U.S. patent application number 15/874807 was filed with the patent office on 2018-08-30 for methods for inhibiting atherosclerosis by administering an inhibitor of pcsk9.
The applicant listed for this patent is REGENERON PHARMACEUTICALS, INC., SANOFI BIOTECHNOLOGY. Invention is credited to Viktoria Gusarova, Anusch Peyman, William J. Sasiela, Hans-ludwig Schaefer, Uwe Schwahn.
Application Number | 20180244801 15/874807 |
Document ID | / |
Family ID | 52008606 |
Filed Date | 2018-08-30 |
United States Patent
Application |
20180244801 |
Kind Code |
A1 |
Sasiela; William J. ; et
al. |
August 30, 2018 |
METHODS FOR INHIBITING ATHEROSCLEROSIS BY ADMINISTERING AN
INHIBITOR OF PCSK9
Abstract
The present invention provides methods and compositions for
inhibiting atherosclerotic plaque formation in a subject. In
certain embodiments, the methods of the present invention comprise
selecting a subject who has, or is at risk of developing,
atherosclerosis, and administering to the subject a pharmaceutical
composition comprising a proprotein convertase subtilisin/kexin
type 9 (PCSK9) inhibitor. In certain embodiments, this PCSK9
inhibitor is an anti-PCSK9 antibody, or antigen binding
protein.
Inventors: |
Sasiela; William J.;
(Tarrytown, NY) ; Gusarova; Viktoria; (Tarrytown,
NY) ; Peyman; Anusch; (Frankfurt Am Main, DE)
; Schaefer; Hans-ludwig; (Frankfurt Am Main, DE) ;
Schwahn; Uwe; (Frankfurt Am Main, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENERON PHARMACEUTICALS, INC.
SANOFI BIOTECHNOLOGY |
Tarrytown
Paris |
NY |
US
FR |
|
|
Family ID: |
52008606 |
Appl. No.: |
15/874807 |
Filed: |
January 18, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14896196 |
Dec 4, 2015 |
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PCT/US14/41204 |
Jun 6, 2014 |
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15874807 |
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61832459 |
Jun 7, 2013 |
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61892215 |
Oct 17, 2013 |
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61944855 |
Feb 26, 2014 |
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62002508 |
May 23, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 9/10 20180101; C07K
16/40 20130101; Y02A 50/30 20180101; A61K 31/4015 20130101; A61K
39/3955 20130101; A61K 2039/505 20130101; C07K 2317/565 20130101;
C07K 2317/76 20130101; C07K 2317/21 20130101; A61P 43/00 20180101;
C07K 14/4703 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 31/4015 20060101 A61K031/4015; A61K 39/395
20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 7, 2013 |
EP |
13305762.0 |
Oct 18, 2013 |
EP |
13306436.0 |
Claims
1-39. (canceled)
40. A method of inhibiting atherosclerotic plaque formation, the
method comprising: (a) selecting a subject who has atherosclerosis;
and (b) administering to the subject a pharmaceutical composition
comprising an antibody or antigen-binding fragment thereof that
specifically binds to proprotein convertase subtilisin/kexin type 9
(PCSK9), wherein the antibody or antigen-binding fragment thereof
comprises heavy and light chain CDR amino acid sequences having SEQ
ID NOs:86, 87, 88, 90, 91, and 92, such that the administration of
the antibody or antigen-binding fragment thereof inhibits an
increase in the number, total area, severity, or instability of
atherosclerotic plaques in the subject.
41. The method of claim 40, wherein the subject has suffered a
stroke or myocardial infarction.
42. The method of claim 40, wherein the subject has one or more of
the following conditions: a) a disease or disorder selected from
the group consisting of type I diabetes mellitus, type II diabetes
mellitus, Kawasaki disease, chronic inflammatory disease, and
hypertension; b) heterozygous Familial Hypercholesterolemia (heFH);
and c) a form of hypercholesterolemia that is not Familial
Hypercholesterolemia (nonFH).
43. The method of claim 40, wherein the subject has an elevated
level of an inflammatory marker.
44. The method of claim 43, wherein the inflammatory marker is
C-reactive protein or an inflammatory cytokine.
45. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof comprises an HCVR having at least 95% sequence
identity to the amino acid sequence of SEQ ID NO:85 and an LCVR
having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO:89.
46. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof inhibits the increase of the number, total area,
severity, or instability of atherosclerotic plaques in the subject
by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
47. The method of claim 40, wherein the subject is on another
lipid-modifying agent before and/or during administration of the
antibody or antigen-binding fragment thereof.
48. The method of claim 47, wherein the therapeutic lipid-modifying
agent is selected from the group consisting of a statin, ezetimibe,
a fibrate, niacin, an omega-3 fatty acid, and a bile acid
resin.
49. The method of claim 48, wherein the statin is selected from the
group consisting of cerivastatin, atorvastatin, simvastatin,
pitavastatin, rosuvastatin, fluvastatin, lovastatin and
pravastatin.
50. The method of claim 40, wherein the subject is not on another
lipid-modifying agent before and/or during administration of the
antibody or antigen-binding fragment thereof.
51. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof is administered subcutaneously.
52. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof inhibits an increase in the number of
atherosclerotic plaques.
53. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof inhibits an increase in the total area of
atherosclerotic plaques.
54. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof inhibits an increase in the severity of
atherosclerotic plaques.
55. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof inhibits an increase in the instability of
atherosclerotic plaques.
56. The method of claim 40, wherein the antibody or antigen-binding
fragment thereof inhibits an increase in the number, total area,
severity, and instability of atherosclerotic plaques.
57. A method of inhibiting atherosclerotic plaque formation, the
method comprising: (a) selecting a subject who has atherosclerosis
and hypercholesterolemia; and (b) administering to the subject a
pharmaceutical composition comprising an antibody or
antigen-binding fragment thereof that specifically binds to PCSK9,
wherein the antibody or antigen-binding fragment thereof comprises
heavy and light chain CDR amino acid sequences having SEQ ID
NOs:86, 87, 88, 90, 91, and 92, such that the administration of the
antibody or antigen-binding fragment thereof inhibits an increase
in the number, total area, severity, or instability of
atherosclerotic plaques in the subject.
58. The method of claim 57, wherein the antibody or antigen-binding
fragment thereof comprises an HCVR having at least 95% sequence
identity to the amino acid sequence of SEQ ID NO:85 and an LCVR
having at least 95% sequence identity to the amino acid sequence of
SEQ ID NO:89.
59. A method of inhibiting atherosclerotic plaque formation, the
method comprising: (a) selecting a subject who has atherosclerosis
and hypercholesterolemia; and (b) administering to the subject
subcutaneously a pharmaceutical composition comprising an antibody
or antigen-binding fragment thereof that specifically binds to
PCSK9, wherein the antibody or antigen-binding fragment thereof
comprises: (i) an HCVR comprising heavy chain CDR amino acid
sequences having SEQ ID NOs:86, 87, and 88, and having at least 95%
sequence identity to the amino acid sequence of SEQ ID NO:85; and
(ii) an LCVR comprising light chain CDR amino acid sequences having
SEQ ID NOs: 90, 91, and 92, and having at least 95% sequence
identity to the amino acid sequence of SEQ ID NO:89, such that the
administration of the antibody or antigen-binding fragment thereof
inhibits an increase in the number, total area, severity, or
instability of atherosclerotic plaques in the subject.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Application No. 61/832,459, filed Jun. 7, 2013;
European Patent Application No. 13305762.0, filed Jun. 7, 2013;
U.S. Provisional Application No. 61/892,215, filed Oct. 17, 2013;
European Patent Application No. 13306436.0, filed Oct. 18, 2013;
U.S. Provisional Application No. 61/944,855, filed Feb. 26, 2014;
and U.S. Provisional Application No. 62/002,508, filed May 23,
2014. The content of each of the aforementioned applications is
hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of therapeutic
treatments for atherosclerosis. More specifically, the invention
relates to the administration of proprotein convertase
subtilisin/kexin type 9 (PCSK9) inhibitors to inhibit
atherosclerotic plaque formation in a subject.
