35.60) Absolute change from
Baseline to Week -2.0 -0.9 -0.8 -2.2 -1.4 11 (2.26) (1.61) (1.72)
(1.64) (1.76) Week 12 NRS Score 3.5 5.2 4.8 3.5 4.4 (1.48) (2.37)
(2.47) (2.37) (2.44) % Change from Baseline to Week 12 -28.9 -25.4
-17.9 -35.5 -25.4 (29.54) (25.39) (33.42) (33.02) (32.53) Absolute
change from Baseline to Week -1.5 -1.7 -1.0 -1.7 -1.3 12 (1.66)
(1.50) (1.77) (1.73) (1.71)
TABLE-US-00022 TABLE 22 Summary of Percentage and Absolute Change
in IGA Score from Baseline - all values represented as Mean (SD)
mAb1 All Doses Placebo 75 mg 150 mg 300 mg Combined No. Patients 16
8 22 21 51 Baseline IGA Score 3.6 4.1 3.9 3.5 3.8 (0.62) (0.72)
(0.35) (0.68) (0.51) Day 4 IGA Score 3.6 4.1 3.9 3.3 3.7 (0.65)
(0.73) (0.35) (0.71) (0.48) % Change from Baseline to Day 4 -1.6
0.0 -1.1 -3.6 -2.0 (6.79) (6.25) (0.00) (5.33) (8.96) Absolute
change from Baseline to Day 4 -0.1 0.0 0.0 -0.1 -0.1 (0.27) (0.25)
(0.00) (0.21) (0.36) Day 8 IGA Score 3.3 4.0 3.6 3.1 3.5 (0.73)
(0.90) (0.00) (0.85) (0.54) % Change from Baseline to Day 8 -5.6
-2.5 -7.3 -10.3 -7.8 (12.46) (21.28) (7.07) (12.55) (13.67)
Absolute change from Baseline to Day 8 -0.2 -0.1 -0.3 -0.4 -0.3
(0.46) (0.68) (0.35) (0.46) (0.50) Day 15 IGA Score 3.4 3.6 3.0 2.9
3.1 (0.83) (0.99) (0.52) (0.97) (0.70) % Change from Baseline to
Day 15 -2.8 -11.3 -23.7 -16.3 -18.5 (17.55) (28.98) (16.20) (16.69)
(18.16) Absolute change from Baseline to Day 15 -0.1 -0.5 -0.9 -0.6
-0.7 (0.65) (0.92) (0.76) (0.64) (0.60) Day 22 IGA Score 3.1 3.4
2.7 2.3 2.7 (0.80) (0.67) (0.52) (0.73) (0.80) % Change from
Baseline to Day 22 -9.0 -17.5 -30.8 -32.5 -29.4 (19.17) (19.61)
(15.35) (12.76) (23.99) Absolute change from Baseline to Day 22
-0.3 -0.8 -1.2 -1.1 -1.1 (0.68) (0.65) (0.710 (0.52) (0.79) Day 25
IGA Score 3.0 3.1 2.5 2.2 2.5 (0.86) (0.89) (0.35) (0.87) (0.89) %
Change from Baseline to Day 25 -12.1 -23.8 -34.5 -35.7 -33.2
(21.05) (29.43) (10.94) (18.64) (25.16) Absolute change from
Baseline to Day 25 -0.5 -1.0 -1.4 -1.2 -1.2 (0.77) (0.93) (0.53)
(0.790 (0.83) Day 29 IGA Score 2.9 3.0 2.4 2.3 2.4 (0.89) (1.08)
(0.53) (0.99) (0.85) % Change from Baseline to Day 29 -16.0 -26.3
-38.0 -34.9 -34.8 (21.68) (24.48) (16.20) (24.02) (21.18) Absolute
change from Baseline to Day 29 -0.5 -1.1 -1.5 -1.2 -1.3 (0.85)
(0.80) (0.83) (1.00) (0.68) Day 36 IGA Score 2.9 3.0 2.2 2.4 2.4
(0.70) (1.20) (0.58) (0.76) (0.50) % Change from Baseline to Day 36
-16.7 -26.4 -44.1 -33.3 -37.1 (16.97) (26.35) (17.49) (19.38)
(9.13) Absolute change from Baseline to Day 36 -0.5 -1.1 -1.7 -1.2
-1.4 (0.74) (0.85) (0.90) (0.81) (0.40) Day 43 IGA Score 2.8 3.3
2.3 2.2 2.4 (0.97) (1.06) (0.76) (1.02) (0.83) % Change from
Baseline to Day 43 -21.1 -19.3 -40.8 -39.0 -36.6 (24.53) (26.91)
(21.88) (28.04) (19.85) Absolute change from Baseline to Day 43
-0.8 -0.9 -1.6 -1.3 1.4 (0.89) (0.97) (1.07) (1.04) (0.58) Day 50
IGA Score 2.7 3.3 2.4 2.1 2.4 (1.00) (1.19) (0.82) (1.07) (0.80) %
Change from Baseline to Day 50 -18.9 -18.3 -37.2 -40.7 -36.0
(24.57) (30.98) (23.80) (25.87) (21.93) Absolute change from
Baseline to Day 50 -0.6 -0.8 -1.5 -1.4 -1.3 (0.95) (1.12) (1.17)
(1.07) (0.70) Day 57 IGA Score 2.8 3.2 2.5 2.2 2.5 (1.00) (1.20)
(0.75) (1.03) (0.97) % Change from Baseline to Day 57 -17.6 -22.5
-34.8 -36.3 -33.7 (24.60) (33.45) (22.08) (25.21) (24.99) Absolute
change from Baseline to Day 57 -0.6 -1.0 -1.4 -1.2 -1.3 (0.97)
(1.01) (1.10) (1.07) (0.83) Day 64 IGA Score 2.7 3.5 2.7 2.1 2.6
(1.06) (0.79) (1.05) (1.08) (0.81) % Change from Baseline to Day 64
-18.9 -14.2 -30.9 -38.5 -31.5 (25.22) (21.44) (29.23) (26.08)
(20.61) Absolute change from Baseline to Day 64 -0.6 -0.7 -1.2 -1.3
-1.2 (0.98) (0.67) (1.37) (1.06) (0.70) Day 71 IGA Score 2.6 3.4
2.8 2.1 2.5 (1.10) (0.81) (0.89) (0.86) (1.15) % Change from
Baseline to Day 71 -22.0 -17.0 -25.5 -41.7 -32.0 (30.18) (20.84)
(26.36) (27.32) (31.65) Absolute change from Baseline to Day 71
-0.7 -0.8 -1.1 -1.5 -1.2 (1.15) (0.65) (1.300 (1.18) (1.10) Day 85
IGA Score 2.6 3.2 2.8 2.6 2.8 (0.96) (1.17) (0.84) (0.99) (0.96) %
Change from Baseline to Day 85 -20.8 -22.0 -25.6 -24.6 -24.7
(28.77) (36.69) (24.65) (31.31) (28.66) Absolute change from
Baseline to Day 85 -0.7 -1.0 -1.1 -0.8 -1.0 (1.07) (1.16) (1.22)
(1.23) (0.90)
TABLE-US-00023 TABLE 23 Number (%) of subjects achieving EASI-50 at
Day 29 and every study visit - LOCF All Doses Number and % Placebo
75 mg 150 mg 300 mg Combined subjects with EASI50 (N = 16) (N = 8)
(N = 22) (N = 21) (N = 51) Week 4, Day 29 3 (18.8%) 3 (37.5%) 12
(54.5%) 15 (71.4%) 30 (58.8%) Week 2, Day 15 0 0 6 (27.3%) 11
(52.4%) 17 (33.3%) Week 5, Day 36 3 (18.8%) 5 (62.5%) 16 (72.7%) 15
(71.4%) 36 (70.6%) Week 6, Day 43 3 (18.8%) 2 (25.0%) 14 (63.6%) 16
(76.2%) 32 (62.7%) Week 8, Day 57 5 (31.3%) 2 (25.0%) 12 (54.5%) 13
(61.9%) 27 (52.9%) Week 10, Day 71 6 (37.5%) 1 (12.5%) 13 (59.1%)
16 (76.2%) 30 (58.8%) Week 12, Day 85 3 (18.8%) 1 (12.5%) 12
(54.5%) 17 (81.0%) 30 (58.8%)
TABLE-US-00024 TABLE 24 Number (%) of subjects achieving EASI-25 at
Day 29 and every study visit - LOCF All Doses Number and % Placebo
75 mg 150 mg 300 mg Combined subjects with EASI25 (N = 16) (N = 8)
(N = 22) (N = 21) (N = 51) Week 4, Day 29 4 (25.0%) 7 (87.5%) 16
(72.7%) 18 (85.7%) 41 (80.4%) Week 2, Day 15 3 (18.8%) 5 (62.5%) 13
(59.1%) 16 (76.2%) 34 (66.7%) Week 5, Day 36 6 (37.5%) 7 (87.5%) 19
(86.4%) 18 (85.7%) 44 (86.3%) Week 6, Day 43 7 (43.8%) 5 (62.5%) 19
(86.4%) 18 (85.7%) 42 (82.4%) Week 8, Day 57 8 (50.0%) 4 (40.0%) 16
(72.7%) 17 (81.0%) 37 (72.5%) Week 10, Day 71 8 (50.0%) 3 (37.5%)
17 (77.3%) 19 (90.5%) 39 (76.5%) Week 12, Day 85 9 (56.3%) 3
(37.5%) 16 (72.7%) 20 (95.2%) 39 (76.5%)
TABLE-US-00025 TABLE 25 Number (%) of subjects achieving EASI-75 at
Day 29 and every study visit -LOCF All Doses Number and % Placebo
75 mg 150 mg 300 mg Combined subjects with EASI25 (N = 16) (N = 8)
(N = 22) (N = 21) (N = 51) Week 4, Day 29 1 (6.3%) 1 (12.5%) 6
(27.3%) 8 (38.1%) 15 (29.4%) Week 2, Day 15 0 0 1 (4.5%) 1 (4.8%) 2
(3.9%) Week 5, Day 36 1 (6.3%) 1 (12.5%) 9 (40.9%) 7 (33.3%) 17
(33.3%) Week 6, Day 43 1 (6.3%) 1 (12.5%) 8 (36.4%) 6 (28.6%) 15
(29.4%) Week 8, Day 57 2 (12.5%) 1 (12.5%) 9 (40.9%) 6 (28.6%) 16
(31.4%) Week 10, Day 71 2 (12.5%) 1 (12.5%) 6 (27.3%) 11 (52.4%) 18
(35.5%) Week 12, Day 85 2 (12.5%) 1 (12.5%) 6 (27.3%) 7 (33.3%) 14
(27.5%)
[0390] mAb1 was well-tolerated and effective in adults with
moderate-to-severe AD. mAb1 administration significantly improved
AD disease activity and severity. At 4 weeks, 150 mg and 300 mg
mAb1 achieved significant improvements vs. placebo for change from
baseline in % BSA (p<0.05) (FIG. 15), IGA (p<0.001) (FIG.
16), EASI (p<0.001) (FIG. 17), and pruritus NRS (p<0.01, 300
mg) (FIG. 18). More patients had .gtoreq.50% reduction in EASI
score with 150 mg mAb1 (54.5%) and with 300 mg (71.4%) vs. placebo
(18.8%; p<0.05 for both) (FIGS. 19 and 20). More patients
achieved EASI-25, EASI-50, and EASI-75 with mAb1 over placebo at
week 4 (FIG. 21).
[0391] For 300 mg mAb1, significant improvement was seen within 2
weeks in % BSA (p<0.02), IGA (p<0.05), and EASI
(p<0.0001). Improvements for BSA, IGA and EASI (p<0.05 vs.
placebo) were maintained for 8 weeks. The proportion of patients
with IGA 0 or 1 at week 4 was higher than placebo, but not
statistically significant (FIG. 22).
[0392] The most common treatment-emergent adverse events (AEs) with
mAb1 administration were nasopharyngitis (19.6% vs. 12.5% for
placebo) and headache (11.8% vs. 6.3% for placebo).
Example 9
Parallel-Group, Dose-Ranging Clinical Trial of Subcutaneously
Administered Anti-IL-4R Antibody (mAb1) in Adult Patients with
Moderate-to-Severe Atopic Dermatitis
A. Study Design
[0393] This study was a 32-week, randomized, double-blind,
placebo-controlled, parallel group study to assess the dose
response profile of weekly doses of mAb1 in adults with
moderate-to-severe AD. The primary objective of the study was to
assess the efficacy of multiple mAb1 dose regimens, compared to
placebo, in adult patients with moderate-to-severe AD. The
secondary objectives were: (1) to assess the safety of multiple
mAb1 dose regimens, compared to placebo, in adult patients with
moderate-to-severe AD; (2) to assess the pharmacokinetics (PK) of
multiple mAb1 dose regimens in adult patients with
moderate-to-severe AD; and (3) to assess the potential immune
response across multiple mAb1 dose regimens, and to compare to
placebo, in adult patients with moderate-to-severe AD.
[0394] The target population included adults with
moderate-to-severe AD which could not be adequately controlled with
topical medications or for whom topical treatment is otherwise
inadvisable (e.g., side effects or safety risks). Approximately 240
to 288 patients were enrolled. Eligible patients were randomized in
a 1:1:1:1:1:1 ratio to receive 1 of 6 weekly treatment regimens (5
active, 1 placebo). Randomization was stratified by disease
severity (moderate vs. severe AD) and region (Japan vs. rest of
world). The dosing schedule followed is given in Table
TABLE-US-00026 TABLE 26 All loading doses = 2 injections of 2 mL
each; All week 1-week 15 doses = 1 injection of 2 mL Subsequent
dosing through week 15 Day 1 (loading dose) Cumulative N 1
injection of each Total dose Dose regimen monthly dose 40 300 mg
600 mg 300 mg qw 1200 mg 300 mg 40 300 mg 600 mg 300 mg q2w 600 mg
300 mg 40 300 mg 600 mg 300 mg q4w 300 mg 300 mg 40 200 mg 400 mg
200 mg q2w 400 mg 200 mg 40 200 mg 400 mg 100 mg q4w 100 mg 200 mg
40 Placebo 0 mg PBO qw 0 mg Placebo
[0395] All patients received 2 injections (a loading dose) on day
1, followed by weekly injections. For every 2 weeks (q2w) and every
4 weeks (q4w) dose-regimens, the next dose of study drug was
administered at week 2 and week 4, respectively. Patients assigned
to q2w and q4w dose-regimens received volume-matched placebo every
week when mAb1 was not administered. After providing informed
consent, patients were assessed for study eligibility at the
screening visit. Patients who met eligibility criteria underwent
day 1/baseline assessments, randomization, and then received a
weekly injection of study drug from day 1 through week 15. During
this time, patients returned for weekly clinic visits with some
weeks requiring only a telephone contact. Patients (and/or
caregivers) were trained on injecting study drug at visits 2, 3, 4,
5, and 6, and self-injected study drug at later study visits that
required only a telephone contact. Patients were closely monitored
at the study site for a minimum of 1 hour after each of the first 5
weekly injections. Safety, laboratory, and clinical effect
assessments were performed at specified clinic visits. The end of
treatment period visit occurred at week 16, 1 week after the last
dose of study drug, when the primary endpoint was assessed.
Follow-up visits occurred every 2 weeks from week 18 through week
32. The end of study visit occurred at week 32. Rescue treatment
for AD (medication and/or phototherapy) was provided to study
patients, if necessary, at the discretion of the investigator.
Patients who needed rescue treatment were immediately discontinued
from study drug, but were asked to continue to follow the schedule
of study assessments. Efficacy measurements were obtained
(Investigator's Global Assessment [IGA], Eczema Area and Severity
Index [EASI], etc.) immediately before administering any rescue
treatment. One sample for DNA analysis and multiple samples for RNA
analysis were collected from patients who consent to participate in
the optional genomic sub-study.
[0396] Study Treatment:
[0397] mAb1 administered subcutaneously: 300 mg weekly (qw), 300 mg
q2w, 300 mg q4w, 200 mg q2w, or 100 mg q4w, from day 1 through week
150R once weekly subcutaneous dose of placebo from day 1 through
week 15. A basic bland topical emollient was applied twice daily
from day -7 through day 8.
[0398] Endpoints of the Study:
[0399] The primary endpoint of the study was the percent change in
EASI score from baseline to week 16. The secondary endpoints
included: (1) proportion of patients achieving IGA 0 (clear) or 1
(almost clear) at week 16; (2) proportion of patients achieving IGA
score reduction of .gtoreq.2 at week 16; (3) absolute change in
EASI scores from baseline to week 16; (4) proportion of patients
achieving EASI-50, EASI-75, and EASI-90 (50, 75 and 90% reduction
from baseline in EASI score) at week 16; (6) proportion of patients
achieving SCORAD-50, SCORAD-75, and SCORAD-90 (50, 75 and 90%
reduction from baseline in SCORAD score) at week 16; (7) absolute
and percent change from baseline in pruritus scores (NRS and
4-point categorical scale); (8) absolute and percent change from
baseline in POEM scores; (9) changes from baseline in GISS
components (erythema, infiltration/population, excoriations, and
lichenification); 910) changes from baseline in GISS cumulative
score; (11) incidence of treatment-emergent adverse events (TEAEs)
from baseline through week 32; and (12) pharmacokinetic profile of
multiple mAb1 dose regimens.
[0400] The other exploratory endpoints included: (1) distribution
of disease severity scores (eg, IGA, EASI, SCORAD) and change from
baseline to various time points through week 16; (2) changes in
Pruritus NRS, Pruritus categorical scale, SCORAD (pruritus VAS and
sleep disturbance VAS), patient global assessment of disease
status, patient global assessment of treatment effect, DLQI, POEM,
EQ-5D, Itchy QOL, and HADS from baseline to various time points
through week 16; (3) absolute and percent change in % BSA, SCORAD
score, EASI and Pruritus NRS, from baseline to various time points
through week 16; (4) proportion of patients who achieve reduction
of IGA score by .gtoreq.2 from baseline to various time points
through week 16; (5) proportion of patients who achieve reduction
of IGA score by .gtoreq.3 from baseline to various time points
through week 16; (6) changes in efficacy parameters from week 16 to
week 32; (7) Incidence and profile (titers over time) of mAb1 ADAs;
(8) effect of mAb1 plasma concentration on ADA formation and
persistence; (9) effect of ADA on mAb1 plasma concentration; (14)
effect of ADA on clinical outcomes (safety and efficacy); (10)
effect of PK parameters (C.sub.max and AUC) on clinical outcomes;
and (11) effect of body weight on drug exposure and clinical
outcomes.
[0401] Rationale for Study Design:
[0402] The purpose of this study was to find an optimal
dose-regimen, which will be further investigated in confirmatory
phase 3 studies. The design of this phase 2b study was informed by
results from a previous mAb1 study, which investigated the safety
and efficacy of a 300 mg dose of mAb1 administered weekly (qw) for
12 consecutive weeks in patients with moderate-to-severe AD. The
selection of phase 2b dose-regimens was also supported by observed
and simulated correlations between pharmacokinetic (PK) and
pharmacodynamics (PD) parameters (PK/PD models) from earlier
clinical trials. Using 300 mg qw (ie, the dose-regimen studied in
phase 2a) as the high anchor of the dose range, the goal was to
identify the lowest dose-regimen with maximal or near-maximal
efficacy and/or, depending on mAb1's emerging safety profile, find
the dose-regimen with the best benefit/risk ratio. Accordingly, 5
mAb1 dose-regimens were selected to reasonably cover the spectrum
between a potentially supra-therapeutic dose-regimen (ie, the high
anchor) and a dose-regimen with clearly sub-optimal efficacy (ie,
the low anchor). The protocol also included a placebo arm to allow
comparison of each active dose-regimen to a control.
[0403] Use of Loading Doses:
[0404] Most patients received a loading dose on day 1, consisting
of a doubling of the nominal dose that was administered at
subsequent visits. This allowed systemic concentrations of mAb1 to
reach steady state and the targeted systemic concentration faster,
and potentially reducing the time to clinical benefit. Study
treatment was administered for 16 weeks so that systemic
concentrations of functional mAb1 could stabilize for all
dose-regimens investigated. Pharmacokinetic modeling suggested that
`every-4-weeks` (q4w) dose-regimens could result in declining
trough concentrations following an initial loading dose.
Consequently, the immunogenic potential of these regimens may not
be fully expressed within a shorter treatment course. After the
last dose of study drug, all patients were followed for 16
additional weeks, which ensured that mAb1 clearance was virtually
complete (plasma concentrations below the lower limit of
quantification) before the end of study visit.
[0405] Rationale for Dose Selection:
[0406] The highest mAb1 dose-regimen administered in this study was
300 mg qw. When given as a short (4-week) treatment course, this
dose-regimen was safe and appeared to be the most efficacious in
earlier phase 1b clinical trials, in which it was investigated
alongside lower dose-regimens (150 mg qw and 75 mg qw).
Pharmacokinetic modeling suggested that 300 mg qw may be
supra-therapeutic in the long run: mAb1 plasma concentrations did
not reach steady state by week 4, and were projected to stabilize
at levels considerably above those required to saturate the target,
ie, the membrane-bound alpha subunit of the IL-4 receptor. However,
this needed to be confirmed by comparing 300 mg qw with lower
dose-regimens in the context of a longer study treatment (12 weeks
or longer), so that plasma concentrations could reach steady state
for all dose-regimens investigated. Although 300 mg qw was
administered for 12 weeks in an earlier study (e.g., phase 2a
proof-of-concept), this dose-regimen was repeated in phase 2b to
confirm phase 2a results and enabled a direct comparison with lower
dose-regimens within the same study. Therefore, 300 mg qw was the
high anchor of the dose range in the present study.
[0407] The low anchor of the dose-regimen range was 100 mg
administered q4w. Based on PK/PD modeling, the resulting mAb1
plasma concentrations at steady state were expected to be
consistently below target mediated clearance (ie, at levels low
enough such that mAb1 elimination was achieved primarily via its
binding to the IL-4 receptor), suggesting that the clinical
response associated with this dose-regimen was incomplete. Three
other dose-regimens were selected between the high and low anchors.
A summary of these dose-regimens and the main rationale for their
selection is provided below: [0408] 300 mg qw: High anchor. Same
dose-regimen studied in phase 2a. [0409] 300 mg every 2 weeks
(q2w): High probability of success based on PK/PD data and models.
Could be sufficient to maintain therapeutic drug levels over
multiple dosing intervals. [0410] 300 mg q4w: PK modeling indicated
that mAb1 plasma levels climbed rapidly to >60 mg/L after the
administration of a loading dose, which was associated with a fast
onset of action, q4w dosing could be sufficient to maintain the
therapeutic effect over time. Since the 300 mg dose is the highest
available, this regimen had the best chance to demonstrate efficacy
for q4w administration. [0411] 200 mg q2w: Some efficacy expected
without reaching the maximum therapeutic effect. Useful for
dose-response assessment and further PK/PD modeling. Helped
evaluate a full spectrum of q2w regimens. [0412] 100 mg q4w: Low
anchor. Likely non-optimally efficacious dose. [0413] Placebo:
Provided a reliable reference for any apparent drug effects.
[0414] Inclusion and Exclusion Criteria:
[0415] A patient had to meet the following criteria to be eligible
for inclusion in the study: (1) Male or female, 18 years or older;
(2) Chronic AD, (according to the AAD Consensus Criteria,
[Eichenfeld 2004]), that has been present for at least 3 years
before the screening visit; (3) EASI score.gtoreq.16 at the
screening and baseline visits; (4) IGA score.gtoreq.3 (on the 0-4
IGA scale) at the screening and baseline visits; (5) .gtoreq.10%
body surface area (BSA) of AD involvement at the screening and
baseline visits; (6) Patients with documented recent history
(within 3 months before the screening visit) of inadequate response
to outpatient treatment with topical medications, or for whom
topical treatments are otherwise inadvisable (eg, because of
important side effects or safety risks)*; (7) Patients must have
applied a stable dose of an additive-free, basic bland emollient
twice daily for at least 7 days before the baseline visit; (8)
Willing and able to comply with all clinic visits and study-related
procedures; (9) Able to understand and complete study-related
questionnaires; and (10) Provide signed informed consent. *NOTE:
For the purpose of this protocol, inadequate response represented
failure to achieve and maintain remission or a low disease activity
state (eg, IGA 0=clear to 2=mild) despite treatment with topical
corticosteroids of medium to high potency (.+-.topical calcineurin
inhibitors as appropriate), applied daily for at least 28 days or
for the maximum duration recommended by the product prescribing
information (eg, 14 days for super-potent topical corticosteroids),
whichever is shorter. Important side effects or safety risks are
those that outweigh the potential treatment benefits (eg,
hypersensitivity reactions, significant skin atrophy, systemic
effects, etc., or imminence thereof), as assessed by the
investigator or by patient's treating physician.
[0416] A patient who met any of the following criteria was
ineligible to participate in this study: (1) Prior treatment with
mAb1; (2) Treatment with an investigational drug within 8 weeks or
within 5 half-lives (if known), whichever is longer, before the
baseline visit; (3) The following treatments within 4 weeks before
the baseline visit, or any condition that will likely require such
treatment(s) during the first 4 weeks of study treatment: systemic
corticosteroids, immunosuppressive/immunomodulating drugs (eg,
cyclosporine, mycophenolate-mofetil, IFN-.gamma., azathioprine or
methotrexate), or phototherapy for AD; (4) Treatment with topical
corticosteroids, tacrolimus, and/or pimecrolimus within 1 week
before the baseline visit; (5) Treatment with biologics as follows:
any cell-depleting agents including but not limited to rituximab:
within 6 months before the baseline visit, or until lymphocyte and
CD 19+ lymphocyte count returns to normal, whichever is longer,
infliximab, adalimumab, golimumab, certolizumab pegol, abatacept,
etanercept, anakinra: within 16 weeks before the baseline visit for
any indication, or within 5 years for dermatological indications,
or other biologics: within 5 half-lives (if known) or 16 weeks,
whichever is longer; (6) Treatment of AD with prescription
moisturizers classified as medical device (eg, Atopiclair.RTM.,
MimyX.RTM., Epicerum.RTM., Cerave.RTM., etc.) within 1 week before
the baseline visit; (7) Regular use (more than 2 visits per week)
of a tanning booth/parlor within 4 weeks before the baseline visit;
(8) Planned or anticipated use of any prohibited medications and
procedures (including, but not limited to, topical tacrolimus and
pimecrolimus; corticosteroids; prescription moisturizers classified
as medical devices such as Atopiclair.RTM., MimyX.RTM.,
Epicerum.RTM., Cerave.RTM., etc; allergen immunotherapy; systemic
treatment for AD with an immunosuppressive/immunomodulating
substance; treatment with a live (attenuated) vaccine or with an
investigational drug (other than mAb1); major elective surgeries)
during study treatment; (9) Treatment with a live (attenuated)
vaccine within 12 weeks before the baseline visit; (10) Chronic or
acute infection requiring treatment with antibiotics, antivirals,
antiparasitics, antiprotozoals, or antifungals within 4 weeks
before the screening visit, or superficial skin infections within 1
week before the screening visit; (11) Known or suspected
immunosuppression, including history of invasive opportunistic
infections (eg, histoplasmosis, listeriosis, coccidioidomycosis,
pneumocystosis, aspergillosis) despite infection resolution, or
otherwise recurrent infections of abnormal frequency, or prolonged
infections suggesting an immune-compromised status, as judged by
the investigator; (12) Known history of human immunodeficiency
virus (HIV) infection or HIV seropositivity at the screening visit;
(13) Positive or indeterminate hepatitis B surface antigen (HBsAg),
hepatitis B core antibody (HBcAb), or hepatitis C antibody at the
screening visit; (14) Elevated transaminases (ALT and/or AST) more
than 3 times the upper limit of normal (>3.times.ULN) at the
screening visit; (15) History of clinical endoparasitosis within 12
months before the baseline visit, other than treated vaginal
trichomoniasis; (16) Presence of skin comorbidities that may
interfere with study assessments; (17) History of malignancy within
5 years before the baseline visit, except completely treated in
situ carcinoma of the cervix, completely excised non-metastatic
squamous or basal cell carcinoma of the skin; (18) History of
non-malignant lymphoproliferative disorders; (19) High risk of
parasite infection, such as residence within or recent travel
(within 12 months before the baseline visit) to areas endemic for
endoparasitoses, where the circumstances are consistent with
parasite exposure (eg, extended stay, rural or slum areas, lack of
running water, consumption of uncooked, undercooked, or otherwise
potentially contaminated food, close contact with carriers and
vectors, etc.), unless subsequent medical assessments (eg, stool
exam, blood tests, etc.) have ruled out the possibility of parasite
infection/infestation; (20) History of alcohol or drug abuse within
2 years before the screening visit; (21) Severe concomitant
illness(es) that, in the investigator's judgment, would adversely
affect the patient's participation in the study. Examples include,
but are not limited to patients with short life expectancy,
patients with uncontrolled diabetes (HbA1c.gtoreq.9%), patients
with cardiovascular conditions (eg, stage III or IV cardiac failure
according to the New York Heart Association classification), severe
renal conditions (eg, patients on dialysis) hepato-biliary
conditions (eg, Child-Puig class B or C), neurological conditions
(eg, demyelinating diseases), active major autoimmune diseases (eg,
lupus, inflammatory bowel disease, rheumatoid arthritis, etc.),
other severe endocrinological, gastrointestinal, metabolic,
pulmonary, or lymphatic diseases. The specific justification for
patients excluded under this criterion will be noted in study
documents (chart notes, case report forms [CRF], etc); (22) Any
other medical or psychological condition including relevant
laboratory abnormalities at screening that, in the opinion of the
investigator, suggest a new and/or insufficiently understood
disease, may present an unreasonable risk to the study patient as a
result of his/her participation in this clinical trial, may make
patient's participation unreliable, or may interfere with study
assessments. The specific justification for patients excluded under
this criterion will be noted in study documents (chart notes, CRF,
etc.); (23) Planned major surgical procedure during the patient's
participation in this study; (24) Patient is a member of the
investigational team or his/her immediate family; (25) Pregnant or
breast-feeding women; and (26) Unwilling to use adequate birth
control, if of reproductive potential and sexually active. Adequate
birth control is defined as agreement to consistently practice an
effective and accepted method of contraception throughout the
duration of the study and for 16 weeks after last dose of study
drug.
B. Safety
[0417] Safety was assessed throughout the study by monitoring
Adverse Events and Serious Adverse Events.
[0418] An Adverse Event (AE) is any untoward medical occurrence in
a subject or clinical investigation subject administered a
pharmaceutical product. An AE can, therefore, be any unfavorable
and unintended sign (including abnormal laboratory finding),
symptom, or disease temporally associated with the use of a
medicinal product, whether or not considered related to the
medicinal (investigational) product. AEs also include: any
worsening (i.e., any clinically significant change in frequency
and/or intensity) of a pre-existing condition that is temporally
associated with the use of the study drug; abnormal laboratory
findings considered by the Investigator to be clinically
significant; and any untoward medical occurrence.
[0419] A Serious Adverse Event (SAE) is any untoward medical
occurrence that at any dose results in death; is life-threatening;
requires in-patient hospitalization or prolongation of existing
hospitalization; results in persistent or significant
disability/incapacity; is a congenital anomaly/birth defect; or is
an important medical event.
[0420] In addition, laboratory safety variables, vital sign
variables, 12-lead electrocardiography (ECG) variables, and
physical examination variables were measured throughout the
study.
[0421] The clinical laboratory data consists of hematology, blood
chemistry and urinalysis. Blood samples for hematology testing were
collected at every study visit; blood samples for serum chemistry
testing and urine samples for urinalysis were collected to measure
overall patient health at screening, day 1/baseline (pre-dose), day
15, day 29, day 43, day 57, day 71, day 85, day 99, day 113, day
141, day 169, and day 197 (end-of-study) or early termination if
subject is discontinued from the study.
[0422] Vital sign parameters include respiratory rate (bpm), pulse
rate (bpm), systolic and diastolic blood pressure (mmHg) and body
temperature (.degree. C.). Vital signs were collected (pre-dose, on
dosing days) at screening and day 1/baseline, and days 4, 8, 15,
22, 25, 29, 43, 64, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197
and 211 (end of study) or early termination. Vital signs were taken
at 1 and 2 hours post-injection following the study drug dose on
days 1, 8, 15, and 22.
[0423] 12-Lead ECG parameters include: Ventricular HR, PR interval,
QRS interval, corrected QT interval (QTcF=QT/[RR.sup.0.33] and
QTcB=QT/[RR.sup.0.5]) ECG status: normal, abnormal not clinical
significant or abnormal clinical significant. A standard 12-lead
ECG was performed at screening, day 29, and day 113 (end of
treatment) or early termination.
[0424] A thorough and complete physical examination was performed
at screening, day 29, and day 113 (end of treatment) or early
termination.
C. Efficacy Variables
[0425] The efficacy variables IGA, BSA, EASI, SCORAD, 5-D Pruritus
scale, and Pruritus NRS rating have been described elsewhere herein
(see, e.g., Example 7).
[0426] The IGA, BSA, EASI and SCORAD scores were assessed at every
clinic visit. Patients underwent 5-D pruritus assessment at the
following visits: screening, day 1/baseline (pre-dose), day 113
(end of treatment), and day 211 (end of study) or early
termination. Patients used the IVRS to record their Pruritus NRS
score twice daily through the last study visit.
[0427] In addition, other variables such as Global Individual Signs
Score (GISS), Pruritus Categorical Scale, Patient Oriented Eczema
Measure (POEM), Dermatology Life Quality Index (DLQI), Itchy QOL,
EQ-50, HADS, and Patient Global Assessment of Disease Status and
Treatment Effect were also assessed.
[0428] Baseline for efficacy variable was defined as the last
non-missing value on or before the date of randomization. For the
patient who had no value on or before his/her randomization date,
the last non-missing value on or before the date of first dose
injection was used as baseline.
Example 10
Repeat-Dose Clinical Trial of Subcutaneously Administered
Anti-IL-4R Antibody (mAb1) in Adult Patients with
Moderate-to-Severe Atopic Dermatitis
A. Study Design
[0429] This study was a 28-week randomized, double-blind,
placebo-controlled study of the anti-IL-4R mAb, referred herein as
"mAb1", administered subcutaneously in patients with
moderate-to-severe atopic dermatitis. The treatment period was 12
weeks in duration with the patients followed for a further 16 weeks
after end of the treatment.
[0430] 109 patients were included and randomized in the ratio of
1:1 for the study (54 in placebo and 55 for 300 mg of the
antibody). 43 patients (30 in placebo and 13 in 300 mg group)
withdrew from the study. Randomization was stratified according to
IgE levels (IgE<150 kU/L vs. .gtoreq.150 kU/L at the screening
visit) to test the efficacy of mAb1 in patients with extrinsic or
intrinsic form of AD. Patients who met eligibility criteria
underwent day 1/baseline assessments, randomization, and then
received 300 mg of mAb1 or placebo SC. Each weekly dose of study
drug was given as one 2-mL injection, or was split into two 1-mL
injections. Patients returned for weekly clinic visits and received
an injection of study drug on days 8, 15, 22, 29, 36, 43, 50, 57,
64, 71, and 78. Patients were closely monitored at the study site
for a minimum of 2 hours after each dose of study drug. The end of
the treatment period was day 85. Follow-up visits occurred on days
92, 99, 106, 113, 120, 127, 134, 141, 148, 155, 162, 169, 176, 183,
190, and the end of study visit on day 197.
[0431] Inclusion criteria for the study were as follows: (1) Male
or female 18 years or older; (2) Chronic AD, diagnosed by the
Eichenfield revised criteria of Hannifin and Rajka, that has been
present for at least 3 years before the screening visit; (3) EASI
score.gtoreq.16 at the screening and baseline visits; (4) IGA
score.gtoreq.3 at the screening and baseline visits; (5)
.gtoreq.10% BSA of AD involvement at the screening and baseline
visits; (6) history of inadequate response to a stable 1 month)
regimen of topical corticosteroids or calcineurin inhibitors as
treatment for AD within the last 3 months before the screening
visit; (7) Patients must have applied a stable dose of an
additive-free, basic bland emollient twice-daily for at least 7
days before the baseline visit; and (8) Willingness, commitment,
and ability to return for all clinic visits and complete all
study-related procedures and willing and able to sign the informed
consent form (ICF).
[0432] Exclusion criteria for the study were as follows: (1) Prior
treatment with mAb1; (2) Presence of any of the following
laboratory abnormalities at the screening visit: white blood cell
count<3.5.times.10.sup.3/.mu.L; platelet
count<125.times.10.sup.3/.mu.L; neutrophil
count<1.75.times.10.sup.3/.mu.L; aspartate aminotransferase
(AST)/alanine aminotransferase (ALT)>1.5.times. the ULN; and
CPK>2.times. the ULN; (3) Positive or indeterminate results at
the screening visit for hepatitis B surface antigen, hepatitis B
core antibody or hepatitis C antibody; (4) Onset of a new exercise
routine or major change to a previous exercise routine within 4
weeks prior to screening (visit 1). Subjects had to be willing to
maintain a similar level of exercise for the duration of the study
and to refrain from unusually strenuous exercise for the duration
of the trial; (5) Treatment with an investigational drug within 8
weeks or within 5 half-lives, if known, whichever is longer, before
the baseline visit; (6) Treatment with a live (attenuated) vaccine
within 12 weeks before the baseline visit; (7) Treatment with
allergen immunotherapy within 6 months before the baseline visit;
(8) Treatment with leukotriene inhibitors within 4 weeks before the
baseline visit; (9) Treatment with systemic corticosteroids within
4 weeks before the baseline visit; (10) Treatment with topical
corticosteroids, tacrolimus, and/or pimecrolimus within 1 week
before the baseline visit; (11) Systemic treatment for AD with an
immunosuppressive/immunomodulating substance, eg. Cyclosporine,
mycophenolate-mofetil, IFN-.gamma., phototherapy, (narrow band uvB,
uvB, uvA1, psoralen+uvA), azathioprine, methotrexate, or biologics,
within 4 weeks before the baseline visit; (12) three or more bleach
baths during any week within the 4 weeks before the baseline visit;
(13) Treatment of AD with a medical device (eg. Atopiclair.RTM.,
MimyX.RTM., Epicerum.RTM., Cerave.RTM., etc) within 1 week before
the baseline visit; (14) Chronic or acute infection requiring
treatment with oral or IV antibiotics, antivirals, anti-parasitics,
anti-protozoals, or anti-fungals within 4 weeks before the
screening visit, or superficial skin infections within 1 week
before the screening visit; (15) Known history of HIV infection;
(16) History of hypersensitivity reaction to doxycycline or related
compounds; (17) History of clinical parasite infection, other than
vaginal trichomoniasis; (18) History of malignancy within 5 years
before the baseline visit, with the following exceptions; patients
with a history of completely treated carcinoma in situ of cervix,
and non-metastatic squamous or basal cell carcinoma of the skin are
allowed; (19) Planned surgical procedure during the length of the
patient's participation in the study; (20) Use of a tanning
booth/parlor within 4 weeks before the screening visit; (21)
Significant concomitant illness or history of significant illness
such as psychiatric, cardiac, renal, neurological,
endocrinological, metabolic or lymphatic disease, or any other
illness or condition that would have adversely affected the
subject's participation in this study; (22) Pregnant or
breast-feeding women; and/or (23) Unwilling to use adequate birth
control. Adequate birth control is defined as agreement to
consistently practice an effective and accepted method of
contraception throughout the duration of the study and for 16 weeks
after last dose of study drug. For females, adequate birth control
methods are defined as: hormonal contraceptives, intrauterine
device (IUD), or double barrier contraception (ie,
condom+diaphragm, condom or diaphragm+spermicidal gel or foam). For
males, adequate birth control methods are defined as: double
barrier contraception (ie, condom+diaphragm, condom or
diaphragm+spermicidal gel or foam). For females, menopause is
defined as 24 months without menses; if in question, a
follicle-stimulating hormone of .gtoreq.25 U/mL must be documented.
Hysterectomy, bilateral oophorectomy, or bilateral tubal ligation
must be documented, as applicable.
B. Efficacy Variables
[0433] The primary endpoint was the percent change in EASI score
from baseline to week 12. The secondary endpoints measured in this
study included: (1) proportion of patients who achieved an
investigator's global assessment (IGA) score of 0 or 1 at week 12;
(2) proportion of patients who achieved .gtoreq.50% overall
improvement in EASI score (also called EASI 50) from baseline to
week 12; (3) change in EASI score from baseline to week 12; (4)
change and percent change in IGA score, body surface area
involvement of atopic dermatitis (BSA), eczema area and severity
index (EASI), SCORAD, Pruritus NRS and 5-D pruritus scale from
baseline to week 12; (5) Incidence of TEAEs from baseline through
week 28; (6) change from baseline in eosinophils, TARC,
Phadiatop.TM. results, and total IgE associated with response; (7)
change in QoLIAD from baseline to week 12; (8) proportion of
patients who achieve reduction of IGA score of .gtoreq.2 from
baseline to week 12; (9) proportion of patients who achieve
reduction of IGA score of .gtoreq.3 from baseline to week 12; and
(10) PD response of circulating eosinophils, TARC and total
IgE.
[0434] Baseline for efficacy variable is defined as the last
non-missing value on or before the date of randomization. For the
patient who has no value on or before his/her randomization date
the last non-missing value on or before the date of first dose
injection will be used as baseline.
Investigation Procedures
[0435] The efficacy variables IGA, BSA, EASI, SCORAD, 5-D Pruritus
scale, and Pruritus NRS rating have been described elsewhere herein
(see, e.g., Example 7).