BACKGROUND
[0003] Atherosclerosis represents the major cause of death and
cardiovascular morbidity in the western world. Risk factors for
atherosclerosis include high low density lipoprotein (LDL)
cholesterol levels, low high density lipoprotein (HDL) cholesterol
levels, hypertension, diabetes mellitus, family history, male
gender, cigarette smoke, and high serum cholesterol.
[0004] Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a
proprotein convertase belonging to the proteinase K subfamily of
the secretory subtilase family. It is a serine protease involved in
LDL metabolism that is mainly expressed in the liver, kidney, and
intestines. Evidence suggests that PCSK9 increases plasma LDL
cholesterol by promoting degradation of the LDL receptor, which
mediates LDL endocytosis in the liver, the major route of LDL
clearance from circulation.
[0005] The use of PCSK9 inhibitors (anti-PCSK9 antibodies) to
reduce serum total cholesterol, LDL cholesterol and serum
triglycerides have been described in U.S. Pat. Nos. 8,062,640,
8,357,371, and US Patent Application Publication No. 2013/0064834.
Nonetheless, PCSK9 inhibitors have not been reported to reduce or
inhibit progression of atherosclerotic plaque formation in a
subject. There remains a need in the art for therapeutic methods of
inhibiting atherosclerotic plaque formation.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention addresses the need in the art for
therapeutic methods by providing methods and compositions for
inhibiting atherosclerotic plaque formation in a subject. In
certain aspects, the methods and compositions of the present
invention generally comprise selecting a subject who has, or is at
risk of developing, atherosclerosis, and administering to the
subject a pharmaceutical composition comprising a proprotein
convertase subtilisin/kexin type 9 (PCSK9) inhibitor (e.g., an
anti-PCSK9 antibody or antigen binding protein). In other aspects,
the invention provides a pharmaceutical composition comprising a
PCSK9 inhibitor for use in the inhibition of atherosclerotic plaque
formation in a subject. In yet other aspects, the invention
provides a method of inhibiting atherosclerotic plaque formation in
a subject by administering to the subject a pharmaceutical
composition comprising a PCSK9 inhibitor.
[0007] In certain embodiments, the subject is nonhyperlipidemic. In
other embodiments, the subject is apparently healthy.
[0008] In another aspect, the invention provides a method of
treating or inhibiting progression of atherosclerosis in a subject,
the method comprising: (a) selecting a subject who has suffered a
stroke or myocardial infarction; and (b) administering to the
subject a pharmaceutical composition comprising a PCSK9 inhibitor,
thereby treating or inhibiting progression of atherosclerosis. In
one embodiment, the subject is nonhyperlipidemic.
[0009] In another aspect, the invention provides a pharmaceutical
composition comprising a PCSK9 inhibitor for use in treating or
inhibiting progression of atherosclerosis in a subject who has
suffered a stroke or myocardial infarction.
[0010] In another aspect, the invention provides a method of
treating or inhibiting progression of atherosclerosis in a
nonhyperlipidemic subject, the method comprising (a) selecting a
subject who has, or is known to be at risk of developing,
atherosclerosis, wherein the subject is nonhyperlipidemic; and (b)
administering to the subject a pharmaceutical composition
comprising a PCSK9 inhibitor, thereby treating or inhibiting
progression of atherosclerosis. In one embodiment, the subject is
apparently healthy. In another embodiment, the subject is
nonhypercholesterolemic. In another embodiment, the subject is
nonhypertriglyceridemic.
[0011] In another aspect, the invention provides a pharmaceutical
composition comprising a PCSK9 inhibitor for use in treating or
inhibiting progression of atherosclerosis in a nonhyperlipidemic
subject, wherein the subject has, or is known to be at risk of
developing, atherosclerosis.
[0012] In certain embodiments, the subject has a disease or
disorder selected from the group consisting of type I diabetes
mellitus, type II diabetes mellitus, Kawasaki disease, chronic
inflammatory disease, and hypertension. In another embodiment, the
subject has elevated levels of an inflammatory marker. In a further
embodiment, the inflammatory marker is C-reactive protein. In
another further embodiment, the inflammatory marker is an
inflammatory cytokine.
[0013] In certain exemplary embodiments, the PCSK9 inhibitor is an
antibody, or antigen binding protein, that specifically binds to
PCSK9. In other embodiments, the antibody comprises heavy and light
chain CDR amino acid sequences having SEQ ID NOs 12, 13, 14, 16,
17, and 18. In certain embodiments, the antibody or antigen-binding
protein comprises an HCVR having the amino acid sequence of SEQ ID
NO:11 and an LCVR having the amino acid sequence of SEQ ID NO:15.
In certain embodiments, the antibody or antigen-binding protein
comprises heavy and light chain CDR amino acid sequences having SEQ
ID NOs 2, 3, 4, 7, 8, and 10. In certain embodiments, the antibody
or antigen-binding protein comprises an HCVR having the amino acid
sequence of SEQ ID NO:1 and an LCVR having the amino acid sequence
of SEQ ID N0:6. In one embodiment, the antibody or antigen-binding
protein binds to the same epitope on
[0014] PCSK9 as an antibody comprising the heavy and light variable
domain amino acid sequences forth in SEQ ID NOs: 1 and 6; or 11 and
15, respectively. In some embodiments, the antibody or
antigen-binding protein competes for binding to PCSK9 with an
antibody comprising the heavy and light chain variable domain amino
acid sequences forth in SEQ ID NOs: 1 and 6; or 11 and 15,
respectively.
[0015] In another embodiment, the PCSK9 inhibitor reduces
atherosclerotic plaque formation in the subject by at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.
[0016] In some embodiments, the subject has heterozygous Familial
Hypercholesterolemia (heFH). In other embodiments, the subject has
a form of hypercholesterolemia that is not Familial
Hypercholesterolemia (nonFH).
[0017] In certain embodiments, the subject is on another
lipid-modifying agent before and/or during administration of the
antibody or antigen-binding protein. Therapeutic lipid-modifying
agents include statins, cholesterol uptake inhibitors, ezetimibe,
fibrates, niacin, omega-3 fatty acids, and bile acid resins.
Statins include cerivastatin, atorvastatin, simvastatin,
pitavastatin, rosuvastatin, fluvastatin, lovastatin and
pravastatin.
[0018] In some embodiments, the antibody or antigen binding protein
is administered subcutaneously.