[0436] The IGA, BSA, EASI and SCORAD scores were assessed at every
clinic visit. Patients underwent 5-D pruritus assessment at the
following visits: screening, day 1/baseline (pre-dose), and days
15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183 and 197
(end of study) or early termination. Patients used the IVRS to
record their Pruritus NRS score twice daily through the last study
visit.
[0437] Quality of Life Index for Atopic Dermatitis (QoLIAD):
[0438] The QoLIAD is a 25-item, validated questionnaire used in
clinical practice and clinical trials to assess the impact of AD
disease symptoms and treatment on QoL. The format is a simple
yes/no response to 25 items with a scoring system of 0 to 25; a
high score is indicative of a poor QoL. The questionnaire was
administered at screening and day 1/baseline (pre-dose), and days
29, 57, 85, 99, 113, 127, 141, 155, 169, 183, and 197 (end of
study) or early termination.
C. Investigational Treatment
[0439] mAb1 drug product was supplied as a lyophilized powder in a
5 ml glass vial for SC administration. When delivered SC, the mAb1
drug product was reconstituted with 2.5 ml of sterile water for
injection, yielding a solution containing 150 mg/mL of mAb1. The
dose level of mAb1 tested was 300 mg for SC administration. mAb1 or
placebo was administered as 1 (2 mL) or 2 (1 mL) SC injections in
the clinic on day 1/baseline and days 8, 15, 22, 29, 36, 43, 50,
57, 64, 71, and 78. Although it was preferred that each weekly dose
of study drug be given as one 2-mL injection, each weekly dose
could be split into two 1-mL injections. Subcutaneous injection
sites were alternated between the following sites: back of arms,
abdomen (except the navel or waist area), and upper thighs.
Administration to the extremities was not allowed due to the
possibility of different absorption and bioavailability. If
administration of multiple injections were required on the same
day, each injection was delivered at a different injection site
(e.g., 1 injection administered in the right lower quadrant of the
abdomen and the other in the left lower quadrant of the abdomen).
Subcutaneous injection sites were alternated so that the same sites
were not injected for 2 consecutive weeks.
[0440] Placebo matching mAb1 was prepared in the same formulation
as mAb1, but without addition of antibody.
[0441] Patients were monitored at the study site for a minimum of 2
hours after each dose of study drug.
[0442] In addition, patients were required to apply stable doses of
an additive-free, basic bland emollient twice daily for at least 7
days before the baseline visit and throughout study participation.
Patients reported compliance with background treatment during the
study using the IVRS or IWRS. The system prompted patients to
answer the following question about emollient use: "Did you use a
moisturizer approved by the study doctor on the affected areas of
your skin?"
D. Safety Assessment
[0443] Safety was assessed throughout the study by monitoring
Adverse Events and Serious Adverse Events.
[0444] An Adverse Event (AE) is any untoward medical occurrence in
a subject or clinical investigation subject administered a
pharmaceutical product. An AE can, therefore, be any unfavorable
and unintended sign (including abnormal laboratory finding),
symptom, or disease temporally associated with the use of a
medicinal product, whether or not considered related to the
medicinal (investigational) product. AEs also include: any
worsening (i.e., any clinically significant change in frequency
and/or intensity) of a pre-existing condition that is temporally
associated with the use of the study drug; abnormal laboratory
findings considered by the Investigator to be clinically
significant; and any untoward medical occurrence.
[0445] A Serious Adverse Event (SAE) is any untoward medical
occurrence that at any dose results in death; is life-threatening;
requires in-patient hospitalization or prolongation of existing
hospitalization; results in persistent or significant
disability/incapacity; is a congenital anomaly/birth defect; or is
an important medical event.
[0446] In addition, laboratory safety variables, vital sign
variables, 12-lead electrocardiography (ECG) variables, and
physical examination variables were measured throughout the
study.
[0447] The clinical laboratory data consists of hematology, blood
chemistry and urinalysis. Blood samples for hematology testing were
collected at every study visit; blood samples for serum chemistry
testing and urine samples for urinalysis were collected to measure
overall patient health at screening, day 1/baseline (pre-dose), day
15, day 29, day 43, day 57, day 71, day 85, day 99, day 113, day
141, day 169, and day 197 (end-of study) or early termination if
subject is discontinued from the study.
[0448] Vital sign parameters include respiratory rate (bpm), pulse
rate (bpm), systolic and diastolic blood pressure (mmHg) and body
temperature (.degree. C.). Vital signs were collected (pre-dose, on
dosing days) at screening and day 1/baseline, and days 8, 15, 22,
29, 36, 43, 50, 57, 64, 71, 78, 85, 99, 113, 141, 169 and 197 (end
of study) or early termination. Vital signs were taken at 1 and 2
hours post-injection following the study drug dose on days 1, 8,
15, 22, 29, 36, 43, 50, 57, 64, 71 and 78.
[0449] 12-Lead ECG parameters include: Ventricular HR, PR interval,
QRS interval, corrected QT interval (QTcF=QT/[RR.sup.0.33] and
QTcB=QT/[RR.sup.0.5]) ECG status: normal, abnormal not clinical
significant or abnormal clinical significant. A standard 12-lead
ECG was performed at screening, day 141, and day 197 (end of study)
or early termination.
[0450] Research samples (serum/RNA/plasma) were collected at
screening and day 1/baseline (pre-dose), and days 8, 15, 22, 29,
57, 85, and 197 (end of study) or early termination, and at
unscheduled visits.
[0451] A thorough and complete physical examination was performed
at screening, day 85, and day 197 (end of study) or early
termination.
E. Data Analysis
[0452] 1. Analyses of Exploratory Efficacy Variables
[0453] All categorical variables were analyzed using the Fisher's
Exact test with nominal p-value and confidence intervals reported.
All continuous variables were analyzed by the ANalysis of
COVAriance (ANCOVA) using baseline IgE stratum (<150 kU/L vs.
.gtoreq.150 kU/L at the screening visit). Unless otherwise
specified, assessments of changes from baseline and construction of
confidence intervals for continuous measures were based on an
ANCOVA model which includes treatment as the main factor and
baseline value as covariates. Point estimate and 95% CI of the
difference in adjusted mean change from baseline between two
treatment groups are provided. Missing values will be imputed by
the last observation carried forward (LOCF) approach. In the event
that the model assumptions are not warranted, the Rank-based
analysis of covariates will be used.
[0454] 2. Analysis of Safety Data
[0455] The safety analysis is based on the reported AEs, clinical
laboratory evaluations, vital signs, and 12-lead ECG. Thresholds
for Potentially Clinically Significant Values (PCSV) in laboratory
variables, vital signs and ECG are defined in SAP. The time
interval to detect any event or abnormality is between the infusion
of study medication and end of study. Data collected outside this
interval are excluded from the calculation of descriptive
statistics and identification of abnormalities for laboratory
evaluations, vital signs and ECG.
F. Safety: Results
[0456] mAb1 was generally well-tolerated with a favorable safety
profile. The overall adverse event (AE) profile was characteristic
of a healthy population. No deaths were reported. There were 8
patients with SAEs, of which 1 was in mAb1 group (facial bones
fracture) and 7 were in the placebo group (angina pectoris,
cellulitis, eczema herpeticum, skin bacterial infection, renal
failure, asthmatic crisis, lung disorder and atopic dermatitis).
There were 8 patients with TEAE resulting in discontinuation from
study drug, of which 1 was in the mAb1 group and 7 in the placebo
group. There were 87 patients with at least one TEAE (n=43 [78.2%]
in mAb1 vs. 44 [81.5%] in placebo group). The most frequent TEAEs
were nasopharyngitis infections in subjects dosed with mAb1 (n=22
[40%] vs. 10 [18.5%] for placebo). Other TEAEs in the treatment
group included eye infections, nervous system disorders, and
general disorders and administration site conditions. No other
clinically significant laboratory test results (blood chemistry,
hematology, or urinalysis) were reported during the study. No
trends were seen in mean/median baseline in any laboratory
parameter. There were no significant trends in mean or median
changes from baseline in temperature or pulse throughout the study.
No clinically significant abnormalities were seen on physical
examination results, ECGs or vital signs.
[0457] Subcutaneous administration of mAb1 to adult patients with
moderate-to-severe AD was generally safe and well-tolerated.
G. Efficacy: Results
[0458] The baseline and exploratory efficacy results obtained from
the study are summarized in FIGS. 23-33 and Tables 27-35. As noted
above, patients were treated with 300 mg subcutaneous mAb1 once a
week for 12 weeks, or with placebo.
TABLE-US-00027 TABLE 27 Summary of Baseline Characteristics - all
values represented as Mean (SD) mAb1 All Subjects Placebo 300 mg
Combined Patients 54 55 109 Age (years) Mean (SD) 39.4 (12.29) 33.7
(10.41) 36.5 (11.69) Ethnicity n (%) Hispanic or Latino 1 (1.9%) 3
(5.5%) 4 (3.7%) Not Hispanic or Latino 53 (98.1%) 52 (94.5%) 105
(96.3%) Gender n (%) Male 27 (50.0%) 31 (56.4%) 58 (53.2%) Female
27 (50.0%) 24 (43.6%) 51 (46.8%) Height (cm) Mean (SD) 171.2 (9.89)
173.4 (9.88) 172.3 (9.90) Weight (kg) Mean (SD) 72.41 (17.539)
78.13 (17.416) 75.30 (17.632) BMI (kg/m.sup.2) Mean (SD) 24.51
(4.639) 25.89 (4.837) 25.20 (4.768) Chronic Atopic Dermatitis 14.4
(18.35) 6.6 (10.53) 10.5 (15.37) Diagnosis Age BSA 50.8 (24.14)
46.8 (24.55) 48.8 (24.32) EASI Score 30.8 (13.63) 28.4 (13.57) 29.6
(13.59) IGA Score 4.0 (0.69) 3.9 (0.67) 3.9 (0.68) NRS Score 5.8
(1.93) 6.1 (1.34) 5.9 (1.66) SCORAD Score 69.1 (13.38) 66.7 (13.82)
67.9 (13.59) Pruritus 5-D Scale 18.7 (3.50) 18.4 (3.04) 18.5
(3.26)
TABLE-US-00028 TABLE 28 Summary of Percentage and Absolute Change
in EASI Score from Baseline to Week 12 and Each Visit during
Follow-up period - all values represented as Mean (SD) Placebo 300
mg mAb1 No. Patients 54 55 Baseline EASI Score 30.8 (13.63) 28.4
(13.57) Day 85 EASI Score 24.4 (19.01) 8.5 (12.15) % Change from
Baseline to Day 85 -23.3 (49.26) -74.0 (26.94) Absolute change from
Baseline to -6.4 (14.85) -19.9 (11.52) Day 85 Day 99 EASI Score
24.2 (19.15) 8.4 (11.86) % Change from Baseline to Day 99 -23.2
(49.42) -73.5 (27.21) Absolute change from Baseline to -6.6 (15.20)
-20.0 (12.24) Day 99 Day 113 EASI Score 24.1 (18.80) 9.1 (12.13) %
Change from Baseline to Day 113 -23.4 (47.75) -71.4 (27.03)
Absolute change from Baseline to -6.7 (14.96) -19.4 (11.42) Day 113
Day 127 EASI Score 24.5 (18.91) 9.2 (12.41) % Change from Baseline
to Day 127 -22.1 (47.11) -71.2 (27.39) Absolute change from
Baseline to -6.3 (14.98) -19.2 (11.15) Day 127 Day 141 EASI Score
23.8 (18.47) 9.4 (12.18) % Change from Baseline to Day 141 -23.9
(47.01) -70.8 (26.91) Absolute change from Baseline to -7.0 (14.77)
-19.0 (10.86) Day 141 Day 155 EASI Score 24.0 (18.27) 9.9 (12.40) %
Change from Baseline to Day 155 -23.0 (46.22) -68.8 (27.35)
Absolute change from Baseline to -6.7 (14.49) -18.5 (10.74) Day 155
Day 169 EASI Score 23.5 (18.22) 11.0 (12.76) % Change from Baseline
to Day 169 -24.2 (46.66) -64.4 (29.19) Absolute change from
Baseline to -7.3 (14.93) -17.5 (10.82) Day 169 Day 183 EASI Score
23.5 (18.57) 10.8 (13.00) % Change from Baseline to Day 183 -24.6
(47.35) -65.0 (29.21) Absolute change from Baseline to -7.3 (15.12)
-17.6 (10.93) Day 183 Day 197 EASI Score 23.4 (18.59) 11.0 (13.13)
% Change from Baseline to Day 197 -25.0 (48.57) -64.0 (30.80)
Absolute change from Baseline to -7.4 (15.23) -17.4 (11.88) Day
197
TABLE-US-00029 TABLE 29 Summary of Percentage and Absolute Change
in IGA Score from Baseline to Week 12 and Each Visit during
Follow-up period - all values represented as Mean (SD) Placebo 300
mg mAb1 No. Patients 54 55 Baseline IGA Score 4.0 (0.69) 3.9 (0.67)
Day 85 IGA Score 3.4 (1.19) 2.0 (1.15) % Change from Baseline to
-14.7 (27.37) -49.5 (25.94) Day 85 Absolute change from Baseline to
-0.6 (1.07) -1.9 (0.98) Day 85 Day 99 IGA Score 3.4 (1.16) 2.1
(1.17) % Change from Baseline to -14.0 (27.03) -45.8 (26.98) Day 99
Absolute change from Baseline to -0.6 (1.06) -1.7 (1.06) Day 99 Day
113 IGA Score 3.3 (1.20) 2.2 (1.08) % Change from Baseline to Day
113 -15.9 (27.82) -43.1 (25.53) Absolute change from Baseline to
-0.6 (1.12) -1.7 (1.06) Day 113 Day 127 IGA Score 3.4 (1.16) 2.2
(1.16) % Change from Baseline to Day 127 -14.5 (26.66) -44.1
(27.06) Absolute change from Baseline to -0.6 (1.07) -1.7 (1.07)
Day 127 Day 141 IGA Score 3.4 (1.15) 2.2 (1.12) % Change from
Baseline to Day 141 -15.0 (26.52) -42.8 (26.01) Absolute change
from Baseline to -0.6 (1.05) -1.6 (1.01) Day 141 Day 155 IGA Score
3.4 (1.14) 2.3 (1.08) % Change from Baseline to Day 155 -14.2
(25.89) -41.5 (25.20) Absolute change from Baseline to -0.6 (1.02)
-1.6 (1.01) Day 155 Day 169 IGA Score 3.3 (1.17) 2.5 (1.07) %
Change from Baseline to Day 169 -15.9 (26.96) -36.0 (25.87)
Absolute change from Baseline to -0.6 (1.08) -1.4 (1.03) Day 169
Day 183 IGA Score 3.3 (1.18) 2.4 (1.10) % Change from Baseline to
Day 183 -16.3 (27.33) -37.2 (26.93) Absolute change from Baseline
to -0.7 (1.10) -1.5 (1.09) Day 183 Day 197 IGA Score 3.3 (1.29) 2.3
(1.09) % Change from Baseline to Day 197 -16.5 (30.18) -39.0
(27.42) Absolute change from Baseline to -0.7 (1.20) -1.5 (1.10)
Day 197
TABLE-US-00030 TABLE 30 Summary of Absolute Change in BSA Score
from Baseline to Week 12 and Each Visit during Follow-up period -
all values represented as Mean (SD) Placebo 300 mg mAb1 No.
Patients 54 55 Baseline BSA Score 50.8 (24.13) 46.8 (24.55) Day 85
BSA Score 41.8 (30.44) 19.4 (23.43) Absolute change from Baseline
to -9.0 (21.07) -27.4 (22.81) Day 85 Day 99 BSA Score 41.7 (30.85)
19.9 (22.85) Absolute change from Baseline to -9.2 (21.85) -26.9
(22.74) Day 99 Day 113 BSA Score 41.3 (30.52) 20.8 (23.16) Absolute
change from Baseline to -9.5 (21.34) -26.0 (21.90) Day 113 Day 127
BSA Score 42.1 (30.41) 21.4 (23.48) Absolute change from Baseline
to -8.7 (20.72) -25.4 (21.29) Day 127 Day 141 BSA Score 41.5
(29.85) 21.3 (22.88) Absolute change from Baseline to -9.4 (20.57)
-25.5 (21.50) Day 141 Day 155 BSA Score 41.5 (29.61) 22.1 (23.05)
Absolute change from Baseline to -9.3 (20.26) -24.6 (21.55) Day 155
Day 169 BSA Score 41.2 (29.28) 24.6 (24.15) Absolute change from
Baseline to -9.6 (20.35) -22.2 (21.50) Day 169 Day 183 BSA Score
41.0 (30.28) 24.1 (24.15) Absolute change from Baseline to -9.9
(21.35) -22.7 (22.86) Day 183 Day 197 BSA Score 40.5 (29.95) 24.9
(25.70) Absolute change from Baseline to -10.4 (21.40) -21.9
(24.11) Day 197
TABLE-US-00031 TABLE 31 Summary of Absolute Change in SCORAD Score
from Baseline to Week 12 and Each Visit during Follow-up period -
all values represented as Mean (SD) Placebo 300 mg mAb1 No.