[0019] The invention also provides a unit dosage form comprising
the pharmaceutical compositions of PCSK9 inhibitors. In certain
embodiments, the PCSK9 inhibitor is an antibody or antigen-binding
fragment and the unit dosage form comprises 75 mg, 150 mg, 200 mg,
or 300 mg of the antibody or antigen-binding fragment. The unit
dosage form may be a pre-filled syringe, a pre-filled autoinjector,
a vial, a cartridge, a reusable syringe, or a reusable
autoinjector; the unit dosage form may be hermetically sealed, and
may further indicate the dosage.
[0020] The invention also provides an article of manufacture or kit
comprising the pharmaceutical composition of a PCSK9 inhibitor and
a container, and in some embodiments also includes instructions for
use.
[0021] The invention also provides a use of composition comprising
a PCSK9 inhibitor in the manufacture of a medicament for: (a)
inhibition of atherosclerotic plaque formation in a subject; (b)
treating or inhibiting progression of atherosclerosis in a subject
who has suffered a stroke or myocardial infarction; or (c) treating
or inhibiting progression of atherosclerosis in a nonhyperlipidemic
subject, wherein the subject has, or is known to be at risk of
developing, atherosclerosis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-D shows a series of graphs depicting the effect of
mAb316P, atorvastatin, and their combination on several parameters
after an 18 week treatment period. FIG. 1A shows the effect on
average plasma total cholesterol; FIG. 1B shows the effect on
triglyceride levels. FIGS. 1C and 1D show the lipoprotein profiles
for cholesterol as assessed by FPLC lipoprotein separation after 12
weeks of treatment to study the effects of mAb316P alone (FIG. 1C)
and in combination with atorvastatin (FIG. 10). ***P<0.0045 as
compared to control; .dagger..dagger..dagger.P<0.0045 as
compared to atorvastatin;
.dagger-dbl..dagger-dbl..dagger-dbl.P<0.0045 compared 3 mg/kg
mAb316P to 10 mg/kg mAb316P (n=15 per group).
[0023] FIG. 2A shows a graph of the effect of mAb316P,
atorvastatin, and their combination on hepatic low density
lipoprotein receptor protein levels. ***P<0.0045 as compared to
control; .dagger.P<0.05; .dagger..dagger.P<0.01;
.dagger..dagger..dagger.P<0.0045 as compared to atorvastatin;
.dagger-dbl.P<0.05 compared 3 mg/kg mAb316P to 10 mg/kg mAb316P
(n=8 per group). FIG. 2B shows a graph of the effect of mAb316P,
atorvastatin, and their combination on non-HDL-cholesterol levels.
***P<0.001 as compared to control; #P<0.05; ##P<0.01;
###P<0.001 as compared to atorvastatin.
[0024] FIGS. 3A-F is a series of photos depicting the effect of
mAb316P, atorvastatin, and their combination on plaque morphology.
Representative images of hematoxylin-phloxine-saffron-stained
atherosclerotic lesions in a cross section of the aortic root area
for the control (FIG. 3A), 3 mg/kg mAb316P (FIG. 3B), 10 mg/kg
mAb316P (FIG. 3C), atorvastatin (FIG. 3D), 3 mg/kg
mAb316P+atorvastatin (FIG. 3E) and 10 mg/kg mAb316P+atorvastatin
(FIG. 3F) groups, respectively, after 18 weeks of treatment.
[0025] FIGS. 4A-D shows a series of graphs depicting the effect of
mAb316P, atorvastatin, and their combination on atherosclerosis
development in aortic root and arch. After 18 weeks of treatment,
the total lesion area (FIG. 4A) and number of lesions (FIG. 4B) per
cross section were assessed. Lesion severity was assessed and
categorized as no lesions, mild (type I-III) and severe (type IV-V)
lesions (FIG. 4C). The total plaque load in the aortic arch (FIG.
4D) was analyzed after oil-red O-staining. Data are expressed as
percentage of the stained area. *P<0.05; ***P<0.0045 as
compared to control; .dagger.P<0.05; .dagger..dagger.P<0.01;
.dagger..dagger..dagger.P<0.0045 as compared to atorvastatin;
.dagger-dbl.P<0.05; .dagger-dbl..dagger-dbl.P<0.01;
.dagger-dbl..dagger-dbl..dagger-dbl.P<0.0045 compared 3 mg/kg
mAb316P to 10 mg/kg mAb316P (n=15 per group in the root area and
n=6-7 in the arch).
[0026] FIG. 5 is a graph depicting the correlation between average
plasma total cholesterol and atherosclerotic lesion area. The
square root of the lesion area was plotted against average total
cholesterol. Linear regression analysis was performed.
[0027] FIG. 6 is a series of photos depicting the effect of
mAb316P, atorvastatin, and their combination on lesion composition.
Representative images of immunostaining with Mac-3 for macrophages
followed by sirius red staining for collagen and alpha actin for
smooth muscle cells (SMC) for the control and after 18 weeks of
treatment with mAb316P alone and in combination with
atorvastatin.
[0028] FIGS. 7A-C is a series of graphs depicting the effect of
mAb316P, atorvastatin, and their combination on lesion composition.
Macrophage content (FIG. 7A left) and necrotic content (FIG. 7A
right), including cholesterol clefts, as destabilization factors,
and SMC content (FIG. 7B left) and collagen content (FIG. 7B right)
as stabilization factors were determined in the severe (type IV-V)
lesions after correcting for lesion size. *P<0.05, **P<0.01,
***P<0.001 as compared to control; #P<0.05, ##P<0.01,
###P<0.001 as compared to atorvastatin. The plaque stability
index was calculated as the ratio of the stabilization factors to
the destabilization factors (FIG. 7C). *P<0.05, ***P<0.0045
as compared to control; .dagger.P<0.05,
.dagger..dagger..dagger.P<0.0045 as compared to atorvastatin;
.dagger-dbl.P<0.05 for 3 mg/kg mAb316P compared to 10 mg/kg
mAb316P (n=15 per group).
[0029] FIGS. 8A-C is a series of graphs depicting the effect of
mAb316P, atorvastatin, and their combination on markers of vascular
inflammation. The number of monocytes adhering to the endothelium
(FIG. 8A) and the number of T-cells in the aortic root area (FIG.
8B) were determined per cross section. In addition, intercellular
adhesion molecule 1 (ICAM-1) was determined as percentage of the
stained area (FIG. 8C). Representative images are included.
*P<0.05, **P<0.01, ***P<0.0045 as compared to control;
.dagger.P<0.05, .dagger..dagger..dagger.P<0.0045 as compared
to atorvastatin (n=15 per group).
DETAILED DESCRIPTION
[0030] 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.
[0031] 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.).
[0032] Although any methods and materials similar or equivalent to
those described herein can be used in the practice of the present
invention, exemplary methods and materials are now described. All
publications mentioned herein, and the sequence listing filed
concurrently herewith, are incorporated herein by reference in
their entirety.
Methods for Inhibiting Atherosclerosis
[0033] The methods of the present invention comprise selecting
subjects that have, or are at risk of developing, atherosclerosis,
and administering to these subjects a pharmaceutical composition
comprising a PCSK9 inhibitor.
[0034] Risk factors for atherosclerosis are well known in the art
and include, without limitation, high low density lipoprotein (LDL)
cholesterol levels, low high density lipoprotein (HDL) cholesterol
levels, hypertension, diabetes mellitus, family history, male
gender, cigarette smoking, and high serum cholesterol. Methods of
assessing these risk factors for a given subject are also well
known in the art.
[0035] In certain embodiments, the selected subject is
nonhyperlipidemic. A "non hyperlipidemic" is a subject that is a
nonhypercholesterolemic and/or a nonhypertriglyceridemic subject. A
"nonhypercholesterolemic" subject is one that does not fit the
current criteria established for a hypercholesterolemic subject. A
"nonhypertriglyceridemic" subject is one that does not fit the
current criteria established for a hypertriglyceridemic subject
(See, e.g., Harrison's Principles of Experimental Medicine, 13th
Edition, McGraw-Hill, Inc., N.Y). A hypercholesterolemic subject
has an LDL level of >160 mg/dL, or >130 mg/dL and at least
two risk factors selected from the group consisting of male gender,
family history of premature coronary heart disease, cigarette
smoking (more than 10 per day), hypertension, low HDL (<35
mg/dL), diabetes mellitus, hyperinsulinemia, abdominal obesity,
high lipoprotein (a), and personal history of cerebrovascular
disease or occlusive peripheral vascular disease. A
hypertriglyceridemic subject has a triglyceride (TG) level of
>250 mg/dL. Thus, a nonhyperlipidemic subject is defined as one
whose cholesterol and triglyceride levels are below the limits set
as described above for both the hypercholesterolemic and
hypertriglyceridemic subjects. In certain embodiments the selected
subject is neither nonhyperlipidemic nor receiving treatment for
hyperlipidemia.
[0036] In certain embodiments, the selected subject is apparently
healthy. "Apparently healthy," as used herein, means individuals
who have not previously had an acute, adverse cardiovascular event
such as a myocardial infarction (i.e., individuals who are not at
an elevated risk of a second adverse cardiovascular event due to a
primary adverse cardiovascular event). Apparently healthy
individuals also do not otherwise exhibit symptoms of disease.
[0037] In certain embodiments, the selected subject has previously
suffered an acute adverse cardiovascular event such as a myocardial
infarction, stroke, angina pectoris and/or peripheral
arteriovascular disease. In one embodiment, the selected subject
has previously suffered an acute adverse cardiovascular event such
as a myocardial infarction, stroke, angina pectoris and/or
peripheral arteriovascular disease, but is nonhyperlipidemic. In
one embodiment, the selected subject has previously suffered an
acute adverse cardiovascular event such as a myocardial infarction,
stroke, angina pectoris and/or peripheral arteriovascular disease,
but is neither nonhyperlipidemic nor receiving treatment for
hyperlipidemia.
[0038] In certain embodiments, the selected subject has a disease
or disorder selected from the group consisting of type I diabetes
mellitus, type II diabetes mellitus, Kawasaki disease, chronic
inflammatory disease, and hypertension. In one embodiment, the
selected subject has a disease or disorder selected from the group
consisting of type I diabetes mellitus, type II diabetes mellitus,
Kawasaki disease, chronic inflammatory disease, and hypertension,
but is nonhyperlipidemic. In one embodiment, the selected subject
has a disease or disorder selected from the group consisting of
type I diabetes mellitus, type II diabetes mellitus, Kawasaki
disease, chronic inflammatory disease, and hypertension, but is
neither nonhyperlipidemic nor receiving treatment for
hyperlipidemia.
[0039] In certain embodiments, the selected subject has elevated
levels of an inflammatory marker. In one embodiment, the selected
subject has elevated levels of an inflammatory marker, but is
nonhyperlipidemic. Any marker of systemic inflammation can be
utilized for the purposes of the present invention. Suitable
inflammatory markers include, without limitation, C-reactive
protein (see e.g., U.S. Pat. No. 7,964,614, which is incorporated
by reference herein in its entirety), cytokines (e.g., Il-6, IL-8,
and/or IL-17), and cellular adhesion molecules (e.g., ICAM-1,
ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, and PECAM).
[0040] The level of an inflammatory marker can be obtained by any
art-recognized assay. Typically, the level is determined by
measuring the level of the marker in a body fluid, for example,
blood, lymph, saliva, urine and the like. The level can be
determined by ELISA, or immunoassays or other conventional
techniques for determining the presence of the marker. To determine
if levels of the inflammatory marker are elevated, the level of the
marker measured in a subject can be compared to a suitable control
(e.g., a predetermined value and/or a value obtained from a matched
healthy subject).
[0041] In one embodiment, present invention provides a
pharmaceutical composition comprising a PCSK9 inhibitor for use in
the inhibition of atherosclerotic plaque formation in a subject, as
well as in treating or inhibiting progression of atherosclerosis in
a subject.
[0042] The present invention also provides uses of a pharmaceutical
composition comprising a PCSK9 inhibitor for the manufacture of a
medicament for any of the methods described herein, including
inhibition of atherosclerotic plaque formation, and treating or
inhibiting progression of atherosclerosis in a subject.
PCSK9 Inhibitors
[0043] In certain aspects, the methods of the present invention
comprise administering to a subject a therapeutic composition
comprising a PCSK9 inhibitor.
[0044] As used herein, a "PCSK9 inhibitor" is any agent which binds
to or interacts with human PCSK9 and inhibits the normal biological
function of PCSK9 in vitro or in vivo. Non-limiting examples of
categories of PCSK9 inhibitors include small molecule PCSK9
antagonists, antagonistic nucleic acid molecules (e.g., RNAi
molecules) peptide-based PCSK9 antagonists (e.g., "peptibody"
molecules), and antibodies or antigen-binding fragments of
antibodies that specifically bind human PCSK9.
[0045] As used herein, the term "proprotein convertase
subtilisin/kexin type 9" or "PCSK9" refers to PCSK9 having the
nucleic acid sequence shown in SEQ ID NO:197 and the amino acid
sequence of SEQ ID NO:198, or a biologically active fragment
thereof.
[0046] In certain embodiments, administration of the PCSK9
inhibitor reduces atherosclerotic plaque formation in the subject
(e.g., a human subject) by at least 10% (e.g., 10% 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 100%) relative to atherosclerotic
plaque formation in an untreated subject.
[0047] In certain embodiments, the PCSK9 inhibitor is an antibody
or antigen-binding protein that specifically binds to PCSK9. As
used herein, the term "antibody" refers 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 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-PCSK9 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.
[0048] The term "antibody," as used herein, also includes
antigen-binding fragments of full antibody molecules and
antigen-binding proteins. The terms "antigen-binding portion" of an
antibody, "antigen-binding fragment" of an antibody,
"antigen-binding protein," 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.
[0049] Non-limiting examples of antigen-binding fragments or
antigen-binding proteins 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 expressions
"antigen-binding fragment" and `antigen-binding proteins" as used
herein.
[0050] 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.
[0051] 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-CH3, (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-CH2-CH3;
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, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,
35, 40, 50, 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)).
[0052] 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, including the exemplary bispecific
antibody formats disclosed herein, 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.
[0053] 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.
[0054] 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.
[0055] 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
VH 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.
[0056] 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.
[0057] 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 (see e.g., 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, CH2 or CH3 region which may be desirable,
for example, in production, to improve the yield of the desired
antibody form.
[0058] 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.
[0059] The term "specifically binds," or the like, means that an
antibody or antigen-binding protein 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" PCSK9, as used in the context of
the present invention, includes antibodies that bind PCSK9 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 PCSK9 can, however, have cross-reactivity to other
antigens, such as PCSK9 molecules from other (non-human)
species.