Patients 54 55 Baseline SCORAD Score 69.1 (13.38) 66.7 (13.82) Day
85 SCORAD Score 59.3 (23.44) 31.7 (22.08) Absolute change from
Baseline to -9.8 (20.53) -35.0 (19.43) Day 85 Day 99 SCORAD Score
58.8 (23.35) 32.5 (20.99) Absolute change from Baseline to -10.3
(21.33) -34.3 (18.94) Day 99 Day 113 SCORAD Score 59.1 (22.30) 34.0
(2051) Absolute change from Baseline to -10.0 (20.89) -32.7 (18.48)
Day 113 Day 127 SCORAD Score 59.9 (22.36) 34.0 (21.25) Absolute
change from Baseline to 09.2 (20.59) -32.7 (18.23) Day 127 Day 141
SCORAD Score 59.0 (21.85) 33.9 (20.51) Absolute change from
Baseline to -10.1 (20.12) -32.8 (17.97) Day 141 Day 155 SCORAD
Score 59.0 (22.50) 35.1 (20.16) Absolute change from Baseline to
-10.0 (20.17) -31.6 (17.99) Day 155 Day 169 SCORAD Score 58.5
(22.33) 37.1 (20.82) Absolute change from Baseline to -10.6 (20.90)
-29.6 (19.15) Day 169 Day 183 SCORAD Score 58.7 (22.47) 37.5
(20.89) Absolute change from Baseline to -10.4 (20.86) -29.2
(19.50) Day 183 Day 197 SCORAD Score 57.8 (23.82) 38.8 (22.04)
Absolute change from Baseline to -11.3 (22.05) -27.9 (21.70) Day
197
TABLE-US-00032 TABLE 32 Summary of Absolute Change in 5-D Pruritus
Scale from Baseline to Week 12 and Each Week during Follow-up
period - all values represented as Mean (SD) Placebo 300 mg mAb1
No. Patients 54 55 Baseline 5-D Pruritus Score 18.7 (3.50) 18.4
(3.04) Day 85 5-D Pruritus Score 16.9 (5.33) 11.0 (4.22) Absolute
change from Baseline to -1.9 (4.28) -7.4 (4.33) Day 85 Day 99 5-D
Pruritus Score 16.7 (5.28) 11.3 (3.96) Absolute change from
Baseline to -2.0 (4.63) -7.0 (4.41) Day 99 Day 113 5-D Pruritus
Score 16.5 (5.57) 11.7 (4.05) Absolute change from Baseline to -2.2
(4.91) -6.7 (4.21) Day 113 Day 127 5-D Pruritus Score 16.7 (5.44)
11.5 (4.07) Absolute change from Baseline to -2.0 (4.72) -6.9
(4.24) Day 127 Day 141 5-D Pruritus Score 16.4 (5.67) 11.8 (4.19)
Absolute change from Baseline to -2.3 (5.12) -6.6 (4.56) Day 141
Day 155 5-D Pruritus Score 16.6 (5.53) 12.0 (4.21) Absolute change
from Baseline to -2.1 (4.90) -6.4 (4.49) Day 155 Day 169 5-D
Pruritus Score 16.8 (5.35) 12.7 (4.20) Absolute change from
Baseline to -1.9 (4.78) -5.7 (4.58) Day 169 Day 183 5-D Pruritus
Score 16.6 (5.59) 12.8 (4.56) Absolute change from Baseline to -2.1
(5.02) -5.6 (4.90) Day 183 Day 197 5-D Pruritus Score 16.6 (5.50)
13.1 (4.85) Absolute change from Baseline to -2.1 (5.12) -5.3
(5.06) Day 197
TABLE-US-00033 TABLE 33 Summary of Absolute Change in Average NRS
Score from Baseline to Week 12 and Each Week during Follow-up
period - all values represented as Mean (SD) Placebo 300 mg mAb1
No. Patients 54 55 Baseline NRS Score 5.8 (1.93) 6.1 (1.34) Day 85
NRS Score 4.9 (2.53) 2.6 (1.67) Absolute change from Baseline to
-0.9 (2.07) -3.5 (2.00) Day 85 Day 92 NRS Score 4.8 (2.57) 2.8
(1.68) Absolute change from Baseline to -1.0 (2.07) -3.4 (2.12) Day
92 Day 99 NRS Score 4.7 (2.54) 2.7 (1.72) Absolute change from
Baseline to -1.0 (2.06) -3.4 (2.17) Day 99 Day 106 NRS Score 4.8
(2.59) 2.7 (1.63) Absolute change from Baseline to -1.0 (2.15) -3.4
(2.08) Day 106 Day 113 NRS Score 4.9 (2.69) 2.7 (1.63) Absolute
change from Baseline to -0.9 (2.21) -3.4 (2.00) Day 113 Day 120 NRS
Score 4.8 (2.61) 2.7 (1.68) Absolute change from Baseline to -1.0
(2.18) -3.4 (2.07) Day 120 Day 127 NRS Score 4.8 (2.68) 2.8 (1.79)
Absolute change from Baseline to -1.0 (2.24) -3.3 (2.20) Day 127
Day 134 NRS Score 4.7 (2.75) 2.8 (1.78) Absolute change from
Baseline to -1.1 (2.24) -3.3 (2.18) Day 134 Day 141 NRS Score 4.7
(2.73) 2.9 (1.89) Absolute change from Baseline to -1.1 (2.26) -3.2
(2.28) Day 141 Day 148 NRS Score 4.7 (2.75) 2.9 (1.89) Absolute
change from Baseline to -1.1 (2.28) -3.2 (2.28) Day 148 Day 155 NRS
Score 4.7 (2.75) 2.9 (1.86) Absolute change from Baseline to -1.1
(2.30) -3.2 (2.19) Day 155 Day 162 NRS Score 4.7 (2.75) 3.0 (1.93)
Absolute change from Baseline to -1.1 (2.29) -3.1 (2.28) Day 162
Day 169 NRS Score 4.7 (2.75) 3.2 (1.99) Absolute change from
Baseline to -1.1 (2.28) -3.0 (2.43) Day 169 Day 176 NRS Score 4.7
(2.74) 3.2 (2.01) Absolute change from Baseline to -1.1 (2.27) -3.0
(2.49) Day 176 Day 183 NRS Score 4.7 (2.75) 3.1 (1.97) Absolute
change from Baseline to -1.1 (2.28) -3.0 (2.41) Day 183 Day 190 NRS
Score 4.7 (2.78) 3.1 (1.91) Absolute change from Baseline to -1.1
(2.31) -3.1 (2.25) Day 190 Day 197 NRS Score 4.7 (2.75) 3.1 (1.95)
Absolute change from Baseline to -1.1 (2.28) -3.0 (2.28) Day
197
TABLE-US-00034 TABLE 34 Summary of Subjects achieving an IGA score
of 0 or 1 to Week 12 and each visit during Follow-up period Number
and proportion of subjects achieving an IGA Placebo 300 mg mAb1
score of 0 or 1 (N = 54) (N = 55) Week 12, Day 85 4 (7.4%) 22
(40.0%) Week 14, Day 99 4 (7.4%) 22 (40.0%) Week 16, Day 113 5
(9.3%) 18 (32.7%) Week 18, Day 127 3 (5.6%) 20 (36.4%) Week 20, Day
141 4 (7.4%) 17 (30.9%) Week 22, Day 155 3 (5.6%) 17 (30.9%) Week
24, Day 169 3 (5.6%) 13 (23.6%) Week 26, Day 183 3 (5.6%) 15
(27.3%) Week 28, Day 197 6 (11.1%) 16 (29.1%)
TABLE-US-00035 TABLE 35 Summary of Subjects achieving an EASI 50
Week 12 and each visit during Follow-up period Number and
proportion of subjects achieving an EASI score percent Placebo 300
mg mAb1 decrease of 50% (N = 54) (N = 55) Week 12, Day 85 19
(35.2%) 47 (85.5%) Week 14, Day 99 19 (35.2%) 46 (83.6%) Week 16,
Day 113 18 (33.3%) 46 (83.6%) Week 18, Day 127 18 (33.3%) 45
(81.8%) Week 20, Day 141 18 (33.3%) 46 (83.6%) Week 22, Day 155 16
(29.6%) 43 (78.2%) Week 24, Day 169 18 (33.3%) 40 (72.7%) Week 26,
Day 183 19 (35.2%) 41 (74.5%) Week 28, Day 197 23 (42.6%) 40
(72.7%)
H. Conclusions
[0459] Subcutaneous administration of an anti-IL-4R antibody (mAb1)
to adult patients with moderate-to-severe atopic dermatitis was
generally safe and well tolerated after 12 weekly doses of 300 mg.
Administration of mAb1 at 300 mg resulted in significant
improvement in IGA, EASI, BSA, SCORAD and NRS pruritus through day
85 in both mean and absolute and percent change, as compared to
baseline (see Tables 27-33). The proportion of patients achieving
an IGA score of 0 or 1 at Day 85 for the 300 mg group was 40.0%,
while the same number for placebo was 7.4% (Table 34). At Day 85,
the proportion of patients who achieved an EASI score percent
decrease of 50% ("EASI-50") was 85.5% for the 300 mg group, whereas
the EASI-50 for placebo-treated patients at Day 85 was 35.2% (Table
35). The percent change in EASI score from baseline to week 12 of
mAb1 was statistically significant from placebo group (-74.0% vs.
-23.0%, p-value<0.0001). The treatment group was statistically
significantly different from placebo group in all of the secondary
efficacy endpoints. The following were the p-values for: IGA
responder (0 or 1) (<0.0001), EASI responder (<0.0001), EASI
absolute change from baseline (<0.0001), absolute change of IGA
from baseline (<0.0001), percent change of IGA from baseline
(<0.0001), absolute change in BSA (<0.0001), absolute change
in SCORAD (<0.0001), absolute change in Pruritus NRS
(<0.0001), and absolute change in 5-D pruritus scale from
baseline to week 12 (<0.0001) respectively.
Example 11
Clinical Trial to Assess the Safety of mAb1 Administered
Concomitantly with Topical Corticosteroids to Patients with
Moderate-to-Severe AD
A. Study Design
[0460] This study was a randomized, double-blind, parallel-group,
placebo-controlled study to assess the safety and explore the
efficacy of repeated subcutaneous doses of mAb1 administered
concomitantly with topical corticosteroids (TCS) to treat AD in
patients with moderate-to-severe AD. Patients were randomized 2:1
to receive 300 mg mAb1 or placebo once weekly for 4 consecutive
weeks via subcutaneous injections (on days 1, 8, 15, and 22). All
patients received concomitant open-label, daily treatment for up to
28 days with a potent TCS product (50-100 times as potent as
hydrocortisone), such as methylprednisolone aceponate 0.1%,
mometasone furoate 0.1%, or betamethasone valerate 0.1%. Other
topical medications, such as a lower potency TCS or topical
calcineurin inhibitors (TCI), were used to treat AD lesions located
on the face, flexural and genital areas.
[0461] Starting with the screening visit, patients began applying
an additive-free, basic bland emollient twice daily for at least 7
days before the baseline visit and continued its use throughout the
study (once daily on treatment days in areas of TCS application).
Environmental control measures and non-pharmacologic treatment
modalities such as allergen avoidance and bleach baths were allowed
at the discretion of the investigator.
[0462] Screening occurred between day -21 and -1. Patients received
their first drug injection (300 mg mAb1, or placebo) on day 1 and
returned to the clinic for additional drug injections on days 8, 15
and 22 (+/-1 day) for a total of 4 weekly doses. Starting on day 1,
patients applied the topical medication(s) described above once
daily in the evening, and continued the applications to all
AD-affected areas (i.e., areas with active AD lesions) until
control was achieved for up to 28 days. After control was achieved,
TCS application to AD-prone areas without active lesions (i.e.,
areas from which lesions have cleared) was limited to 2 days every
week until study day 28. After day 28, topical treatment of any
residual AD lesions continued as needed. Throughout the study,
patients continued to apply an additive-free, basic bland
emollient, twice daily (once daily on topical treatment days in
areas treated with topical medication). Patients returned for
clinic visits on days 29, 36, 50, 64 and 78 (end of study).
[0463] The inclusion criteria for the study were: (1) male/female
patients aged 18 years or older; (2) chronic AD as diagnosed by the
Eichenfield revised criteria of Hannifin and Rajka, that had been
present for at least 2 years before screening; (3) AD activity as
assessed by IGA score.gtoreq.3 and SCORAD>20 at the screening
and baseline visits, with one or more active AD lesions for which
treatment with potent TCS is indicated; (4) at least 10% BSA
affected by AD at the screening and baseline visits; (5) patients
must be applying an additive-free, basic bland emollient twice
daily for at least 7 days before the baseline visit; (6) willing
and able to comply with clinic visits and study-related procedures;
and (7) able to read and understand, and willing to sign the
consent form.
[0464] The exclusion criteria for the study were: (1) prior
treatment with mAb1; (2) hypersensitivity to corticosteroids or to
any other ingredients contained in the TCS product used during the
study; (3) AD lesions located predominantly (.gtoreq.50% of the
cumulative lesional area) on face, flexural and genital areas; (4)
presence of skin comorbidities that may interfere with study
assessments; (5) the following treatments within 4 weeks before the
baseline visit or any conditions that may require such treatment(s)
during the study: systemic corticosteroids, immunosuppressive or
immunomodulating drugs, eg, cyclosporine, mycophenolate-mofetil,
IFN-gamma, azathioprine or methotrexate; (6) treatment with
biologics as follows: (a) any cell-depleting agents, including but
not limited to, rituximab; within 6 months prior to the baseline
visit, or until lymphocyte and CD19+ lymphocyte count return to
normal, whichever is larger, (b) infliximab, adalimumab, golimumab,
certolizumab pegol, abatacept, etanercept, anakinra: within 8 weeks
prior to the baseline visit, and 9c) other biologics: within 5
half-lives (if known) or 8 weeks, whichever is longer; (7) any
phototherapy for skin disease (such as narrow band UVB, UVB, UVA1,
psolaren+UVA within 4 weeks before baseline; (8) regular use (more
than 2 visits per week) of a tanning booth/parlor within 4 weeks
before the baseline visit; (9) treatment with a live attenuated
vaccine within 12 weeks before the baseline visit; (10) treatment
with an investigational drug within 8 weeks or within 5 half-lives,
whichever is longer, before the baseline visit; (11) chronic or
acute infection requiring treatment with oral or IV antibiotics,
antivirals, antiparasitics, antiprotozoals, or antifungals within 4
weeks before the screening, or superficial skin infections within 1
week before the screening visit; (12) history of invasive
opportunistic infections such as histoplasmosis, listeriosis,
coccidioidomycosis, candidiasis, pneumocystis jiroveci,
aspergillosis, despite resolution, JC virus (progressive multifocal
leukoencephalopathy; (13) known history of HIV infection; (14)
positive or indeterminate hepatitis B surface antigen, hepatitis B
core antibody, or hepatitis C antibody at the screening visit; (15)
presence of any of the following laboratory abnormalities at the
screening visit: creatine phosphokinase>2.times. upper limit
normal (ULN); aspartate aminotransferase (AST) and/or alanine
aminotransferase (ALT)>2.times.ULN; neutrophil
count<1.75.times.10.sup.3/ul; platelet
count<100.times.10.sup.3/ul; (16) onset of a new exercise
routine or major change to a previous exercise routine within 2
weeks before randomization, or unwillingness to maintain (without
increase) the current level of physical activity throughout the
length of participation in the study; (17) history of a
hypersensitivity reaction to doxycycline or other tetracyclines;
(18) history of a clinical endoparasite infection within 12 months
of the baseline visit, other than treated vaginal trichomoniasis;
(19) history of malignancy within 5 years before the baseline
visit, except completely treated in situ carcinoma of the cervix,
completely excised non-metastatic squamous or basal cell carcinoma
of the skin; (20) history of non-malignant lymphoproliferative
disorders; (21) pregnant or breast-feeding women; (22) men or women
of child-bearing potential who are unwilling to practice
contraceptive measures; (23) history of alcohol or drug abuse
within 2 years of the screening visit; (24) recent travel (within
12 months of randomization) to areas endemic for parasitic
infections, such as developing countries in Africa or the
tropical/subtropical regions of Asia; (25) prior or current history
of significant concomitant illness(es) that would adversely affect
the patient's participation in the study, e.g., stage III or IV
cardiac failure, severe renal, neurological, endocrinological, GI,
hepato-biliary, metabolic, pulmonary or lymphatic disease; and (26)
any other conditions that may present an unreasonable risk to the
study patient, or may make the patient's participation unreliable,
or may interfere with study assessments.
[0465] The primary endpoint of the study was the incidence and
severity of adverse events. The secondary endpoints were
exploratory in nature and included: (1) EASI50 index--Binary
response variable of whether or not .gtoreq.50% reduction in EASI
is achieved from baseline to day 29, and other post-baseline
observation time points; (2) achieving IGA scores of .ltoreq.1
(clear or almost clear) at day 29, and at other post-baseline
observation time points; (3) time to IGA.ltoreq.1 and to EASI50;
(4) changes in IGA, EASI, and SCORAD scores from baseline to day
29, and to other post-baseline observation time points; and (5)
proportion of patients with IGA.ltoreq.1 at week 4 who remain
relapse-free through the end of the observation period.
B. Efficacy Variables
[0466] The efficacy variables IGA, BSA, EASI, SCORAD, and Pruritus
NRS rating have been described elsewhere herein (see, e.g., Example
7). The IGA, BSA, EASI, pruritus NRS and SCORAD scores were
assessed at every clinic visit.
C. Procedures and Assessments
[0467] Safety was assessed by evaluating the incidence of adverse
events (described elsewhere herein) (AEs) from day 1 to day 78, and
by detailed medical history, thorough physical examination, vital
signs, electrocardiograms (ECGs), and clinical laboratory testing.
Blinded safety data was reviewed on an ongoing basis. Concomitant
medications and procedures were collected from screening to day 78
(end of study) or early termination, if applicable. Safety,
laboratory, and efficacy assessments were performed at each clinic
visit. Blood samples were collected for the determination of
systemic trough concentrations of functional mAb1 at every study
visit prior to treatment starting at baseline (day 1). Blood
samples were collected for the analysis of anti-mAb1 antibody
levels at predetermined time points. Research samples and samples
for exploratory biomarker analysis were also collected. Efficacy of
mAb1 was assessed by the EASI, the IGA, the SCORAD, the Pruritus
numerical rating scale (NRS), and % body surface area (BSA) of AD
involvement. Blood samples were collected for pharmacokinetic (PK)
analyses, and analysis of anti-mAb1 antibody levels at
predetermined time points. Research samples and samples for
exploratory biomarker analysis were also collected.
D. Statistical Methods
[0468] As all statistical analyses described in this section were
exploratory in nature, there was no multiplicity adjustment for the
type I error. Each test was at the 5% significance level. All
categorical variables (EASI-50 and IGA responders at each
post-baseline visit, IGA responders at day 29 without later
relapse) were analyzed using the Fisher's Exact test with
calculated nominal p-value from comparison between mAb1 and placebo
groups. The point estimates and confidence intervals of proportions
were presented. The graphs of the proportions over time were
provided. All continuous variables (change or percent change in
IGA, EASI and SCORAD, NRS from baseline to each post-baseline
visit) were analyzed by the ANalysis of COVAriance (ANCOVA). Unless
otherwise specified, assessments of changes from baseline and
construction of confidence intervals for continuous measures were
based on an ANCOVA model which included treatment as the main
factor and baseline value as covariates. The point estimate and 95%
CI of the difference in adjusted mean change from baseline between
two treatment groups was provided. The nominal p-value from
comparison between mAb1 and placebo groups will be provided. In the
event that the model assumptions were not warranted, the Rank-based
analysis of covariates was used. Graphs of mean change from
baseline over time were provided. Time-to-event variables (time to
EASI50 and time to IGA response) were analyzed with a log-rank test
to compare mAb1 with placebo group. Kaplan-Meier survival curves
across two treatment groups were provided. The following analysis
approaches were implemented for this study: (a) Censored LOCF: The
efficacy data was set to missing after prohibited medication was
used or after the patient was discontinued from the study. Then all
missing values were imputed by simple LOCF. (b) Simple Observed
Case (OC) approach: Only observed cases were analyzed.
E. Safety
[0469] Overall, mAb1 was safe and well tolerated in this study. No
deaths were reported. A single serious adverse event (SAE) was
recorded for a patient in the placebo group, who experienced loss
of consciousness, and who withdrew from the study as a result. No
other patients experienced adverse events leading to treatment
discontinuation. A total of 19 out of 31 patients enrolled in the
study reported at least one treatment emergent adverse event
(TEAE)--7 patients (70%) in the placebo group and 12 patients (57%)
in the mAb1 group. By system and organ class (SOC), the most
frequent TEAE reported for mAb1 treatment group were infections and
infestations, 12 patients (57%) vs. 3 patients (30%) for placebo).
The most frequent infection was nasopharyngitis--5 patients (24%)
in the mAb1 group vs. 2 patients (20%) for placebo. There were no
serious or opportunistic infections. Other TEAEs reported in more
than a single patient included nonspecific symptoms such as
headache--3 patients (14%) in the mAb1 group vs. 1 patient (10%) in
the placebo group, somnolence--2 patients (9.5%) in the mab1 group
vs. 0% in the placebo group, oropharyngeal pain--3 patients (14%)
in the mAb1 group vs. 1 patient (10%) in the placebo group, and
cough--2 patients (9.5%) in the mAb1 group vs. 0% in the placebo
group. Most AEs were mild to moderate and generally resolved within
2 weeks. A single severe AE was reported in the mAb1 group:
bacterial bronchitis, with onset on Day 63 (last dose of study drug
on Day 22), which was considered unrelated to the study treatment.
There were no adverse events in the mAb1 group suggestive of
untoward drug-drug (mAb1-TCS) interactions at the skin level. The
analysis of on-treatment potential clinically significant values
(PCSV) for safety laboratory tests, vital signs, and ECC showed
that the rate of PCSVs was generally balanced between the two study
groups, with no systematic distribution or distinct trends,
suggesting that PCSV occurrence was incidental and not related to
the study treatment.
F. Results
[0470] In this study, mAb1 was administered concomitantly with TCS
to patients with moderate to severe AD. Consistent with the current
standard of care in AD, a controlled TCS regimen was required
during the first 4 weeks (i.e., concomitantly with the study
treatment), as described elsewhere herein. Table 36 lists the TCS
medications used by the patients participating in the study.
Patients were to apply TCS to all active lesions, once daily, every
day, until lesion clearance, followed by applications to
lesion-prone areas (from which lesions had cleared) once daily, two
days per week. A potent TCS (Class III) was required to be applied
to at least 50% of lesions. For lesions located on face, skin
folds, or genital areas (where potent TCS are usually not
indicated) lower potency TCS (Class I or II) were allowed. The
amount of TCS used each week was measured by weighing the TCS
containers at the time they were dispensed to patients and upon
their return to the clinic at the next study visit. Tables 37 and
38 summarize the TCS use from day 1 through day 29.
TABLE-US-00036 TABLE 36 TCS medications Placebo 300 mg mAb1 All
patients (N = 10) (N = 21) (N = 31) Subjects with at least one 10
(100%) 21 (100%) 31 (100%) TCS med Corticosteroids, 10 (100%) 21
(100%) 31 (100%) Dermatological Preparations Corticosteroids,
Potent 10 (100%) 21 (100%) 31 (100%) (Group III) Mometasone
(furoate) 6 (60%) 10 (47.6%) 16 (51.6%) Methylprednisolone 6 (60%)
8 (38.1%) 14 (45.2%) (aceponate) Fluticasone propionate 0 4 (19.0%)
4 (12.9%) Corticosteroids, Moderately 0 2 (9.5%) 2 (6.5%) potent
(Group II) Hydrocortisone butyrate 0 2 (9.5%) 2 (6.5%)
Corticosteroids, Weak 2 (20.0%) 1 (4.8%) 3 (9.7%) (Group I)
Hydrocortisone 2 (20.0%) 1 (4.8%) 3 (9.7%)
TABLE-US-00037 TABLE 37 TCS used from Day 1 through Day 29 - all
values in Mean (SD) 300 mg All Placebo (N = 10) mAb1 (N = 21)
patients (N = 31) Total (g) 99.4 (152.49) 48.7 (40.27) 65.0 (92.94)
Visit 3 (week 1) 26.7 (37.53) 14.0 (13.13) 18.1 (23.96) Visit 4
(week 2) 2.5 (40.03) 12.7 (9.96) 16.5 (24.04) Visit 5 (week 3) 26.6
(37.05) 11.5 (12.28) 16.3 (23.75) Visit 6 (week 4) 26.9 (43.61)
11.1 (14.71) 15.6 (26.43)
TABLE-US-00038 TABLE 38 TCS in Class III used from Day 1 through
Day 29 300 mg All Placebo (N = 10) mAb1 (N = 21) patients (N = 31)
Total (g) 93.4 (153.51) 47.2 (39.78) 62.1 (92.77) Visit 3 (week 1)
25.2 (37.83) 13.5 (12.86) 17.3 (23.89) Visit 4 (week 2) 23.0
(40.26) 12.4 (9.76) 15.8 (23.98) Visit 5 (week 3) 25.0 (37.34) 11.0
(12.23) 15.5 (23.71) Visit 6 (week 4) 25.2 (44.00) 11.4 (14.92)
15.5 (26.78)
[0471] The demographic and disease characteristics were for the
most part homogeneous between the two treatment groups (Tables 39
and 40). The mean baseline AD disease severity scores (IGA, EASI,
SCORAD, BSA, and pruritus NRS) were reasonably balanced, as
well.