[0060] Exemplary, non-limiting, PCSK9 inhibitors are set forth
herein and include for example, inhibitors described in US Patent
Application Publication Nos. US20130115223, US20130071405,
US20130064834, US20130064825, US20120122954, US20120015435,
US20110313024, US 20110015252, US20110230542, US20110009628, and
U.S. Ser. No. 61/682,349, which are each incorporated herein by
reference in their entireties. In some embodiments, the PCSK9
inhibitor is an antibody, including an antibody having VH/VL
sequences of Ab "LGT209" as described in International Publication
No. WO2011/072263; an antibody having VH/VL sequences of Ab "L1L3"
as described in International Publication No. WO2010/029513; an
antibody having VH/VL sequences of Ab "5L1721H23_6L3" as described
in International Publication No. WO2011/111007; an antibody having
VH/VL sequences of Ab "5L1721H23_6L3H3" as described in
International Publication No. WO2011/111007; an antibody having
VH/VL sequences of Ab "31H4" as described in International
Publication No. WO2009/026558; an antibody having VH/VL sequences
of Ab "1B20" as described in US Patent Application Publication No.
US 2009/0232795; an antibody having VH/VL sequences of Ab "21B12"
described in US Patent Application Publication No. US 2009/0142352;
an antibody having VH/VL sequences of Ab "508.20.28" as described
in US Patent Application Publication No. US 2012/0195910; or an
antibody having VH/VL sequences of Ab "508.20.33" as described in
US Patent Application Publication No. US 2012/0195910.
[0061] Anti-PCSK9 antibodies useful in the methods and compositions
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 VH 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.
[0062] The present invention also includes methods involving the
use of anti-PCSK9 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-PCSK9 antibodies having HCVR;
LCVR; CDR; HCVR and LCVR; HCVR and CDR; LCVR and CDR; or HCVR, LCVR
and CDR amino acid sequences with, e.g., 10 or fewer, 9 or fewer, 8
or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or
fewer, 2 or fewer, or 1 conservative amino acid substitutions
relative to any of the HCVR, LCVR, and/or CDR amino acid sequences
disclosed herein. In certain embodiments, the substitutions are in
CDR amino acid sequences, e.g. CDR1, CDR2 and/or CDR3. In other
embodiments, other embodiments, the substitutions are in CDR3 amino
acid sequences.
[0063] 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.).
[0064] The term "K.sub.D", as used herein, is intended to refer to
the equilibrium dissociation constant of a particular
antibody-antigen interaction.
[0065] 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.
[0066] According to certain embodiments, the anti-PCSK9 antibody
used in the methods of the present invention is an antibody with
pH-dependent binding characteristics. As used herein, the
expression "pH-dependent binding" means that the antibody or
antigen-binding protein exhibits "reduced binding to PCSK9 at
acidic pH as compared to neutral pH" (for purposes of the present
disclosure, both expressions may be used interchangeably). For the
example, antibodies "with pH-dependent binding characteristics"
includes antibodies and antigen-binding fragments thereof that bind
PCSK9 with higher affinity at neutral pH than at acidic pH. In
certain embodiments, the antibodies and antigen-binding fragments
of the present invention bind PCSK9 with at least 3, 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or
more times higher affinity at neutral pH than at acidic pH.
[0067] According to this aspect of the invention, the anti-PCSK9
antibodies with pH-dependent binding characteristics may possess
one or more amino acid variations relative to the parental
anti-PCSK9 antibody. For example, an anti-PCSK9 antibody with
pH-dependent binding characteristics may contain one or more
histidine substitutions or insertions, e.g., in one or more CDRs of
a parental anti-PCSK9 antibody. Thus, according to certain
embodiments of the present invention, methods are provided
comprising administering an anti-PCSK9 antibody which comprises CDR
amino acid sequences (e.g., heavy and light chain CDRs) which are
identical to the CDR amino acid sequences of a parental anti-PCSK9
antibody, except for the substitution of one or more amino acids of
one or more CDRs of the parental antibody with a histidine residue.
The anti-PCSK9 antibodies with pH-dependent binding may possess,
e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more histidine substitutions,
either within a single CDR of a parental antibody or distributed
throughout multiple (e.g., 2, 3, 4, 5, or 6) CDRs of a parental
anti-PCSK9 antibody. For example, the present invention includes
the use of anti-PCSK9 antibodies with pH-dependent binding
comprising one or more histidine substitutions in HCDR1, one or
more histidine substitutions in HCDR2, one or more histidine
substitutions in HCDR3, one or more histidine substitutions in
LCDR1, one or more histidine substitutions in LCDR2, and/or one or
more histidine substitutions in LCDR3, of a parental anti-PCSK9
antibody.
[0068] As used herein, the expression "acidic pH" means a pH of 6.0
or less (e.g., less than about 6.0, less than about 5.5, less than
about 5.0, etc.). The expression "acidic pH" includes pH values of
about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5,
5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As
used herein, the expression "neutral pH" means a pH of about 7.0 to
about 7.4. The expression "neutral pH" includes pH values of about
7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.
Preparation of Human Antibodies
[0069] 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 PCSK9.
[0070] 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 PCSK9 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] Specific examples of human antibodies or antigen-binding
fragments of antibodies that specifically bind PCSK9 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:1 and 11, or a
substantially similar sequence thereof having at least 90%, at
least 95%, at least 98% or at least 99% sequence identity.
Alternatively, specific examples of human antibodies or
antigen-binding fragments of antibodies that specifically bind
PCSK9 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 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141,
149, 157, 165, 173, 181, and 189, or a substantially similar
sequence thereof having at least 90%, at least 95%, at least 98% or
at least 99% sequence identity. 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 6 and 15, or a substantially similar sequence thereof having at
least 90%, at least 95%, at least 98% or at least 99% sequence
identity. Alternatively, 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 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145,
153, 161, 169, 177, 185, and 193, or a substantially similar
sequence thereof having at least 90%, at least 95%, at least 98% or
at least 99% sequence identity.
[0075] Sequence identity between two amino acids sequences is
determined over the entire length of the reference amino acid
sequence, i.e. the amino acid sequence identified with a SEQ ID NO,
using the best sequence alignment and/or over the region of the
best sequence alignment between the two amino acid sequences,
wherein the best sequence alignment can be obtained with art known
tools, e.g. Align, using standard settings, preferably
EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend
0.5.
[0076] In certain embodiments of the present invention, the
antibody or antigen-binding protein 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:1/6 and 11/15.
Alternatively, in certain embodiments of the present invention, the
antibody or antigen-binding protein 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:37/41, 45/49,
53/57, 61/65, 69/73, 77/81, 85/89, 93/97, 101/105, 109/113,
117/121, 125/129, 133/137, 141/145, 149/153, 157/161, 165/169,
173/177, 181/185, and 189/193.
[0077] In certain embodiments of the present invention, the
anti-PCSK9 antibody, or antigen-binding protein, that can be used
in the methods of the present invention has
HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 amino acid sequences selected
from SEQ ID NOs: 2/3/4/7/8/10 (mAb316P) and 12/13/14/16/17/18
(mAb300N) (See US Patent App. Publ No. 2010/0166768) and
12/13/14/16/17/18, wherein SEQ ID NO:16 comprises a substitution of
histidine for leucine at amino acid residue 30 (L3OH).
[0078] In certain embodiments of the present invention, the
antibody or antigen-binding protein comprises HCVR/LCVR amino acid
sequence pairs selected from the group consisting of SEQ ID NOs:1/6
and 11/15. In certain exemplary embodiments, the antibody or
antigen-binding protein comprises an HCVR amino acid sequence of
SEQ ID NO:1 and an LCVR amino acid sequence of SEQ ID NO:6. In
certain exemplary embodiments, the antibody or antigen-binding
protein comprises an HCVR amino acid sequence of SEQ ID NO:11 and
an LCVR amino acid sequence of SEQ ID NO:15. In certain exemplary
embodiments, the antibody or antigen-binding protein comprises an
HCVR amino acid sequence of SEQ ID NO:11 and an LCVR amino acid
sequence of SEQ ID NO:15 comprising a substitution of histidine for
leucine at amino acid residue 30 (L30H).
[0079] In certain embodiments, the antibody or antigen-binding
protein exhibits one or more or the following properties when
administered by weekly subcutaneous injection to an
APOE*3Leiden.CETP mouse: [0080] a. reduces total cholesterol levels
relative to untreated controls by about 37% when administered at 3
mg/kg; [0081] b. reduces total cholesterol levels relative to
untreated controls by about 46% when administered at 10 mg/kg;
[0082] c. reduces total cholesterol levels relative to untreated
controls by about 48% when administered at 3 mg/kg in combination
with 3.6 mg/kg/day atorvastin; [0083] d. reduces total cholesterol
levels relative to untreated controls by about 58% when
administered at 10 mg/kg in combination with 3.6 mg/kg/day
atorvastin; [0084] e. reduces total cholesterol levels by about 36%
when administered at 3 mg/kg in combination with 3.6 mg/kg/day
atorvastin, relative to treatment with 3.6 mg/kg/day atorvastin
alone; [0085] f. reduces total cholesterol levels by about 48% when
administered at 10 mg/kg in combination with 3.6 mg/kg/day
atorvastin, relative to treatment with 3.6 mg/kg/day atorvastin
alone; [0086] g. reduces triglyceride levels relative to untreated
controls by about 33% when administered at 3 mg/kg; [0087] h.
reduces triglyceride levels relative to untreated controls by about
36% when administered at 10 mg/kg; [0088] i. reduces triglyceride
levels by about 40% when administered at 3 mg/kg in combination
with 3.6 mg/kg/day atorvastin, relative to treatment with 3.6
mg/kg/day atorvastin; [0089] j. reduces triglyceride levels by
about 51% when administered at 10 mg/kg in combination with 3.6
mg/kg/day atorvastin, relative to treatment with 3.6 mg/kg/day
atorvastin alone; [0090] k. increases hepatic LDLR expression
relative to untreated controls by about 88% when administered at 3
mg/kg; [0091] l. increases hepatic LDLR expression relative to
untreated controls by about 178% when administered at 10 mg/kg;
[0092] m. increases hepatic LDLR expression by about 71% when
administered at 3 mg/kg in combination with 3.6 mg/kg/day
atorvastin, relative to treatment with 3.6 mg/kg/day atorvastin
alone; [0093] n. increases hepatic LDLR expression by about 140%
when administered at 10 mg/kg in combination with 3.6 mg/kg/day
atorvastin, relative to treatment with 3.6 mg/kg/day atorvastin
alone; [0094] o. decreases atherosclerotic lesion size relative to
untreated controls by about 70% when administered at 3 mg/kg;
[0095] p. decreases atherosclerotic lesion size relative to
untreated controls by about 87% when administered at 10 mg/kg;
[0096] q. decreases atherosclerotic lesion size relative to
untreated controls by about 88% when administered at 3 mg/kg in
combination with 3.6 mg/kg/day atorvastin; [0097] r. decreases
atherosclerotic lesion size relative to untreated controls by about
98% when administered at 10 mg/kg in combination with 3.6 mg/kg/day
atorvastin; [0098] s. decreases atherosclerotic lesion size
relative by about 82% when administered at 3 mg/kg in combination
with 3.6 mg/kg/day atorvastin, relative to treatment with 3.6
mg/kg/day atorvastin alone; [0099] t. decreases atherosclerotic
lesion size relative by about 97% when administered at 10 mg/kg in
combination with 3.6 mg/kg/day atorvastin, relative to treatment
with 3.6 mg/kg/day atorvastin alone; [0100] u. reduces triglyceride
levels by about 72% when administered at 3 mg/kg, relative to
treatment with 3.6 mg/kg/day atorvastatin alone; [0101] v. reduces
triglyceride levels by about 79% when administered at 10 mg/kg,
relative to treatment with 3.6 mg/kg/day atorvastatin alone; [0102]
w. reduces total cholesterol levels by about 22% when administered
at 3 mg/kg, relative to treatment with 3.6 mg/kg/day atorvastatin
alone; [0103] x. reduces total cholesterol levels by about 34% when
administered at 10 mg/kg, relative to treatment with 3.6 mg/kg/day
atorvastatin alone; [0104] y. increases the percentage of
undiseased aortic segments by about 236% when administered at 3
mg/kg, relative to untreated control; [0105] z. increases the
percentage of undiseased aortic segments by about 549% when
administered at 10 mg/kg, relative to untreated control; [0106] aa.
increases the percentage of undiseased aortic segments by about
607% when administered at 3 mg/kg in combination with 3.6 mg/kg/day
atorvastatin, relative to control; and [0107] bb. increases the
percentage of undiseased aortic segments by about 1118% when
administered at 3 mg/kg in combination with 3.6 mg/kg/day
atorvastatin, relative to control.
Pharmaceutical Compositions and Methods of Administration
[0108] The present invention includes methods which comprise
administering a PCSK9 inhibitor to a subject, wherein the PCSK9
inhibitor 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.
[0109] 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, inhalation, 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.
[0110] A pharmaceutical composition of the present invention can be
delivered subcutaneously or intravenously with a standard needle
and syringe. In addition, pen delivery devices and autoinjector
delivery devices readily have applications in delivering a
pharmaceutical composition of the present invention. Such delivery
devices can be reusable or disposable, and can be adapted to
administer a variable dose or a fixed dose. A reusable 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 delivery device can then be reused. In a
disposable delivery device, there is no replaceable cartridge.
Rather, the disposable 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. A delivery device adapted for
variable dosing may include a mechanism for setting a dose within a
range of dose volumes (in some cases, the range may be limited to a
value less than a total volume contained in the cartridge). A
delivery device adapted for fixed dosing may include a mechanism
which delivers the total volume of the cartridge when the delivery
device it actuated. In such cases, the cartridge may be a
pre-filled syringe.
[0111] Numerous delivery devices have applications in the 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.sup.1m 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. In some embodiments, the reusable pen or
autoinjector delivers a fixed dose. In other embodiments, the
reusable pen or autoinjector can deliver a variable dose. In
different embodiments, the reusable pen or autoinjector can deliver
a single dose or multiple doses.
[0112] In certain embodiments, the pharmaceutical composition is
delivered in a controlled release system. In certain embodiments, a
pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref.
Biomed. Eng. 14:201), including a micropump. 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.