TABLE-US-00039 TABLE 39 Summary of Demographics 300 mg All Patients
Placebo (N = 10) (N = 21) (N = 31) Mean age, 37.8 (16.73) 36.0
(11.26) 36.6 (13.01) years (SD) Race, % Caucasian 10 (100%) 20
(95.2%) 30 (96.8%) Other 0 1 (4.8%) 1 (3.2%) Ethnicity, %
Non-Hispanic 10 (100%) 21 (100%) 31 (100%) Gender, % Male 5 (50.0%)
8 (38.1%) 13 (41.9%) Female 5 (50.0%) 132 (61.9%) 18 (58.1%) Mean
BMI, 23.92 (3.469) 25.26 (3.257) 24.83 (3.330) kg/m.sup.3 (SD)
TABLE-US-00040 TABLE 40 Summary of Baseline Characteristics - all
values as Mean (SD) mAb1 All Subjects Placebo 300 mg Combined No.
Patients 10 21 31 Chronic Atopic 5.4 (9.26) 5.3 (11.12) 5.4 (10.40)
Dermatitis Diagnosis Age BSA 38.85 (24.052) 40.43 (20.912) 39.92
(21.579) EASI Score 24.10 (12.695) 23.12 (12.354) 23.43 (12.261)
IGA Score 3.35 (0.474) 3.43 (0.598) 3.40 (0.554) NRS Score 5.00
(1.394) 6.43 (2.002) 5.97 (1.928) SCORAD Score 58.20 (13.834) 66.31
(13.013) 63.69 (13.607)
[0472] The exploratory efficacy results obtained from the study are
summarized in FIGS. 34-47 and Tables 41-44. Despite the relatively
small sample size and the limited treatment period, the analysis
showed statistically significant and clinically relevant effects of
mAb1 vs. placebo in key exploratory efficacy endpoints, including
EASI-50 responder rate, as well as change and percent change from
baseline in EASI, SCORAD, IGA, and pruritus NRS, with some
improvements persisting for several weeks after the discontinuation
of study treatment. For EASI-50, 100% of patients in the mAb1 plus
TCS group met the responder criteria on Day 29, vs. 50% in the
placebo plus TCS group (P-value 0.0015). Other endpoints like IGA
0-1 responder rate showed numerical superiority to placebo but did
not reach statistical significance (47.6% vs. 30.0% for placebo).
Notably, patients treated with mAb1 used on average approximately
50% less TCS, which might have underestimated the mAb1 treatment
effect relative to the placebo (TCS alone) comparator group.
TABLE-US-00041 TABLE 41 Summary of Percent and Absolute Change in
EASI score from Baseline to Day 29 Placebo 300 mg mAb1 No. Patients
10 21 Baseline EASI Score Mean (SD) 24.1 (12.70) 23.1 (12.35) Day
29 EASI Score Mean (SD) 14.4 (13.91) 6.2 (6.68) % Change from
Baseline to Day 29 -52.5 (39.53) -75.6 (13.29) Absolute change from
Baseline to -9.7 (8.42) -16.9 (8.06) Day 29
TABLE-US-00042 TABLE 42 Summary of Percent and Absolute Change in
IGA score from Baseline to Day 29 Placebo 300 mg mAb1 No. Patients
10 21 Baseline IGA Score Mean (SD) 3.4 (0.47) 3.4 (0.60) Day 29 IGA
Score Mean (SD) 2.4 (1.43) 1.6 (0.80) % Change from Baseline to Day
29 -30.6 (39.00) -52.5 (21.44) Absolute change from Baseline to
-1.0 (1.17) -1.8 (0.81) Day 29
TABLE-US-00043 TABLE 43 Summary of % and Absolute Change in SCORAD
Score from Baseline to Day 29 Placebo 300 mg mAb1 No. Patients 10
21 Baseline SCORAD Score Mean (SD) 58.2 (13.83) 66.3 (13.01) Day 29
SCORAD Score Mean (SD) 37.1 (25.11) 26.4 (13.53) % Change from
Baseline to Day 29 -40.0 (33.91) -59.8 (18.35) Absolute change from
Baseline to -21.1 (17.99) -39.9 (15.67) Day 29
TABLE-US-00044 TABLE 44 Summary of % and Absolute Change in
Pruritus NRS from Baseline to Day 29 Placebo 300 mg mAb1 No.
Patients 10 21 Baseline NRS Score Mean (SD) 5.0 (1.39) 6.4 (2.00)
Day 29 NRS Score Mean (SD) 3.4 (1.96) 1.8 (1.33) % Change from
Baseline to Day 29 -24.7 (47.30) -70.7 (21.45) Absolute change from
Baseline to -1.6 (2.40) -4.6 (2.01) Day 29
[0473] Subcutaneous administration of mAb1 to adult patients with
moderate-to-severe AD treated concomitantly with TCS was generally
safe and well tolerated. Treatment with mAb1 administered
concomitantly with TCS was associated with significantly superior
outcomes compared with TCS treatment alone. The proportion of
patients achieving an EASI-50 score was numerically much greater
that was seen in recent studies in which mAb1 was used as
monotherapy (the best EASI-50 so far had been 75%) which suggested
that mAb1 and TCS acted additively or synergistically. However, it
might also be in part due to the small sample size and slight
differences in the patient populations between studies.
[0474] The study demonstrated additional efficacy provided by mAb1
in patients with moderate-to-severe AD who received TCS treatment.
These results suggested that combination therapy could provide
additional clinical benefits to patients with moderate-to-severe
AD, compared to either treatment used as monotherapy. The results
also suggested the possibility of a TCS sparing effect of mAb1,
which could potentially lead to safer long term management of
patients with AD.
Example 12
Biomarker Analysis
[0475] Biomarker analysis was conducted on samples taken from
subjects who participated in clinical trials of mAb1. In
particular, IgE and thymus and activation chemokine (TARC) levels
were measured in samples from patients at baseline and at different
time points following initiation of study treatment(s). The
Phadiatop.TM. test was performed to detect antigen-specific IgE. In
addition, molecular profiling was carried out on skin lesions of
patients who participated in clinical trials of mAb1.
A. Administration of mAb1 to Healthy Subjects
[0476] In a first clinical trial, subjects were administered single
intravenous (IV) (1.0, 3.0, 8.0 and 12.0 mg/kg) or subcutaneous
(SC) (150 and 300 mg) doses of mAb, or placebo (see Example 2
herein). Samples for biomarker analysis were collected from the
antibody- and placebo-treated subjects at days 1 (baseline), 8, 29,
and 85 (or early termination). Levels of IgE and TARC were measured
in each sample. A p-value of <0.10 was considered statistically
significant to allow for small sample size. A mixed-effect repeated
measures model was used for mean analyses and non-parametric test
for median analyses. The median percent change in IgE and TARC
levels from patient samples are summarized in Tables 45 and 46,
respectively.
TABLE-US-00045 TABLE 45 Median Percent Change in IgE Level from
Baseline Following mAb1 or Placebo Administration subcutaneous (SC)
intravenous (IV) mAb1 mAb1 150 300 3 8 12 Placebo mg mg Placebo
mg/kg mg/kg mg/kg Baseline -- -- -- -- -- -- -- Day 8 -1.3 -4.4 0.0
-1.3 -3.3 1.3 0.0 Day 29 1.0 -8.5 -15.4 1.0 -10.7 -8.9 -7.9 Day 85
14.7 -16.5 -17.2 14.7 -4.8 -10.7 -25.6
TABLE-US-00046 TABLE 46 Median Percent Change in TARC Level from
Baseline Following mAb1 or Placebo Administration subcutaneous (SC)
intravenous (IV) mAb1 mAb1 150 300 3 8 12 Placebo mg mg Placebo
mg/kg mg/kg mg/kg Baseline -- -- -- -- -- -- -- Day 8 7.7 -16.0
-34.9 7.7 -28.2 -20.0 3.7 Day 29 4.1 -14.8 -21.0 4.1 0.8 -0.4 -25.2
Day 85 -5.7 -29.9 -15.1 -5.7 -0.5 -1.6 -17.0
[0477] Baseline levels of IgE were highly variable as shown in the
comparison of mean and median baseline IgE per treatment group
(FIG. 48A). The laboratory reference range for the utilized IgE
assay is 0-114 kU/L and 15 of the 40 subjects had total IgE
levels>114 kU/L at baseline. As shown in Table 43 and FIG. 48B,
IgE levels generally declined in proportion to the mAb1 dose and
exposure time. By Day 85 after administration, subjects receiving
SC-administered mAb1 exhibited a median decrease in IgE level of
16.5% (150 mg) and 17.2% (300 mg). Significant IgE decreases were
also observed in patients receiving IV-administered mAb1, with
decreases in IgE of 10.7% and 25.6% in the 8 mg/kg and 12 mg/kg
groups, respectively. By contrast, IgE levels increased over time
in placebo treated subjects.
[0478] Median serum TARC levels at baseline were generally
comparable between the treatment groups (FIG. 49A), and were higher
than those reported in the literature. Mean baseline TARC was 616
pg/mL with a range of 134-1327 pg/mL. TARC levels in healthy
subjects have been reported in the range of 106-431 ng/L (Hijnen et
al 2004, J. Allergy Clin. Immunol. 113: 334-340). Significant
reductions in TARC were observed in samples taken from mAb1-treated
subjects compared to placebo with both subcutaneous doses (p=0.044
for 150 mg and p=0.047 for 300 mg) (FIG. 49B and Table 44). For
example, a single SC dose of 300 mg of mAb1 caused a median
decrease in TARC level by almost 35% at day 8 (p=0.052), while TARC
level increased by 7.7% in placebo-treated patients. Significant
decreases in TARC levels were sustained in mAb1-treated patients at
Day 29 through the end of the study (Day 85) in both SC- and
IV-administered groups. When data was pooled for all subjects
treated with mAb1, the overall difference between mAb1 and placebo
for percent change from baseline in TARC was significant (p=0.004)
(FIG. 50). Significant differences were also observed at days 8
(p=0.012) and 29 (p=0.022).
B. Administration of mAb1 to Subjects with Atopic Dermatitis
[0479] Biomarker levels were also measured in samples from two
separate clinical trials involving subjects with atopic dermatitis
(AD). In "Study A", AD subjects were administered either mAb1 (75,
150 or 300 mg) or placebo, on days 1, 8, 15 and 22 of the study
(i.e., four weekly doses). In "Study B", AD subjects were
administered 150 mg or 300 mg of mAb1, or placebo, on days 1, 8, 15
and 22 of the study (i.e., four weekly doses) (see Example 7
herein). All administrations for both studies were subcutaneous
(SC). Samples for biomarker analysis were collected from the
antibody- and placebo-treated subjects from both studies at days 1
(baseline), 4, 8, 15, 22, 25, 29, 36, 43, 50, 57, 64, 71 and 85 (or
early termination). Levels of IgE, TARC, lactate dehydrogenase
(LDH), and antigen-specific IgE (Phadiatop) were measured in each
sample.
[0480] Serum TARC was measured using a validated assay (Human
CCL17/TARC Quantikine ELISA kit, R&D Systems; validation and
assays performed by Quest Diagnostics). Total serum IgE levels were
determined using the ImmunoCAP.RTM. Total IgE test (Thermo
Scientific FDA cleared test; performed by Quest Diagnostics).
Lactate dehydrogenase (LDH) was measured using the Roche Modular
test (FDA cleared; performed by Covance Central Laboratories).
Phadiatop.RTM. (Thermo Scientific FDA cleared test) assays were
performed by Viracor-IBT. Two-sample median test was used to
compare the biomarker changes from baseline with mAb1 to
placebo.
[0481] Mean baseline levels of serum TARC, total IgE and LDH for
all AD patients enrolled in study `B` were higher than the reported
upper limit of normal (ULN) (Table 47 and FIG. 51).
TABLE-US-00047 TABLE 47 Summary of baseline biomarker
characteristics Mean Baseline (SEM) Mean Baseline (SEM) Mean
Baseline (SEM) Mean Baseline (SEM) Biomarker All patients (n = 37)
Placebo (n = 10) 150 mg DPL (n = 14) 300 mg DPL (n = 14) EASI 28.4
(2.56) 25.64 (4.34) 32.55 (4.96) 25.91 (3.71) TARC (pg/mL) 6914.9
(2001.3) 7001 (2669.8) 9162.2 (4851.7) 4601.4 (1957.3) IgE (kU/L)
8038.3 (2052.2) 15026.5 (4748.6) 7231.9 (2634.1) 2931.7 (1383.4)
Phadiatop .RTM. 34/36 patients were + All + All + 2 patients were -
LDH (U/L) 240.4 (13.4) 296.7 (28.8) 226.1 (21) 212.5 (16.1) Eos
(10]cells/L) 0.50 (0.06) 0.65 (0.13) 0.49 (0.09) 0.41 (0.11) Eos
(%) 6.37 (0.71) 8.03 (1.06) 6.17 (1.18) 5.29 (1.32)
[0482] Mean baseline eosinophil levels were at the high end of the
reference range (Table 47). All but 2 patients with available data
tested positive for the Phadiatop test. Both of these patients also
had normal total serum IgE levels. Phadiatop results were
unavailable for one patient.
[0483] A broad spectrum of baseline TARC and IgE was observed in
the enrolled moderate-to-severe AD population. 27/36 of patients
had serum TARC levels>1000 pg/mL (.about.twice the mean levels
reported for healthy volunteers (FIG. 51A). 32/36 of patients had
IgE levels.gtoreq.150 kU/L (a cutoff often cited to distinguish
extrinsic and intrinsic AD) (FIG. 51B). 17/37 had LDH levels above
234 U/L (FIG. 51C). No patients had LDH levels below 100 U/L.
[0484] Using local linear regression, an overall mAb1 treatment
effect (percent change from baseline) on total serum IgE was
observed compared to placebo in both dose groups (p<0.0001)
(FIG. 52). Total serum IgE levels decreased with mAb1 treatment,
while an overall increase was observed at the end of the study in
the placebo treated group.
[0485] The median percent change in IgE levels from baseline for
each group from both studies A and B (combined data) is summarized
in Table 48.
TABLE-US-00048 TABLE 48 Median Percent Change in IgE Level from
Baseline (Study A & B Combined) subcutaneous (SC) mAb1 Placebo
75 mg 150 mg 300 mg *Baseline -- -- -- -- Day 4 2.7 TBD 4.3 0.0
*Day 8 0.2 TBD 17.6 -2.2 *Day 15 25.7 TBD 13.2 0.0 *Day 22 19.0 TBD
-4.4 -2.1 Day 25 28.4 TBD -7.4 -9.5 Day 29 32.0 TBD 0.2 -1.6 Day 36
43.0 TBD -5.5 -12.1 Day 43 43.0 TBD -11.2 -4.8 Day 50 28.9 TBD
-13.7 -17.3 Day 57 54.2 TBD -11.7 -18.3 Day 64 51.2 TBD -21.6 -14.3
Day 71 37.5 TBD -15.4 -22.3 Day 85 41.7 TBD -16.8 -23.9 *Denotes
days when drug or placebo was administered
[0486] As shown in Table 48 and FIG. 52, a statistically
significant decrease in IgE was observed in samples from
mAb1-treated subjects compared to placebo. The median percent
change IgE at day 85 was -23.9% in patients treated with 300 mg
mAb1, compared to a 41.7% increase in the placebo group
(p<0.0001). The median percent change from baseline in the 150
mg group compared to placebo was significant at all time-points
from days 29-85 (p<0.03). The median percent change from
baseline in the 300 mg group compared to placebo was significant at
all time-points from days 15-85 (p<0.04).
[0487] Using local linear regression, an overall treatment effect
was observed for LDH. There was a statistically significant
decrease in LDH in the 300 mg treatment group (p=0.0051) (FIG. 53).
Median percent change was not statistically significant at any
single time point, however, a temporal trend was observed
(p=0.008).
[0488] mAb1 treatment rapidly suppressed serum TARC levels in AD
patients (FIG. 54). The median percent change in TARC levels from
baseline for each group from both studies (combined data) is
summarized in Table 49.
TABLE-US-00049 TABLE 49 Median Percent Change in TARC Level from
Baseline (Study A and B combined) subcutaneous (SC) mAb1 Placebo 75
mg 150 mg 300 mg *Baseline -- -- -- -- Day 4 -7.9 TBD -37.0 -20.8
*Day 8 -1.1 TBD -24.8 -37.5 *Day 15 -19.3 TBD -61.0 -56.6 *Day 22
-5.0 TBD -64.4 -73.1 Day 25 -25.5 TBD -69.5 -78.5 Day 29 -22.7 TBD
-79.9 -70.9 Day 36 -18.3 TBD -78.1 -77.9 Day 43 -35.3 TBD -86.3
-72.3 Day 50 -28.9 TBD -82.2 -67.4 Day 57 -37.4 TBD -55.2 -71.4 Day
64 -33.2 TBD -45.5 -78.1 Day 71 -43.0 TBD -28.6 -60.3 Day 85 -45.2
TBD -28.3 -37.3 *Denotes days when drug or placebo was
administered
[0489] A statistically significant reduction in serum TARC was
observed in patients treated with 300 mg mAb1 compared to placebo
(p<0.0001; local linear regression analysis). Statistically
significant suppression was maintained through day 50 in patients
treated with 300 mg mAb1, approximately one month after the last
dose (administered on study day 21). The 150 mg group achieved
comparable magnitude of suppression, but levels were observed to
increase sooner than in the 300 mg group. Statistically significant
suppression (median percent change TARC from baseline compared to
placebo) was observed at days 36 and 43 in the 150 mg group
(p<0.03), as well as days 22, 25, 29, 36, and 50 with the 300 mg
group (p<0.04).
[0490] Intra-patient variability of TARC levels was observed over
the course of the study in placebo-treated patients. Data from only
4 placebo-treated patients was available at the end of the study,
due to a high dropout rate in that group.
[0491] In conclusion, TARC, IgE and LDH, biomarkers associated with
Th2 inflammation and/or AD disease activity, were all suppressed by
mAb1 treatment in AD patients. mAb1 rapidly decreased serum TARC
levels in AD patients, compared to placebo. Duration of suppression
appeared to be dose-related and data suggested that the effect
might be sustained even after drug discontinuation. Total serum IgE
levels significantly declined in mAb1 treated patients. IgE
continued to decline (median percent change) in the 300 mg group
after the treatment phase, suggesting that maximal IgE suppression
had not yet been achieved. A consistent reduction in LDH levels
from baseline was observed in patients treated with mAb1. A direct
link between LDH and IL-4 and IL-13 is unknown, but its association
with disease severity suggested LDH might be a measure of the
extent of skin damage in AD patients. The suppression of TARC and
IgE demonstrated that mAb1 is a potent inhibitor of Th2
inflammation.
Correlations Among Biomarkers and AD-Associated Parameters
[0492] In Study "B" (see Example 7), patients with severe AD were
given 150 or 300 mg mAb1 or placebo (PBO) weekly for four weeks.
Pruritus was measured using twice-daily pruritus Numeric Rating
Scale (NRS; ranging from 0-10) to generate an average weekly NRS
score & a bi-weekly 5-D Pruritus Scale assessments. The 5-D
scale is a 5 question tool used to assess multiple dimensions of
itch: degree, duration, direction, disability, and distribution.
Mean baseline NRS & 5-D scores were 5.5 & 19, respectively.
The average weekly NRS scores rapidly decreased (mean % change from
baseline) by 31.9% at week 2 (p<0.02), & 55.2% at week 7
(p=0.01) in the 300 mg group vs +1.3% and -17.3% respectively in
the PBO group. Rapid reduction in 5-D scores was also observed in
patients treated with 300 mg mAb1 (mean % change -28.2% at day 15,
p=0.0009; -37.1% at day 29, p=0.0007; -42.5% at day 43, p=0.012;
+3.6%, +8.1% & -9.4% respectively in the PBO group). Serum
levels of CCL17, a marker of IL4/IL13 activity, also rapidly
declined on treatment. Both CCL17 and pruritus were suppressed for
several weeks following the end of treatment. Table 50 shows the
correlation of pruritus (5D and NRS) with outcomes of dermatitis
(EASI) and CCL17.