[0113] The injectable preparations may include dosage forms for
intravenous, subcutaneous, intracutaneous and intramuscular
injections, drip infusions, etc. These injectable preparations may
be prepared by known methods. For example, the injectable
preparations may be prepared, e.g., by dissolving, suspending or
emulsifying the antibody or its salt described above in a sterile
aqueous medium or an oily medium conventionally used for
injections. As the aqueous medium for injections, there are, for
example, physiological saline, an isotonic solution containing
glucose and other auxiliary agents, etc., which may be used in
combination with an appropriate solubilizing agent such as an
alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,
polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,
HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor
oil], etc. As the oily medium, there are employed, e.g., sesame
oil, soybean oil, etc., which may be used in combination with a
solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
The injection thus prepared is filled in an appropriate
ampoule.
[0114] 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. As used
herein, "unit dosage form" refers to physically discrete units
suitable as single dosages for human and/or animal subjects, each
unit containing a predetermined quantity of active material (i.e.,
a PCSK9 inhibitor) calculated to produce the desired therapeutic
effect in association with a pharmaceutical diluent, carrier or
vehicle. In some embodiments, the PCSK9 inhibitor is an antibody or
antigen-binding protein and the unit dosage form comprises 75 mg,
150 mg, 200 mg, or 300 mg of the antibody or antigen-binding
protein. Examples of suitable unit dosage forms for liquid
pharmaceutical compositions include applicators such as vials,
syringes, including pre-filled syringes and reusable syringes,
autoinjectors, including pre-filled autoinjectors and reusable
autoinjectors, cartridges, and ampules; other suitable unit dosage
forms include tablets, pills, capsules, suppositories, wafers,
segregated multiples of any of the foregoing, and other forms as
herein described or generally known in the art. The unit dosage
form may be hermetically sealed. For unit dosage forms containing
the pharmaceutical composition within an applicator, the quantity
of the PCSK9 inhibitor may be indicated on the applicator.
[0115] The present invention includes includes an article of
manufacture or kit comprising (a) one or more unit dosage forms
comprising a pharmaceutical composition of present invention, and
(b) a container or package. The article of manufacture or kit can
further comprise (c) a label or packaging insert with instructions
for use. In some embodiments, the article of manufacture can
further comprise (d) one or more unit dosage forms of a
lipid-modifying therapy (e.g. a blister of tablets comprising as an
active ingredient an HMG-CoA reductase inhibitor).
Dosage and Administration Regimens
[0116] The amount of PCSK9 inhibitor (e.g., anti-PCSK9 antibody)
administered to a subject according to the methods and compositions
of the present invention is, generally, a therapeutically effective
amount. As used herein, the phrase "therapeutically effective
amount" means a dose of PCSK9 inhibitor that results in a
detectable reduction in atherosclerotic lesions. For example,
"therapeutically effective amount" of a PCSK9 inhibitor includes,
e.g., an amount of PCSK9 inhibitor that causes a reduction of at
least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or more in sclerotic lesion area or lesion severity when
administered to a human patient, e.g., as illustrated in the
Examples herein. Alternatively, animal models can be used to
establish whether a particular amount of a candidate PCSK9
inhibitor is a therapeutically effective amount.
[0117] In the case of an anti-PCSK9 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 3mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg,
about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg,
about 75 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-PCSK9 antibody.
[0118] In certain embodiments, the anti-PCSK9 antibody is
administered to a subject at a dose of 75 mg. In certain
embodiments, the anti-PCSK9 antibody is administered to a subject
at a dose of 150 mg. In certain embodiments, the anti-PCSK9
antibody is administered to a subject at a dose of 300 mg.
[0119] The amount of anti-PCSK9 antibody contained within the
individual doses may be expressed in terms of milligrams of
antibody per kilogram of patient body weight (i.e., mg/kg). For
example, the anti-PCSK9 antibody may be administered to a patient
at a dose of about 0.0001 to about 10 mg/kg of patient body
weight.
[0120] According to certain embodiments of the present invention,
multiple doses of a PCSK9 inhibitor 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 a PCSK9 inhibitor. As used herein,
"sequentially administering" means that each dose of PCSK9
inhibitor 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 a PCSK9 inhibitor, followed by one
or more secondary doses of the PCSK9 inhibitor, and optionally
followed by one or more tertiary doses of the PCSK9 inhibitor.
[0121] The terms "initial dose," "secondary doses," and "tertiary
doses," refer to the temporal sequence of administration of the
PCSK9 inhibitor. 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. In certain embodiments, however, the amount of
PCSK9 inhibitor contained in the initial, secondary and/or tertiary
doses will vary from one another (e.g., adjusted up or down as
appropriate) during the course of treatment. The initial,
secondary, and tertiary doses may all contain the same amount of
PCSK9 inhibitor.
[0122] In certain embodiments, each secondary and/or tertiary dose
is administered 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, or more) days after the immediately preceding dose. The
phrase "the immediately preceding dose," as used herein, means, in
a sequence of multiple administrations, the dose of PCSK9 inhibitor
which is administered to a patient prior to the administration of
the very next dose in the sequence with no intervening doses.
[0123] In certain embodiments, the methods comprise administering
to a patient any number of secondary and/or tertiary doses of a
PCSK9 inhibitor. 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.
[0124] 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 29 days 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 1 to 60 days 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.
[0125] In certain embodiments, the anti-PCSK9 antibody is
administered to a subject at an initial dose of at about 75 mg
every two weeks.
[0126] In additional embodiments, the antibody is administered to a
subject at an initial dose of about 150 mg every two weeks.
[0127] In one embodiment, an initial dose is administered during an
initial dosing period, after which the subject's LDL-C value is
monitored determine if the initial dose should be changed to a
secondary dose, by measuring the LDL-C value to determine if it is
at or below a target LDL-C value. For example, a subject may begin
treatment with an initial dose of 75 mg of the antibody, with a
target LDL-C value of 100 milligrams per deciliter (mg/dL). If the
subject's LDL-C reaches a target LDL-C value at the end of the
initial dosing period, then the subject continues treatment on the
initial dose, but changes to a secondary dose if that target value
is not reached. In one embodiment, the initial dose is about 75 mg,
administered every two weeks, the target LDL-C value is 100 mg/dL,
and the secondary dose, if necessary, is 150 mg every two weeks.
For example, the subject may be first monitored after about 8 weeks
of treatment, and if their LDL-C value is above 100 mg/dL, the
subject may be uptitrated to a secondary dose of 150 mg,
administered every two weeks. Monitoring may continue throughout
the treatment period. In another embodiment, the initial dose is 75
mg of antibody every two weeks, the target LDL-C value is 70 mg/dL,
and the secondary dose is 150 mg every two weeks if the target
LDL-C value is not reached.
Prior Therapies and Combination Therapies
[0128] The methods of the present invention, according to certain
embodiments, comprise administering a pharmaceutical composition
comprising an anti-PCSK9 antibody to a subject who is on a
therapeutic regimen for the treatment of hypercholesterolemia
and/or atherosclerosis at the time of, or just prior to,
administration of the pharmaceutical composition of the invention.
For example, a patient who has previously been diagnosed with
hypercholesterolemia and/or atherosclerosis may have been
prescribed and is taking a stable therapeutic regimen of another
lipid modifying therapy prior to and/or concurrent with
administration of a pharmaceutical composition comprising an
anti-PCSK9 antibody. In other embodiments, the subject has not been
previously treated with a lipid modifying therapy.