TABLE-US-00050 TABLE 50 5D correlations NRS correlations EASI CCL17
EASI CCL17 Time Spearman Spearman Spearman Spearman point r P r P r
P r P Actual Values Baseline 0.41 0.0111 0.46 0.0044 0.35 0.0321
0.42 0.0117 Day 29 0.62 0.0001 0.55 0.0024 0.64 <0.0001 0.12
0.5413 Percent change from baseline Day15 0.65 <0.0001 0.46
0.0089 0.51 0.0012 0.32 0.0795 Day29 0.61 <0.0001 0.48 0.0105
0.61 <0.0001 0.11 0.5640
[0493] Overall, for all treatment groups, the 5D score
significantly correlated with CCL17 (r=0.46, p=0.004 at baseline;
r=0.55, p=0.002 at day 29) & EASI scores in this study (r=0.41,
p=0.011 at baseline; 0.62, p<0.0001 at day 29). The percent
change in 5D significantly correlated with the percent change from
baseline in EASI (r=0.65, p<0.0001 for day 15; and r=0.61,
p<0.0001 for day 29) and CCL17 (r=0.46, p=0.0089 for day 15; and
r=0.48, p=0.0105 for day 29) for the overall treatment groups at
Days 15 and 29. Treatment groups were also individually assessed
for correlation of Pruritus 5D with EASI and CCL17. At day 15, only
the 150 mg group demonstrated strong and significant correlation
between the percent change in EASI and percent change in 5D
(r=0.81, p=0.0005). Similarly at day 29, the only significant
correlation was for the 150 mg group (r=0.57, p=0.0036). Although
there was a significant overall correlation between percent change
in CCL17 and percent change in 5D score at both day 15 and day 29,
none of the individual treatment groups showed such a correlation
at either day.
[0494] Pruritus severity, assessed using the NRS, showed moderate
to strong correlations with EASI that were significant. However,
NRS values correlated with CCL17 values only at baseline, with no
significant correlation for percent change from baseline. The rapid
& sustained improvement in pruritus observed in adult AD
patients treated with mAb1 suggests IL-4/IL-13 signaling is a key
mechanism for AD pruritus. The correlation between pruritus and
CCL17 levels highlights the relationship between IL-4/IL-13
mediated inflammation, AD disease activity & pruritus in severe
AD.
C. Repeated Administration of mAb1 to Subjects with
Moderate-to-Severe Atopic Dermatitis
[0495] IgE and TARC levels were measured in samples from a clinical
trial involving subjects with moderate-to-severe atopic dermatitis
(AD). AD subjects were administered 300 mg of mAb1, or placebo, on
days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71 and 78 of the study
(i.e., 12 weekly doses) (see Example 10 herein). All
administrations for both studies were subcutaneous (SC). Serum
samples for biomarker analysis were collected from the antibody-
and placebo-treated subjects from both studies at days 1
(baseline), 8, 15, 22, 25, 29, 36, 43, 50, 57, 64, 71, 85, 99, 113,
127, 141, 155, 169, 183 and 197 (end of study) or early
termination. Levels of IgE, TARC and antigen-specific IgE
(Phadiatop.TM. test) were measured in each sample.
[0496] TARC is a chemokine induced by IL-4/IL-13, shown to be
strongly associated with disease severity of AD, and may be
involved in pathogenesis of the disease. Baseline TARC levels were
assessed for potential predictive value for treatment response.
Post-treatment samples were evaluated for pharmacodynamics effect
of mAb1 on TARC.
[0497] Patients with AD often have elevated IgE. Total IgE levels
have been found to correlate with AD severity and may be involved
in the pathogenesis of the disease. Baseline IgE levels were
assessed for potential predictive value for treatment response.
Post-treatment samples were evaluated for pharmacodynamics effects
of mAb1 on total IgE.
[0498] The Phadiatop.TM. test is an in vitro diagnostic screening
tool used to detect antigen-specific IgE for common inhalants.
Baseline results of the Phadiatop.TM. test were assessed for
potential predictive value for treatment response. Post-treatment
samples were evaluated for pharmacodynamics effects of mAb1 on the
Phadiatop.TM. antigen panel.
[0499] In line with the results obtained from earlier clinical
trials (see sections A and B above), the TARC and IgE levels
decreased and remained suppressed below baseline through the
16-week post-treatment follow-up period (FIGS. 55-56).
[0500] Greater magnitude of IgE suppression was observed with 12
weeks of 300 mg mAb1 treatment during a 16-week followup (median
-57%) as compared 4 weeks of mAb1. Magnitude of TARC suppression
was comparable at the end-of-treatment after 12 weeks (median -83%)
and weeks (median -76%) of mAb1 treatment.
D. Concomitant Administration of mAb1 with Topical Corticosteroids
in Patients with Moderate-to-Severe Atopic Dermatitis
[0501] TARC and IgE modulation was studied in a trial evaluating
the safety and efficacy of mAb1 in combination with topical
corticosteroids (TCS) in adult patients with moderate-to-severe AD.
Two treatment groups were compared (weekly dosing for 4 weeks):(300
mg mAb1+TCS), versus (placebo+TCS). TCS were administered from day
1 up to day 28 (patients stopped TCS treatment if lesions cleared)
(see Example 11 herein). Patients were evaluated at screening,
baseline (day 1), weekly through week 5, then every other week
through week 11. TARC levels decreased in both treatment groups,
with a trend for greater suppression in the mAb1+TCS group compared
to placebo (PBO)+TCS. Differences were statistically significant at
days 22, 29 and 50. IgE levels also decreased in both treatment
groups. There was no statistically significant difference in IgE
suppression between groups.
TABLE-US-00051 TABLE 51 Baseline TARC by treatment group Treatment
group Placebo + TCS 300 mg mAb1 + TCS n 9 20 Mean (SD; pg/mL)
2999.2 (4063.88) 2703.4 (3411.76) Median (pg/mL) 913 1444 Min:max
325:11966 347:14100
[0502] TARC was measured using the R&D Systems human TARC
Quantikine ELISA kit. Table 51 summarizes the baseline TARC levels
by treatment group. The mean and median baseline TARC levels for
both treatment groups were above the normal range of 106-431 pg/mL
(Weihrauch et al 2005; Cancer Res. 65: 13), as well as the observed
baseline levels as in Section A above.
TABLE-US-00052 TABLE 52 Mean percent change TARC from baseline with
standard deviation (SD) Mean % Study change from Treatment group
day n baseline SD Placebo + TCS 8 9 -25.5 36.8 15 9 -15.6 70.5 22 9
-5.9 48.5 29 8 -24.4 23.2 36 8 -39.7 35.7 50 9 -33.1 47.8 64 8
-25.1 53.2 78 9 -20.8 73.0 300 mg mAb1 + TCS 8 20 -43.8 30.9 15 21
-49.6 38.7 22 20 -51.0 44.1 29 21 -55.2 41.3 36 20 -57.9 42.9 50 21
-61.1 35.4 64 21 -36.8 66.8 78 19 -35.3 47.0
[0503] TARC levels decreased from baseline in both treatment
groups, thus TCS alone may lower serum TARC levels in AD patients.
Although the magnitude of median % change TARC from baseline was
consistently larger in the mAb1+TCS group compared to placebo+TCS,
the difference was only statistically significant at days 22, 29
and 50 (least square mean difference estimated from analysis of
covariance) (Table 52).
[0504] The mean and median baseline IgE levels are summarized in
Table 53.
TABLE-US-00053 TABLE 53 Baseline IgE by treatment group Treatment
group Placebo + TCS 300 mg mAb1 + TCS n 9 21 Mean (SD; kU/L)
12016.4 (19659.15) 4197.8 (5293.78) Median (kU/L) 4250 1728 Min:Max
119:61600 61:19790
[0505] IgE levels declined in both treatment groups. After day 29,
there was a trend for the magnitude of median percent change IgE to
be greater in the mAb1+TCS group, there was no statistically
significant difference at any time point in the study (LS mean
difference estimated from analysis of covariance; Table 54).
TABLE-US-00054 TABLE 54 mean percent change from baseline with SD
Mean % Study change from Treatment group day n baseline SD Placebo
+ TCS 8 9 -1.1 8.7 15 9 -10.0 15.7 22 9 -9.5 16.7 29 8 -9.4 23.1 36
8 -13.2 19.2 50 9 -13.0 18.3 64 8 -20.8 15.8 78 9 -21.5 18.3 300 mg
mAb1 + TCS 8 20 0.3 16.1 15 21 8.0 46.4 22 20 0.7 44.0 29 21 -7.3
40.5 36 20 -1.8 49.2 50 21 -10.0 43.7 64 21 -22.2 44.0 78 19 -29.0
39.4
[0506] Approximately 50% of patients achieved at least an EASI50 by
day 29 on placebo+TCS and the suppression of TARC and IgE in this
group was consistent with the clinical improvement observed. All
patients on mAb1+ TCS achieved at least an EASI50 by day 29 (see
Example 11). Trends were observed for greater suppression of TARC
and IgE in the mAb1+TCS compared to placebo+TCS. However, the only
statistically significant differences observed were in TARC
suppression at days 22, 29 and 50.
[0507] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
Sequence CWU 1
1
2751351DNAArtificial SequenceSynthetic 1caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttccgc tcttatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcggtc atatcatatg atggaagtaa taaatattat
180atagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgaat 240ctgcaaatga acagcctgag acttgaggac acggctgtat
attactgtgc gaaagagggg 300agggggggat ttgactactg gggccaggga
atcccggtca ccgtctcctc a 3512117PRTArtificial SequenceSynthetic 2Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr
Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Asn65 70 75 80 Leu Gln Met Asn Ser Leu Arg Leu
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Arg Gly
Gly Phe Asp Tyr Trp Gly Gln Gly Ile Pro 100 105 110 Val Thr Val Ser
Ser 115 324DNAArtificial SequenceSynthetic 3ggattcacct tccgctctta
tggc 2448PRTArtificial SequenceSynthetic 4Gly Phe Thr Phe Arg Ser
Tyr Gly1 5 524DNAArtificial SequenceSynthetic 5atatcatatg
atggaagtaa taaa 2468PRTArtificial SequenceSynthetic 6Ile Ser Tyr
Asp Gly Ser Asn Lys1 5 730DNAArtificial SequenceSynthetic
7gcgaaagagg ggaggggggg atttgactac 30810PRTArtificial
SequenceSynthetic 8Ala Lys Glu Gly Arg Gly Gly Phe Asp Tyr1 5 10
9324DNAArtificial SequenceSynthetic 9gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca
ggtcataaac aattatttag cctggtttca gcagaaacca 120gggaaagtcc
ctaagtccct gatccatgct gcatccagtt tacaaagtgg ggtcccatca
180aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgccaacag tataatagtc
acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa acga
32410108PRTArtificial SequenceSynthetic 10Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr 20 25 30 Leu Ala
Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Ser Leu Ile 35 40 45
His Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser
His Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 1118DNAArtificial SequenceSynthetic 11caggtcataa
acaattat 18126PRTArtificial SequenceSynthetic 12Gln Val Ile Asn Asn
Tyr1 5 139DNAArtificial SequenceSynthetic 13gctgcatcc
9143PRTArtificial SequenceSynthetic 14Ala Ala Ser1
1527DNAArtificial SequenceSynthetic 15caacagtata atagtcaccc gtggacg
27169PRTArtificial SequenceSynthetic 16Gln Gln Tyr Asn Ser His Pro
Trp Thr1 5 17351DNAArtificial SequenceSynthetic 17caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cctctggatt caccttccgc tcttatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcggtc atatcatatg atggaagtaa
taaatattat 180atagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgaat 240ctgcaaatga acagcctgag acttgaggac
acggctgtat attactgtgc gaaagagggg 300agggggggat ttgactactg
gggccaggga accctggtca ccgtctcctc a 35118117PRTArtificial
SequenceSynthetic 18Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser 115 19321DNAArtificial
SequenceSynthetic 19gacatccaga tgacccagtc tccatcctca ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca ggtcataaac aattatttag
cctggtttca gcagaaacca 120gggaaagtcc ctaagtccct gatccatgct
gcatccagtt tacaaagtgg ggtcccatca 180aagttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttatta ctgccaacag tataatagtc acccgtggac gttcggccaa
300gggaccaagg tggaaatcaa a 32120107PRTArtificial SequenceSynthetic
20Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn
Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys
Ser Leu Ile 35 40 45 His Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asn Ser His Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 21351DNAArtificial
SequenceSynthetic 21caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttccgc tcttatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt
atatcatatg atggaagtaa taaatactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agctgaggac acggctgtgt attactgtgc gaaagagggg
300agggggggat ttgactactg gggccaggga accctggtca ccgtctcctc a
35122117PRTArtificial SequenceSynthetic 22Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
23322DNAArtificial SequenceSynthetic 23gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca
ggtcataaac aattatttag cctggtttca gcagaaacca 120gggaaagccc
ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgccaacag tataatagtc
acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa ac
32224107PRTArtificial SequenceSynthetic 24Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr 20 25 30 Leu Ala
Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser
His Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 25351DNAArtificial SequenceSynthetic 25caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cctctggatt caccttcaga agctatggca tacactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa
taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacactgtat 240ctgcaaatga acagcctgat aactgaggac
acggctgtgt attattgtgt gaaagagggg 300aggggggggt ttgactactg
gggccaggga accacggtca ccgtctcctc a 35126117PRTArtificial
SequenceSynthetic 26Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln
Met Asn Ser Leu Ile Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Val Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100
105 110 Val Thr Val Ser Ser 115 2724DNAArtificial SequenceSynthetic
27ggattcacct tcagaagcta tggc 24288PRTArtificial SequenceSynthetic
28Gly Phe Thr Phe Arg Ser Tyr Gly1 5 2924DNAArtificial
SequenceSynthetic 29atatcatatg atggaagtaa taaa 24308PRTArtificial
SequenceSynthetic 30Ile Ser Tyr Asp Gly Ser Asn Lys1 5
3130DNAArtificial SequenceSynthetic 31gtgaaagagg ggaggggggg
gtttgactac 303210PRTArtificial SequenceSynthetic 32Val Lys Glu Gly
Arg Gly Gly Phe Asp Tyr1 5 10 33324DNAArtificial SequenceSynthetic
33gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgtc gggcgagtca ggtcattaat aattatttag cctggtttca gcagaaacca
120gggaaagtcc ctaagtccct gatccatgct gcatccagtt tgcaaagagg
ggtcccatca 180aagttcagcg gcagtggatc tgggacagat ttcactctca
ccatcaacag cctgcagcct 240gaagattttg caacttatta ctgccaacaa
tataatagtt acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa acga
32434108PRTArtificial SequenceSynthetic 34Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn Tyr 20 25 30 Leu Ala
Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys Ser Leu Ile 35 40 45
His Ala Ala Ser Ser Leu Gln Arg Gly Val Pro Ser Lys Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser
Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 3518DNAArtificial SequenceSynthetic 35caggtcatta
ataattat 18366PRTArtificial SequenceSynthetic 36Gln Val Ile Asn Asn
Tyr1 5 379DNAArtificial SequenceSynthetic 37gctgcatcc
9383PRTArtificial SequenceSynthetic 38Ala Ala Ser1
3927DNAArtificial SequenceSynthetic 39caacaatata atagttaccc gtggacg
27409PRTArtificial SequenceSynthetic 40Gln Gln Tyr Asn Ser Tyr Pro
Trp Thr1 5 41351DNAArtificial SequenceSynthetic 41caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cctctggatt caccttcaga agctatggca tacactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagtaa
taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacactgtat 240ctgcaaatga acagcctgat aactgaggac
acggctgtgt attattgtgt gaaagagggg 300aggggggggt ttgactactg
gggccaggga accctggtca ccgtctcctc a 35142117PRTArtificial
SequenceSynthetic 42Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp
Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln
Met Asn Ser Leu Ile Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Val Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100
105 110 Val Thr Val Ser Ser 115 43321DNAArtificial
SequenceSynthetic 43gacatccaga tgacccagtc tccatcctca ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca ggtcattaat aattatttag
cctggtttca gcagaaacca 120gggaaagtcc ctaagtccct gatccatgct
gcatccagtt tgcaaagagg ggtcccatca 180aagttcagcg gcagtggatc
tgggacagat ttcactctca ccatcaacag cctgcagcct 240gaagattttg
caacttatta ctgccaacaa tataatagtt acccgtggac gttcggccaa
300gggaccaagg tggaaatcaa a 32144107PRTArtificial SequenceSynthetic
44Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn
Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Val Pro Lys
Ser Leu Ile 35 40 45 His Ala Ala Ser Ser Leu Gln Arg Gly Val Pro
Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Asn Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asn Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 45351DNAArtificial
SequenceSynthetic 45caggtgcagc tggtggagtc tgggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt caccttcaga agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt
atatcatatg atggaagtaa taaatactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agctgaggac acggctgtgt attactgtgt gaaagagggg
300aggggggggt ttgactactg gggccaggga accctggtca ccgtctcctc a
35146117PRTArtificial SequenceSynthetic 46Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Val Lys Glu Gly Arg Gly Gly Phe Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
47322DNAArtificial SequenceSynthetic 47gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca
ggtcattaat aattatttag
cctggtttca gcagaaacca 120gggaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttatta ctgccaacaa tataatagtt acccgtggac gttcggccaa
300gggaccaagg tggaaatcaa ac 32248107PRTArtificial SequenceSynthetic
48Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Val Ile Asn Asn
Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys
Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asn Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 49375DNAArtificial
SequenceSynthetic 49caggtgcagc tggtggagtc tgggggaggc ttggaacagc
cgggggggtc cttgagactc 60tcctgtgcag gctctggatt cacgtttaga gactatgcca
tgacctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtcgcatcg
attagtggtt ccggtggtaa cacatacttc 180gcagactccg tgaagggccg
gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga
300ctctctataa caattcgccc acgctattat ggtttggacg tctggggcca
agggtccacg 360gtcaccgtct cctca 37550125PRTArtificial
SequenceSynthetic 50Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu
Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly
Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Gly Ser
Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu 100
105 110 Asp Val Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser 115 120
125 5124DNAArtificial SequenceSynthetic 51ggattcacgt ttagagacta
tgcc 24528PRTArtificial SequenceSynthetic 52Gly Phe Thr Phe Arg Asp
Tyr Ala1 5 5324DNAArtificial SequenceSynthetic 53attagtggtt
ccggtggtaa caca 24548PRTArtificial SequenceSynthetic 54Ile Ser Gly
Ser Gly Gly Asn Thr1 5 5554DNAArtificial SequenceSynthetic
55gcgaaagatc gactctctat aacaattcgc ccacgctatt atggtttgga cgtc
545618PRTArtificial SequenceSynthetic 56Ala Lys Asp Arg Leu Ser Ile
Thr Ile Arg Pro Arg Tyr Tyr Gly Leu1 5 10 15 Asp
Val57324DNAArtificial SequenceSynthetic 57gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgttggaga cagagtcacc 60atcacttgtc gggcgagtca
ggccattaac aatcatttag cctggtttca gcagaaacca 120gggaaagccc
ctaagtccct gatctttgct gtatccagtt tgcaaagtgg ggtcccatca
180aagttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgccaacag tataatagtt
acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa acga
32458108PRTArtificial SequenceSynthetic 58Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ala Ile Asn Asn His 20 25 30 Leu Ala
Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45
Phe Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser
Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 5918DNAArtificial SequenceSynthetic 59caggccatta
acaatcat 18606PRTArtificial SequenceSynthetic 60Gln Ala Ile Asn Asn
His1 5 619DNAArtificial SequenceSynthetic 61gctgtatcc
9623PRTArtificial SequenceSynthetic 62Ala Val Ser1
6327DNAArtificial SequenceSynthetic 63caacagtata atagttaccc gtggacg
27649PRTArtificial SequenceSynthetic 64Gln Gln Tyr Asn Ser Tyr Pro
Trp Thr1 5 65372DNAArtificial SequenceSynthetic 65gaggtgcagc
tggtggagtc tgggggaggc ttggaacagc cgggggggtc cttgagactc 60tcctgtgcag
gctctggatt cacgtttaga gactatgcca tgacctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtcgcatcg attagtggtt ccggtggtaa
cacatacttc 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagatcga 300ctctctataa caattcgccc
acgctattat ggtttggacg tctggggcca agggaccacg 360gtcaccgtct cc
37266124PRTArtificial SequenceSynthetic 66Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 67321DNAArtificial SequenceSynthetic
67gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgttggaga cagagtcacc
60atcacttgtc gggcgagtca ggccattaac aatcatttag cctggtttca gcagaaacca
120gggaaagccc ctaagtccct gatctttgct gtatccagtt tgcaaagtgg
ggtcccatca 180aagttcagcg gcagtggatc tgggacagac ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataatagtt acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa a
32168107PRTArtificial SequenceSynthetic 68Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ala Ile Asn Asn His 20 25 30 Leu Ala
Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45
Phe Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser
Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 69373DNAArtificial SequenceSynthetic 69gaggtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacgtttaga gactatgcca tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggtt ccggtggtaa
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagatcga 300ctctctataa caattcgccc
acgctattat ggtttggacg tctggggcca agggaccacg 360gtcaccgtct cct
37370124PRTArtificial SequenceSynthetic 70Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 71322DNAArtificial SequenceSynthetic
71gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgtc gggcgagtca ggccattaac aatcatttag cctggtttca gcagaaacca
120gggaaagccc ctaagtccct gatctatgct gtatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataatagtt acccgtggac gttcggccaa 300gggaccaagg tggaaatcaa ac
32272107PRTArtificial SequenceSynthetic 72Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ala Ile Asn Asn His 20 25 30 Leu Ala
Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45
Tyr Ala Val Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser
Tyr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 73375DNAArtificial SequenceSynthetic 73caggtgcagc
tggtggagtc tgggggaggc ttggaacagc cgggggggtc cttgagactc 60tcctgtgcag
gctctggatt cacgtttaga gactatgcca tgacctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtcgcatcg attagtggtt ccggtggtaa
cacatacttc 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagatcga 300ctctctataa caattcgccc
acgctattat ggtttggacg tctggggcca agggtccacg 360gtcaccgtct cctca
37574125PRTArtificial SequenceSynthetic 74Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Ser Thr Val
Thr Val Ser Ser 115 120 125 7524DNAArtificial SequenceSynthetic
75ggattcacgt ttagagacta tgcc 24768PRTArtificial SequenceSynthetic
76Gly Phe Thr Phe Arg Asp Tyr Ala1 5 7724DNAArtificial
SequenceSynthetic 77attagtggtt ccggtggtaa caca 24788PRTArtificial
SequenceSynthetic 78Ile Ser Gly Ser Gly Gly Asn Thr1 5
7954DNAArtificial SequenceSynthetic 79gcgaaagatc gactctctat
aacaattcgc ccacgctatt atggtttgga cgtc 548018PRTArtificial
SequenceSynthetic 80Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg
Tyr Tyr Gly Leu1 5 10 15 Asp Val81339DNAArtificial
SequenceSynthetic 81gaaatagtgt tgacgcagtc tccactctcc ctgcccgtca
cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg tatagtattg
gatacaacta tttggattgg 120tacctgcaga agtcagggca gtctccacag
ctccttatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt
cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg
aggctgagga tgttgggttt tattactgca tgcaagctct acaaactccg
300tacacttttg gcccggggac caagctggag atcaaacga 33982113PRTArtificial
SequenceSynthetic 82Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu Tyr Ser 20 25 30 Ile Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Ser Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Ala 85 90 95
Leu Gln Thr Pro Tyr Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100
105 110 Arg8333DNAArtificial SequenceSynthetic 83cagagcctcc
tgtatagtat tggatacaac tat 338411PRTArtificial SequenceSynthetic
84Gln Ser Leu Leu Tyr Ser Ile Gly Tyr Asn Tyr1 5 10
859DNAArtificial SequenceSynthetic 85ttgggttct 9863PRTArtificial
SequenceSynthetic 86Leu Gly Ser1 8727DNAArtificial
SequenceSynthetic 87atgcaagctc tacaaactcc gtacact
27889PRTArtificial SequenceSynthetic 88Met Gln Ala Leu Gln Thr Pro
Tyr Thr1 5 89372DNAArtificial SequenceSynthetic 89gaggtgcagc
tggtggagtc tgggggaggc ttggaacagc cgggggggtc cttgagactc 60tcctgtgcag
gctctggatt cacgtttaga gactatgcca tgacctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtcgcatcg attagtggtt ccggtggtaa
cacatacttc 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagatcga 300ctctctataa caattcgccc
acgctattat ggtttggacg tctggggcca agggaccacg 360gtcaccgtct cc
37290124PRTArtificial SequenceSynthetic 90Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Phe Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 91336DNAArtificial SequenceSynthetic
91gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg
120tacctgcaga agtcagggca gtctccacag ctccttatct atttgggttc
taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca
cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttgggttt
tattactgca tgcaagctct acaaactccg 300tacacttttg gcccggggac
caagctggag atcaaa 33692112PRTArtificial SequenceSynthetic 92Asp Ile
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20
25 30 Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln
Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Phe Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr
Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 110
93373DNAArtificial SequenceSynthetic 93gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacgtttaga gactatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggtt ccggtggtaa cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaagatcga 300ctctctataa caattcgccc acgctattat
ggtttggacg tctggggcca agggaccacg 360gtcaccgtct cct
37394124PRTArtificial SequenceSynthetic 94Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 95337DNAArtificial SequenceSynthetic
95gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc
60atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta tttggattgg
120tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc
taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca
cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtt
tattactgca tgcaagctct acaaactccg 300tacacttttg gccaggggac
caagctggag atcaaac 33796112PRTArtificial SequenceSynthetic 96Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30 Ile Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
97375DNAArtificial SequenceSynthetic 97caggtgcagc tggtggagtc
tgagggactc ttggaacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
caactttaga gactttgcca tgacctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcatct attagtggta gtggtagtaa tacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaacca
cacgctgtat 240ctgcgaatga acagcctgag agccgaagac acggccgtgt
attactgtgc gaaagatcga 300ctctctataa caattcgccc acgctattac
ggtctggacg tctggggcca agggtccacg 360gtcaccgtct cctca
37598125PRTArtificial SequenceSynthetic 98Gln Val Gln Leu Val Glu
Ser Glu Gly Leu Leu Glu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Phe 20 25 30 Ala Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn His Thr Leu
Tyr65 70 75 80 Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Ser Thr Val
Thr Val Ser Ser 115 120 125 9924DNAArtificial SequenceSynthetic
99ggattcaact ttagagactt tgcc 241008PRTArtificial SequenceSynthetic
100Gly Phe Asn Phe Arg Asp Phe Ala1 5 10124DNAArtificial
SequenceSynthetic 101attagtggta gtggtagtaa taca 241028PRTArtificial
SequenceSynthetic 102Ile Ser Gly Ser Gly Ser Asn Thr1 5
10354DNAArtificial SequenceSynthetic 103gcgaaagatc gactctctat
aacaattcgc ccacgctatt acggtctgga cgtc 5410418PRTArtificial
SequenceSynthetic 104Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu1 5 10 15 Asp Val105324DNAArtificial
SequenceSynthetic 105gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca ggacattagc aattattttg
cctggtatca gcagaagcca 120gggaaagttc ctaagctcct gatctttgct
gcatccactt tgcatccagg ggtcccatct 180cggttcagtg gcagtggatc
tgggacagat ttcactctca ccattcgcag cctgcagcct 240gaagatgttg
caacttatta ctgtcaaaaa tatgacagtg ccccgtacac ttttggccag
300gggaccaagg tggaaatcaa acga 324106108PRTArtificial
SequenceSynthetic 106Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Phe Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Thr
Leu His Pro Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro65 70 75 80 Glu
Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
10718DNAArtificial SequenceSynthetic 107caggacatta gcaattat
181086PRTArtificial SequenceSynthetic 108Gln Asp Ile Ser Asn Tyr1 5
1099DNAArtificial SequenceSynthetic 109gctgcatcc 91103PRTArtificial
SequenceSynthetic 110Ala Ala Ser1 11127DNAArtificial
SequenceSynthetic 111caaaaatatg acagtgcccc gtacact
271129PRTArtificial SequenceSynthetic 112Gln Lys Tyr Asp Ser Ala
Pro Tyr Thr1 5 113372DNAArtificial SequenceSynthetic 113gaggtgcagc
tggtggagtc tgagggactc ttggaacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt caactttaga gactttgcca tgacctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcatct attagtggta gtggtagtaa
tacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaacca cacgctgtat 240ctgcgaatga acagcctgag agccgaagac
acggccgtgt attactgtgc gaaagatcga 300ctctctataa caattcgccc
acgctattac ggtctggacg tctggggcca agggaccacg 360gtcaccgtct cc
372114124PRTArtificial SequenceSynthetic 114Glu Val Gln Leu Val Glu
Ser Glu Gly Leu Leu Glu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Phe 20 25 30 Ala Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn His Thr Leu
Tyr65 70 75 80 Leu Arg Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 115321DNAArtificial SequenceSynthetic
115gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc gggcgagtca ggacattagc aattattttg cctggtatca
gcagaagcca 120gggaaagttc ctaagctcct gatctttgct gcatccactt
tgcatccagg ggtcccatct 180cggttcagtg gcagtggatc tgggacagat
ttcactctca ccattcgcag cctgcagcct 240gaagatgttg caacttatta
ctgtcaaaaa tatgacagtg ccccgtacac ttttggccag 300gggaccaagc
tggagatcaa a 321116107PRTArtificial SequenceSynthetic 116Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20
25 30 Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu
Ile 35 40 45 Phe Ala Ala Ser Thr Leu His Pro Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Arg Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln
Lys Tyr Asp Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 117373DNAArtificial SequenceSynthetic
117gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt caactttaga gactttgcca tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta
gtggtagtaa tacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaagatcga 300ctctctataa
caattcgccc acgctattac ggtctggacg tctggggcca agggaccacg
360gtcaccgtct cct 373118124PRTArtificial SequenceSynthetic 118Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Phe
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Ser Asn Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser
Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser 115 120 119322DNAArtificial
SequenceSynthetic 119gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca ggacattagc aattatttag
cctggtatca gcagaaacca 120gggaaagttc ctaagctcct gatctatgct
gcatccactt tgcaatcagg ggtcccatct 180cggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagatgttg
caacttatta ctgtcaaaaa tatgacagtg ccccgtacac ttttggccag
300gggaccaagc tggagatcaa ac 322120107PRTArtificial
SequenceSynthetic 120Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu
Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asp Ser Ala Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
121357DNAArtificial SequenceSynthetic 121caggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgtag cttctggatt
cacccttaac aactttgtca tgaactgggt ccgccaggtt 120ccagggaagg
gactggagtg ggtctctttt attagtgcta gtggtggtag tatatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca cttccaagaa
cacattatat 240ctgcaaatga acagcctgag agccgacgac acggccgtct
attactgtgc gaaatccccg 300tataactgga acccctttga ctattggggc
cagggaacca cggtcaccgt ctcctca 357122119PRTArtificial
SequenceSynthetic 122Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser
Gly Phe Thr Leu Asn Asn Phe 20 25 30 Val Met Asn Trp Val Arg Gln
Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Phe Ile Ser Ala
Ser Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Ser Pro Tyr Asn Trp Asn Pro Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr Val Thr Val Ser Ser 115 12324DNAArtificial
SequenceSynthetic 123ggattcaccc ttaacaactt tgtc 241248PRTArtificial
SequenceSynthetic 124Gly Phe Thr Leu Asn Asn Phe Val1 5
12524DNAArtificial SequenceSynthetic 125attagtgcta gtggtggtag tata
241268PRTArtificial SequenceSynthetic 126Ile Ser Ala Ser Gly Gly
Ser Ile1 5 12736DNAArtificial SequenceSynthetic 127gcgaaatccc
cgtataactg gaaccccttt gactat 3612812PRTArtificial SequenceSynthetic
128Ala Lys Ser Pro Tyr Asn Trp Asn Pro Phe Asp Tyr1 5 10
129327DNAArtificial SequenceSynthetic 129gacatccagt tgacccagtc
tccagccacc ctgtctgtgt ctccagggga acgagccacc 60ctctcctgca gggccagtct
gagtgttagc agcaaattag cctggtacca gcagacacct 120ggccaggctc
ccagactcct catctatagt gcctccaccc gggccactgg tatcccagtc
180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag
cctgcagtct 240gaagattttg cggtttatta ctgtcagcag tataatcatt
ggcctccgta cacttttggc 300caggggacca aggtggagat caaacga
327130109PRTArtificial SequenceSynthetic 130Asp Ile Gln Leu Thr Gln
Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Leu Ser Val Ser Ser Lys 20 25 30 Leu Ala
Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn His
Trp Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 13118DNAArtificial SequenceSynthetic 131ctgagtgtta
gcagcaaa 181326PRTArtificial SequenceSynthetic 132Leu Ser Val Ser
Ser Lys1 5 1339DNAArtificial SequenceSynthetic 133agtgcctcc
91343PRTArtificial SequenceSynthetic 134Ser Ala Ser1
13530DNAArtificial SequenceSynthetic 135cagcagtata atcattggcc
tccgtacact 3013610PRTArtificial SequenceSynthetic 136Gln Gln Tyr
Asn His Trp Pro Pro Tyr Thr1 5 10 137357DNAArtificial
SequenceSynthetic 137gaggtgcagc tggtggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgtag cttctggatt cacccttaac aactttgtca
tgaactgggt ccgccaggtt 120ccagggaagg gactggagtg ggtctctttt
attagtgcta gtggtggtag tatatactac 180gcagactccg tgaagggccg
gttcaccatc tccagagaca cttccaagaa cacattatat 240ctgcaaatga
acagcctgag agccgacgac acggccgtct attactgtgc gaaatccccg
300tataactgga acccctttga ctattggggc cagggaaccc tggtcaccgt ctcctca
357138119PRTArtificial SequenceSynthetic 138Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Leu Asn Asn Phe
20 25 30 Val Met Asn Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Phe Ile Ser Ala Ser Gly Gly Ser Ile Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr
Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Tyr Asn
Trp Asn Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 139324DNAArtificial SequenceSynthetic 139gaaatagtga
tgacgcagtc tccagccacc ctgtctgtgt ctccagggga acgagccacc 60ctctcctgca
gggccagtct gagtgttagc agcaaattag cctggtacca gcagacacct
120ggccaggctc ccagactcct catctatagt gcctccaccc gggccactgg
tatcccagtc 180aggttcagtg gcagtgggtc tgggacagag ttcactctca
ccatcagcag cctgcagtct 240gaagattttg cggtttatta ctgtcagcag
tataatcatt ggcctccgta cacttttggc 300caggggacca agctggagat caaa
324140108PRTArtificial SequenceSynthetic 140Glu Ile Val Met Thr Gln
Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Leu Ser Val Ser Ser Lys 20 25 30 Leu Ala
Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Val Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Ser65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn His
Trp Pro Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 141357DNAArtificial SequenceSynthetic 141gaggtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacccttaac aactttgtca tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtgcta gtggtggtag
tatatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaatccccg 300tataactgga acccctttga
ctattggggc cagggaaccc tggtcaccgt ctcctca 357142119PRTArtificial
SequenceSynthetic 142Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Leu Asn Asn Phe 20 25 30 Val Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala
Ser Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Ser Pro Tyr Asn Trp Asn Pro Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115 143325DNAArtificial
SequenceSynthetic 143gaaatagtga tgacgcagtc tccagccacc ctgtctgtgt
ctccagggga aagagccacc 60ctctcctgca gggccagtct gagtgttagc agcaaattag
cctggtacca gcagaaacct 120ggccaggctc ccaggctcct catctatagt
gcctccacca gggccactgg tatcccagcc 180aggttcagtg gcagtgggtc
tgggacagag ttcactctca ccatcagcag cctgcagtct 240gaagattttg
cagtttatta ctgtcagcag tataatcatt ggcctccgta cacttttggc
300caggggacca agctggagat caaac 325144108PRTArtificial
SequenceSynthetic 144Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Val Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Leu Ser Val Ser Ser Lys 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Thr
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn His Trp Pro Pro 85 90
95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
145375DNAArtificial SequenceSynthetic 145caggtgcagc tggtggagtc
tgggggaggc ttggaacagc cgggggggtc cctgagactc 60tcctgtgcag gctctggatt
cacctttaga gactatgcca tgacctgggt ccgccaggct 120ccagggaagg
gactggagtg ggtctcatct attagtggtt ccggtggtaa cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaagatcga 300ctctctataa caattcgccc acgctattat
ggtttggacg tctggggcca agggtccacg 360gtcaccgtct cctca
375146125PRTArtificial SequenceSynthetic 146Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Ser Thr Val
Thr Val Ser Ser 115 120 125 14724DNAArtificial SequenceSynthetic
147ggattcacct ttagagacta tgcc 241488PRTArtificial SequenceSynthetic
148Gly Phe Thr Phe Arg Asp Tyr Ala1 5 14924DNAArtificial
SequenceSynthetic 149attagtggtt ccggtggtaa caca 241508PRTArtificial
SequenceSynthetic 150Ile Ser Gly Ser Gly Gly Asn Thr1 5
15154DNAArtificial SequenceSynthetic 151gcgaaagatc gactctctat
aacaattcgc ccacgctatt atggtttgga cgtc 5415218PRTArtificial
SequenceSynthetic 152Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu1 5 10 15 Asp Val153339DNAArtificial
SequenceSynthetic 153gacatcgtgt tgacccagtc tccactctcc ctgcccgtca
cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg tatagtattg
gatacaacta tttggattgg 120tacctgcaga agtcagggca gtctccacag
ctccttatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt
cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg
aggctgagga tgttgggttt tattactgca tgcaagctct acaaactccg
300tacacttttg gccaggggac caagctggag atcaaacga
339154113PRTArtificial SequenceSynthetic 154Asp Ile Val Leu Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ile Gly
Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ser Gly Gln Ser 35 40 45
Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys
Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 110 Arg15533DNAArtificial
SequenceSynthetic 155cagagcctcc tgtatagtat tggatacaac tat
3315611PRTArtificial SequenceSynthetic 156Gln Ser Leu Leu Tyr Ser
Ile Gly Tyr Asn Tyr1 5 10 1579DNAArtificial SequenceSynthetic
157ttgggttct 91583PRTArtificial SequenceSynthetic 158Leu Gly Ser1
15927DNAArtificial SequenceSynthetic 159atgcaagctc tacaaactcc
gtacact 271609PRTArtificial SequenceSynthetic 160Met Gln Ala Leu
Gln Thr Pro Tyr Thr1 5 161372DNAArtificial SequenceSynthetic
161gaggtgcagc tggtggagtc tgggggaggc ttggaacagc cgggggggtc
cctgagactc 60tcctgtgcag gctctggatt cacctttaga gactatgcca tgacctgggt
ccgccaggct 120ccagggaagg gactggagtg ggtctcatct attagtggtt
ccggtggtaa cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaagatcga 300ctctctataa
caattcgccc acgctattat ggtttggacg tctggggcca agggaccacg
360gtcaccgtct cc 372162124PRTArtificial SequenceSynthetic 162Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Glu Gln Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Arg Asp Tyr
20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser
Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser 115 120 163336DNAArtificial
SequenceSynthetic 163gacatcgtga tgacccagtc tccactctcc ctgcccgtca
cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg tatagtattg
gatacaacta tttggattgg 120tacctgcaga agtcagggca gtctccacag
ctccttatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt
cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg
aggctgagga tgttgggttt tattactgca tgcaagctct acaaactccg
300tacacttttg gccaggggac caagctggag atcaaa 336164112PRTArtificial
SequenceSynthetic 164Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ile Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Ser Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Phe Tyr Tyr Cys Met Gln Ala 85 90
95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 165373DNAArtificial SequenceSynthetic 165gaggtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacctttaga gactatgcca tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggtt ccggtggtaa
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagatcga 300ctctctataa caattcgccc
acgctattat ggtttggacg tctggggcca agggaccacg 360gtcaccgtct cct
373166124PRTArtificial SequenceSynthetic 166Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val
Thr Val Ser 115 120 167337DNAArtificial SequenceSynthetic
167gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga
gccggcctcc 60atctcctgca ggtctagtca gagcctcctg tatagtattg gatacaacta
tttggattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct
atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt
ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga
tgttggggtt tattactgca tgcaagctct acaaactccg 300tacacttttg
gccaggggac caagctggag atcaaac 337168112PRTArtificial
SequenceSynthetic 168Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ile Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90
95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 169375DNAArtificial SequenceSynthetic 169caggtgcagc
tggtggagtc tgggggagtc ttggagcagc ctggggggtc cctgagactc 60tcctgtacag
cctctggatt cacctttaga gactatgcca tgacctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcatct attagtggta gtggtggtaa
tacatactac 180gcagactccg tgaggggccg gttcaccatc tccagagaca
actccaacca cacgctgtat 240ctgcaaatga acagcctgag agccgaagac
acggccgtat attactgtgc gaaagatcga 300ctctccataa caattcgccc
acgctattac ggtttggacg tctggggcca agggtccacg 360gtcaccgtct cctca
375170125PRTArtificial SequenceSynthetic 170Gln Val Gln Leu Val Glu
Ser Gly Gly Val Leu Glu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Thr Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Asn His Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Ser Thr Val
Thr Val Ser Ser 115 120 125 17124DNAArtificial SequenceSynthetic
171ggattcacct ttagagacta tgcc 241728PRTArtificial SequenceSynthetic
172Gly Phe Thr Phe Arg Asp Tyr Ala1 5 17324DNAArtificial
SequenceSynthetic 173attagtggta gtggtggtaa taca 241748PRTArtificial
SequenceSynthetic 174Ile Ser Gly Ser Gly Gly Asn Thr1 5
17554DNAArtificial SequenceSynthetic 175gcgaaagatc gactctccat
aacaattcgc ccacgctatt acggtttgga cgtc 5417618PRTArtificial
SequenceSynthetic 176Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro
Arg Tyr Tyr Gly Leu1 5 10 15 Asp Val177324DNAArtificial
SequenceSynthetic 177gatattgtga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60attacttgcc gggcgagtca ggacattagc aattattttg
cctggtatca gcagaagcca 120gggaaagttc ctaaactcct gatctttgct
gcatccactt tgcatccagg ggtcccatct 180cggttcagtg gcagtggatc
tgggacagat ttcactctca ccattagtag cctgcagcct 240gaagatgttg
caacttatta ctgtcaaaag tataacagtg ccccgtacac ttttggccag
300gggaccaagg tggaaatcaa
acga 324178108PRTArtificial SequenceSynthetic 178Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35
40 45 Phe Ala Ala Ser Thr Leu His Pro Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr
Asn Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 17918DNAArtificial SequenceSynthetic
179caggacatta gcaattat 181806PRTArtificial SequenceSynthetic 180Gln
Asp Ile Ser Asn Tyr1 5 1819DNAArtificial SequenceSynthetic
181gctgcatcc 91823PRTArtificial SequenceSynthetic 182Ala Ala Ser1
18327DNAArtificial SequenceSynthetic 183caaaagtata acagtgcccc
gtacact 271849PRTArtificial SequenceSynthetic 184Gln Lys Tyr Asn
Ser Ala Pro Tyr Thr1 5 185372DNAArtificial SequenceSynthetic
185gaggtgcagc tggtggagtc tgggggagtc ttggagcagc ctggggggtc
cctgagactc 60tcctgtacag cctctggatt cacctttaga gactatgcca tgacctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcatct attagtggta
gtggtggtaa tacatactac 180gcagactccg tgaggggccg gttcaccatc
tccagagaca actccaacca cacgctgtat 240ctgcaaatga acagcctgag
agccgaagac acggccgtat attactgtgc gaaagatcga 300ctctccataa
caattcgccc acgctattac ggtttggacg tctggggcca agggaccacg
360gtcaccgtct cc 372186124PRTArtificial SequenceSynthetic 186Glu
Val Gln Leu Val Glu Ser Gly Gly Val Leu Glu Gln Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Arg Asp Tyr
20 25 30 Ala Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Asn His Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Leu Ser
Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser 115 120 187321DNAArtificial
SequenceSynthetic 187gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60attacttgcc gggcgagtca ggacattagc aattattttg
cctggtatca gcagaagcca 120gggaaagttc ctaaactcct gatctttgct
gcatccactt tgcatccagg ggtcccatct 180cggttcagtg gcagtggatc
tgggacagat ttcactctca ccattagtag cctgcagcct 240gaagatgttg
caacttatta ctgtcaaaag tataacagtg ccccgtacac ttttggccag
300gggaccaagc tggagatcaa a 321188107PRTArtificial SequenceSynthetic
188Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30 Phe Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys
Leu Leu Ile 35 40 45 Phe Ala Ala Ser Thr Leu His Pro Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Lys Tyr Asn Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 189373DNAArtificial
SequenceSynthetic 189gaggtgcagc tggtggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttaga gactatgcca
tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct
attagtggta gtggtggtaa tacatactac 180gcagactccg tgaagggccg
gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agccgaggac acggccgtat attactgtgc gaaagatcga
300ctctccataa caattcgccc acgctattac ggtttggacg tctggggcca
agggaccacg 360gtcaccgtct cct 373190124PRTArtificial
SequenceSynthetic 190Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly
Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Asp Arg Leu Ser Ile Thr Ile Arg Pro Arg Tyr Tyr Gly Leu
100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120
191322DNAArtificial SequenceSynthetic 191gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcgagtca
ggacattagc aattatttag cctggtatca gcagaaacca 120gggaaagttc
ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct
180cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagatgttg caacttatta ctgtcaaaag tataacagtg
ccccgtacac ttttggccag 300gggaccaagc tggagatcaa ac
322192107PRTArtificial SequenceSynthetic 192Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser
Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 193355DNAArtificial SequenceSynthetic 193gaagtgcacc
tggtggaatc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtgagg
cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct
120ccggggaagg gcctggaatg ggtctcaggt cttagtcgga caagtgtcag
tataggctat 180gcggactctg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctccctttat 240ttggaaatga acagtctgag acctgaggac
acggccttat attactgtgc aaaatggggg 300acccgggggt attttgacta
ctggggccag ggaaccctgg tcaccgtctc ctcag 355194118PRTArtificial
SequenceSynthetic 194Glu Val His Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser
Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Leu Ser Arg
Thr Ser Val Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu
Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90
95 Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 19524DNAArtificial
SequenceSynthetic 195ggattcacct ttgatgatta tgcc 241968PRTArtificial
SequenceSynthetic 196Gly Phe Thr Phe Asp Asp Tyr Ala1 5
19724DNAArtificial SequenceSynthetic 197cttagtcgga caagtgtcag tata
241988PRTArtificial SequenceSynthetic 198Leu Ser Arg Thr Ser Val
Ser Ile1 5 19933DNAArtificial SequenceSynthetic 199gcaaaatggg
ggacccgggg gtattttgac tac 3320011PRTArtificial SequenceSynthetic
200Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr1 5 10
201322DNAArtificial SequenceSynthetic 201gacatccaga tgacccagtc
tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60atcacttgtc gggcgagtca
ggatattagt atttggttag cctggtatca gcagagtcca 120gggaaagccc
ctaaactcct gatcaatgtt gcatcccgtt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcaacag
tctgcagcct 240gaagattttg taacttacta ttgtcaacag gctaacagtt
tcccgatcac cttcggccaa 300gggacacgac tggcgaccaa ac
322202107PRTArtificial SequenceSynthetic 202Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ile Trp 20 25 30 Leu Ala
Trp Tyr Gln Gln Ser Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Asn Val Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Val Thr Tyr Tyr Cys Gln Gln Ala Asn Ser
Phe Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Ala Thr Lys
100 105 20318DNAArtificial SequenceSynthetic 203caggatatta gtatttgg
182046PRTArtificial SequenceSynthetic 204Gln Asp Ile Ser Ile Trp1 5
2059DNAArtificial SequenceSynthetic 205gttgcatcc 92063PRTArtificial
SequenceSynthetic 206Val Ala Ser1 20727DNAArtificial
SequenceSynthetic 207caacaggcta acagtttccc gatcacc
272089PRTArtificial SequenceSynthetic 208Gln Gln Ala Asn Ser Phe
Pro Ile Thr1 5 209355DNAArtificial SequenceSynthetic 209gaggtgcagc
tggtggagtc tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtgagg
cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct
120ccggggaagg gcctggaatg ggtctcaggt cttagtcgga caagtgtcag
tataggctat 180gcggactctg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctccctttat 240ttggaaatga acagtctgag acctgaggac
acggccttat attactgtgc aaaatggggg 300acccgggggt attttgacta
ctggggccag ggaaccctgg tcaccgtctc ctcag 355210118PRTArtificial
SequenceSynthetic 210Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser
Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Leu Ser Arg
Thr Ser Val Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu
Glu Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90
95 Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 211322DNAArtificial
SequenceSynthetic 211gacatccaga tgacccagtc tccatcttcc gtgtctgcat
ctgtgggaga cagagtcacc 60atcacttgtc gggcgagtca ggatattagt atttggttag
cctggtatca gcagagtcca 120gggaaagccc ctaaactcct gatcaatgtt
gcatcccgtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcaacag tctgcagcct 240gaagattttg
taacttacta ttgtcaacag gctaacagtt tcccgatcac cttcggccaa
300gggacacgac tggagattaa ac 322212107PRTArtificial
SequenceSynthetic 212Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ile Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Ser
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Asn Val Ala Ser Arg
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro65 70 75 80 Glu
Asp Phe Val Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile 85 90
95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
213355DNAArtificial SequenceSynthetic 213gaagtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttgat gattatgcca tgcactgggt ccggcaagct 120ccagggaagg
gcctggagtg ggtctcaggt cttagtcgga caagtgtcag tataggctat
180gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa
ctccctgtat 240ctgcaaatga acagtctgag agctgaggac acggccttgt
attactgtgc aaaatggggg 300acccgggggt attttgacta ctggggccaa
ggaaccctgg tcaccgtctc ctcag 355214118PRTArtificial
SequenceSynthetic 214Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Leu Ser Arg
Thr Ser Val Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90
95 Ala Lys Trp Gly Thr Arg Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 215322DNAArtificial
SequenceSynthetic 215gacatccaga tgacccagtc tccatcttcc gtgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca ggatattagt atttggttag
cctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgtt
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttacta ttgtcaacag gctaacagtt tcccgatcac cttcggccaa
300gggacacgac tggagattaa ac 322216107PRTArtificial
SequenceSynthetic 216Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ile Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile 85 90
95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
217363DNAArtificial SequenceSynthetic 217gaggtgcagc tgttggagtc
tgggggaggc ttgctacagc cgggggggtc cctgagactc 60tcctgtgcag cctctggaat
cacctttagc acctatgcca tgagctgggt ccgtcaggct 120ccagggaggg
ggctggagtg ggtctcagct attagtggta gtggtgatag cacatcctac
180gcagactccg tgaagggccg gttcaccagc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaagtcata 300gcagctcgtc ctcactggaa cttcgatctc
tggggccgtg gcaccctggt cactgtctcc 360tca 363218121PRTArtificial
SequenceSynthetic 218Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Leu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ile Thr Phe Ser Thr Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser
Gly Ser Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ser Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Val Ile Ala Ala Arg Pro His Trp Asn Phe Asp Leu Trp
Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser 115 120
21924DNAArtificial SequenceSynthetic 219ggaatcacct ttagcaccta tgcc
242208PRTArtificial SequenceSynthetic 220Gly Ile Thr Phe Ser Thr
Tyr Ala1 5 22124DNAArtificial SequenceSynthetic 221attagtggta
gtggtgatag caca 242228PRTArtificial SequenceSynthetic 222Ile Ser
Gly Ser Gly Asp Ser Thr1 5 22342DNAArtificial SequenceSynthetic
223gcgaaagtca tagcagctcg tcctcactgg aacttcgatc tc
4222414PRTArtificial SequenceSynthetic 224Ala Lys Val Ile Ala Ala
Arg Pro His Trp Asn Phe Asp Leu1 5 10 225324DNAArtificial
SequenceSynthetic 225gaaattgtgt tgacacagtc tccagccacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagt agatatttag
cctggtatca acagaaacct 120ggccaggctc ccaggctcct catctatgat
gcatccaaca gggccactgg catcccagcc 180aggttcagtg gcagtgggtc
tgggacagac ttcactctca ccatcagcag cctagagcct 240gaagattttg
gagtttatta ctgtcagcag cgtagtgact ggccgctcac tttcggcgga
300gggaccaagg tggagatcaa acgg 324226107PRTArtificial
SequenceSynthetic 226Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80 Glu
Asp Phe Gly Val Tyr Tyr Cys Gln Gln Arg Ser Asp Trp Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
22718DNAArtificial SequenceSynthetic 227cagagtgtta gtagatat
182286PRTArtificial SequenceSynthetic 228Gln Ser Val Ser Arg Tyr1 5
2299DNAArtificial SequenceSynthetic 229gatgcatcc 92303PRTArtificial
SequenceSynthetic 230Asp Ala Ser1 23127DNAArtificial
SequenceSynthetic 231cagcagcgta gtgactggcc gctcact
272329PRTArtificial SequenceSynthetic 232Gln Gln Arg Ser Asp Trp
Pro Leu Thr1 5 233363DNAArtificial SequenceSynthetic 233gaggtgcagc
tgttggagtc tgggggaggc ttgctacagc cgggggggtc cctgagactc 60tcctgtgcag
cctctggaat cacctttagc acctatgcca tgagctgggt ccgtcaggct
120ccagggaggg ggctggagtg ggtctcagct attagtggta gtggtgatag
cacatcctac 180gcagactccg tgaagggccg gttcaccagc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagtcata 300gcagctcgtc ctcactggaa
cttcgatctc tggggccgtg gcaccctggt cactgtctcc 360tca
363234121PRTArtificial SequenceSynthetic 234Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Leu Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr 20 25 30 Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ser Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Val Ile Ala Ala Arg Pro His Trp Asn
Phe Asp Leu Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser
115 120 235324DNAArtificial SequenceSynthetic 235gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagt agatatttag cctggtatca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg gagtttatta ctgtcagcag
cgtagtgact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa acgg
324236107PRTArtificial SequenceSynthetic 236Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80 Glu Asp Phe Gly Val Tyr Tyr Cys Gln Gln Arg Ser Asp
Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 237363DNAArtificial SequenceSynthetic 237gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc cgggggggtc cctgagactc 60tcctgtgcag
cctctggaat cacctttagc acctatgcca tgagctgggt ccgtcaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtgatag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagtcata 300gcagctcgtc ctcactggaa
cttcgatctc tggggccgtg gcaccctggt cactgtctcc 360tca
363238121PRTArtificial SequenceSynthetic 238Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Thr Tyr 20 25 30 Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Val Ile Ala Ala Arg Pro His Trp Asn
Phe Asp Leu Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser
115 120 239324DNAArtificial SequenceSynthetic 239gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagt agatatttag cctggtatca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagtgact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa acgg
324240108PRTArtificial SequenceSynthetic 240Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asp
Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 241366DNAArtificial SequenceSynthetic 241caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60acctgtgcag
cctctggatt caccttcagt agtaatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcaatt atatcatatg atggaaataa
tcaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagca cacgctgtat 240ctggaaatga acagcctgag agctgaggac
acggctgtgt attactgtac aaaagccatc 300tctataagtg gaacttacaa
ctggttcgat tcctggggcc agggaaccct ggtcaccgtc 360tcctca
366242122PRTArtificial SequenceSynthetic 242Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Thr Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Ile Ile Ser Tyr Asp Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys His Thr Leu
Tyr65 70 75 80 Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Lys Ala Ile Ser Ile Ser Gly Thr Tyr Asn
Trp Phe Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 24324DNAArtificial SequenceSynthetic 243ggattcacct
tcagtagtaa tggc 242448PRTArtificial SequenceSynthetic 244Gly Phe
Thr Phe Ser Ser Asn Gly1 5 24524DNAArtificial SequenceSynthetic
245atatcatatg atggaaataa tcaa 242468PRTArtificial SequenceSynthetic
246Ile Ser Tyr Asp Gly Asn Asn Gln1 5 24745DNAArtificial
SequenceSynthetic 247acaaaagcca tctctataag tggaacttac aactggttcg
attcc 4524815PRTArtificial SequenceSynthetic 248Thr Lys Ala Ile Ser
Ile Ser Gly Thr Tyr Asn Trp Phe Asp Ser1 5 10 15
249324DNAArtificial SequenceSynthetic 249gaaattgtat tgacacagtc
tccagccatc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc aggtacttag cctggtacca acagaaacct 120ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc
180aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag
cctagagcct 240gaagattttg cagtttatta ctgtcaacag cgtagcaact
ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa acgg
324250107PRTArtificial SequenceSynthetic 250Glu Ile Val Leu Thr Gln
Ser Pro Ala Ile Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 25118DNAArtificial SequenceSynthetic 251cagagtgtta gcaggtac
182526PRTArtificial SequenceSynthetic 252Gln Ser Val Ser Arg Tyr1 5
2539DNAArtificial SequenceSynthetic 253gatgcatcc 92543PRTArtificial
SequenceSynthetic 254Asp Ala Ser1 25527DNAArtificial
SequenceSynthetic 255caacagcgta gcaactggcc gctcact
272569PRTArtificial SequenceSynthetic 256Gln Gln Arg Ser Asn Trp
Pro Leu Thr1 5 257366DNAArtificial SequenceSynthetic 257caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60acctgtgcag
cctctggatt caccttcagt agtaatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcaatt atatcatatg atggaaataa
tcaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagca cacgctgtat 240ctggaaatga acagcctgag agctgaggac
acggctgtgt attactgtac aaaagccatc 300tctataagtg gaacttacaa
ctggttcgat tcctggggcc agggaaccct ggtcaccgtc 360tcctca
366258122PRTArtificial SequenceSynthetic 258Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Thr Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Ile Ile Ser Tyr Asp Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys His Thr Leu
Tyr65 70 75 80 Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Lys Ala Ile Ser Ile Ser Gly Thr Tyr Asn
Trp Phe Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 259324DNAArtificial SequenceSynthetic 259gaaattgtat
tgacacagtc tccagccatc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc aggtacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcaacag
cgtagcaact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa acgg
324260107PRTArtificial SequenceSynthetic 260Glu Ile Val Leu Thr Gln
Ser Pro Ala Ile Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 261366DNAArtificial SequenceSynthetic 261caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cctctggatt caccttcagt agtaatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaaataa
tcaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agctgaggac
acggctgtgt attactgtac aaaagccatc 300tctataagtg gaacttacaa
ctggttcgat tcctggggcc agggaaccct ggtcaccgtc 360tcctca
366262122PRTArtificial SequenceSynthetic 262Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn 20 25 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Ser Tyr Asp Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Thr Lys Ala Ile Ser Ile Ser Gly Thr Tyr Asn
Trp Phe Asp Ser Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 263324DNAArtificial SequenceSynthetic 263gaaattgtat
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc aggtacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcaacag
cgtagcaact ggccgctcac tttcggcgga 300gggaccaagg tggagatcaa acgg
324264108PRTArtificial SequenceSynthetic 264Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 2658PRTArtificial SequenceSynthetic 265Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5 2668PRTArtificial SequenceSynthetic 266Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 26718PRTArtificial SequenceSynthetic
267Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15 Xaa Xaa26811PRTArtificial SequenceSynthetic 268Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 2693PRTArtificial
SequenceSynthetic 269Xaa Xaa Xaa1 2709PRTArtificial
SequenceSynthetic 270Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5
271330PRTArtificial SequenceSynthetic 271Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
272327PRTArtificial SequenceSynthetic 272Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
Thr65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160 Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180
185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305
310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 273327PRTArtificial
SequenceSynthetic 273Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80 Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp145 150 155 160 Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215
220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320 Leu
Ser Leu Ser Leu Gly Lys 325 274207PRTHomo sapiens 274Met Lys Val
Leu Gln Glu Pro Thr Cys Val Ser Asp Tyr Met Ser Ile1 5 10 15 Ser
Thr Cys Glu Trp Lys Met Asn Gly Pro Thr Asn Cys Ser Thr Glu 20 25
30 Leu Arg Leu Leu Tyr Gln Leu Val Phe Leu Leu Ser Glu Ala His Thr
35 40 45 Cys Ile Pro Glu Asn Asn Gly Gly Ala Gly Cys Val Cys His
Leu Leu 50 55 60 Met Asp Asp Val Val Ser Ala Asp Asn Tyr Thr Leu
Asp Leu Trp Ala65 70 75 80 Gly Gln Gln Leu Leu Trp Lys Gly Ser Phe
Lys Pro Ser Glu His Val 85 90 95 Lys Pro Arg Ala Pro Gly Asn Leu
Thr Val His Thr Asn Val Ser Asp 100 105 110 Thr Leu Leu Leu Thr Trp
Ser Asn Pro Tyr Pro Pro Asp Asn Tyr Leu 115 120 125 Tyr Asn His Leu
Thr Tyr Ala Val Asn Ile Trp Ser Glu Asn Asp Pro 130 135 140 Ala Asp
Phe Arg Ile Tyr Asn Val Thr Tyr Leu Glu Pro Ser Leu Arg145 150 155
160 Ile Ala Ala Ser Thr Leu Lys Ser Gly Ile Ser Tyr Arg Ala Arg Val
165 170 175 Arg Ala Trp Ala Gln Cys Tyr Asn Thr Thr Trp Ser Glu Trp
Ser Pro 180 185 190 Ser Thr Lys Trp His Asn Ser Tyr Arg Glu Pro Phe
Glu Gln His 195 200 205 275231PRTMacaca fasicularis 275Met Gly Trp
Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Val1 5 10 15 Leu
Leu Gln Val Ala Ser Ser Gly Ser Met Lys Val Leu Gln Glu Pro 20 25
30 Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu Trp Lys Met
35 40 45 Gly Gly Pro Thr Asn Cys Ser Ala Glu Leu Arg Leu Leu Tyr
Gln Leu 50 55 60 Val Phe Gln Ser Ser Glu Thr His Thr Cys Val Pro
Glu Asn Asn Gly65 70 75 80 Gly Val Gly Cys Val Cys His Leu Leu Met
Asp Asp Val Val Ser Met 85 90 95 Asp Asn Tyr Thr Leu Asp Leu Trp
Ala Gly Gln Gln Leu Leu Trp Lys 100 105 110 Gly Ser Phe Lys Pro Ser
Glu His Val Lys Pro Arg Ala Pro Gly Asn 115 120 125 Leu Thr Val His
Thr Asn Val Ser Asp Thr Val Leu Leu Thr Trp Ser 130 135 140 Asn Pro
Tyr Pro Pro Asp Asn Tyr Leu Tyr Asn Asp Leu Thr Tyr Ala145 150 155
160 Val Asn Ile Trp Ser Glu Asn Asp Pro Ala Tyr Ser Arg Ile His Asn
165 170 175 Val Thr Tyr Leu Lys Pro Thr Leu Arg Ile Pro Ala Ser Thr
Leu Lys 180 185 190 Ser Gly Ile Ser Tyr Arg Ala Arg Val Arg Ala Trp
Ala Gln His Tyr 195 200 205 Asn Thr Thr Trp Ser Glu Trp Ser Pro Ser
Thr Lys Trp Tyr Asn Ser 210 215 220 Tyr Arg Glu Pro Phe Glu Gln225
230
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