[0129] The methods of the present invention, according to certain
embodiments, comprise administering as a monotherapy a
pharmaceutical composition comprising an anti-PCSK9 antibody to a
subject, in the absence of any concurrent lipid modifying
therapy.
[0130] The methods of the present invention, according to certain
embodiments, also comprise administering a pharmaceutical
composition comprising an anti-PCSK9 inhibitor to a subject in
combination with another lipid modifying therapy
[0131] As used herein, lipid modifying therapies include, for
example, (1) agents which induce a cellular depletion of
cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl
(HMG)-coenzyme A (CoA) reductase, such as a statin (e.g.,
cerivastatin, atorvastatin, simvastatin, pitavastatin,
rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.); (2)
agents which inhibit cholesterol uptake and or bile acid
re-absorption (such as ezetimibe); (3) agents which increase
lipoprotein catabolism (such as niacin); and/or (4) activators of
the LXR transcription factor that plays a role in cholesterol
elimination such as 22-hydroxycholesterol. Lipid modifying
therapies also include fixed combinations of therapeutic agents
such as ezetimibe plus simvastatin; a statin with a bile resin
(e.g., cholestyramine, colestipol, colesevelam); niacin plus a
statin (e.g., niacin with lovastatin); or with other lipid lowering
agents such as omega-3-fatty acid ethyl esters (for example,
omacor).
EXAMPLES
[0132] 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.
Example 1
Generation of Human Antibodies to Human PCSK9
[0133] Human anti-PCSK9 antibodies were generated as described in
U.S. Pat. No. 8,062,640. The exemplary PCSK9 inhibitor used in the
following Example is the human anti-PCSK9 antibody designated
"mAb316P," also known here as "alirocumab." mAb316P has the
following amino acid sequence characteristics: heavy chain variable
region (HCVR) comprising SEQ ID NO:1; light chain variable domain
(LCVR) comprising SEQ ID NO:6; heavy chain complementarity
determining region 1 (HCDR1) comprising SEQ ID NO:2; HCDR2
comprising SEQ ID NO:3; HCDR3 comprising SEQ ID NO:4; light chain
complementarity determining region 1 (LCDR1) comprising SEQ ID
NO:7; LCDR2 comprising SEQ ID NO:8; and LCDR3 comprising SEQ ID
NO:10.
Example 2
Attainment of Low-Density Lipoprotein Cholesterol Goals in Patients
at High Cardiovascular Risk: Results from a Managed Care Population
Study
[0134] Background: US guidelines support LDL-C goals based on
patient's cardiovascular (CV) risk profile, however, little is
known regarding real-world patterns of LDL-C goal attainment within
specific high CV risk conditions. [0135] Methods: Patients from the
Optumlnsight IMPACT Database (a large US multi-payer claims
database) with an LDL-C measurement during July 2011 to June 2012
and high CV risk conditions were identified. Most recent LDL-C
measurement was defined as the index date and high CV risk
conditions were identified hierarchically during the pre-index
period as follows: recent acute coronary syndrome (ACS, within 6
months pre-index date), coronary events (myocardial infarction,
hospitalization for unstable angina, coronary revascularization),
stroke, and peripheral vascular disease (PVD). [0136] Results: In
total, 110,739 patients met the inclusion criterion. Median (IQR)
age was 59 (53 to 65) years, 53.7% were male, and median (IQR)
LDL-C was 116 (92 to 143) mg/dL. As of index date, 2.7% had a
recent ACS, while 42.1%, 9.2%, and 46.0% had evidence of coronary
events, stroke, and PVD, respectively. The following table (Table
1) represents a summary distribution of patients by LDL-C levels as
of index date for different high CV risk conditions.
TABLE-US-00001 [0136] TABLE 1 Breakdown of patients by LDL-C level
(mg/dL) and risk condition. High CV Risk Condition % Patients by
LDL-C Level (mg/dL) (% Patients; N) <70 .gtoreq.70 to <100
.gtoreq.100 to <130 .gtoreq.130 to <160 .gtoreq.160 Any
Recent ACS (2.7%; 2,966) 17.2% 27.4% 26.7% 16.5% 12.1% 100.0%
Coronary Events 9.5% 25.1% 30.3% 21.4% 13.8% 100.0% (42.1%; 46,616)
Stroke (9.2%; 10,240) 8.0% 23.8% 30.4% 23.5% 14.3% 100.0% PVD
(46.0%; 50,917) 6.9% 23.0% 31.8% 23.8% 14.5% 100.0% Overall (100%;
110,739) 8.3% 24.0% 30.9% 22.6% 14.1% 100.0%
[0137] Conclusions: In a large, contemporary cohort of high CV risk
patients, few achieved LDL-C goals of <70 mg/dL (optional goal
for very high CV risk) or <100 mg/dL. Although LDL-C goal
achievement improved marginally in patients with conditions
signifying higher CV risk, with highest achievement in patients
with recent ACS, the majority of these patients were still above
LDL-C goals. These data show that there is a need for ongoing
efforts to address gaps in LDL-C goal attainment and improving CV
outcomes in high-risk patients.
Example 3
Low-Density Lipoprotein Cholesterol Goal Attainment and
Lipid-Lowering Therapy in a High Cardiovascular Risk Managed Care
Population
[0137] [0138] Background: While US guidelines support statins as
first line therapy to reduce LDL-C, little is known regarding
real-world patterns of LDL-C goal attainment with statins and other
lipid-lowering therapies (LLTs). [0139] Methods: Patients from the
OptumInsight IMPACT Database (a large US multi-payer claims
database) with an LDL-C measurement during July 2011 to June 2012
and high risk CV conditions (coronary events (myocardial
infarction, hospitalization for unstable angina, coronary
revascularization), stroke, and peripheral vascular disease) were
identified. LLT prescription was assessed as of most recent LDL-C
measurement (index date) and categorized as high-potency statin
(atorvastatin 40/80 mg, rosuvastatin 20/40 mg, simvastatin 80 mg),
standard-potency statin (other statins), non-statin LLT (ezetimibe,
niacin, fibrates, bile acid sequestrants), and no LLT. [0140]
Results: In total, 110,739 patients met the inclusion criterion.
Median (IQR) age was 59 (53 to 65) years, 53.7% were male, and
median (IQR) LDL-C was 116 (92 to 143) mg/dL. As of index date,
10.8% were on high-potency statin, 26.9% were on standard-potency
statin, 5.3% were on non-statin LLT, and 57.0% were not on any LLT.
The following table (Table 2) represents a summary distribution of
patients by LDL-C levels as of index date for different LLT
types.
TABLE-US-00002 [0140] TABLE 2 Breakdown of patients by LDL-C level
(mg/dL) and LLT type. LLT Type % Patients by LDL-C Level (mg/dL) (%
Patients; N) <70 .gtoreq.70 to <100 .gtoreq.100 to <130
.gtoreq.130 to <160 .gtoreq.160 Any High-Potency Statin 15.2%
33.7% 26.0% 13.7% 11.4% 100.0% (10.8%; 12,014) Standard-Potency
Statin 11.2% 30.1% 28.5% 18.2% 12.0% 100.0% (26.9%; 29,734)
Non-Statin LLT (5.3%; 12.1% 27.6% 29.8% 19.0% 11.5% 100.0% 5,921)
No LLT (57.0%; 63,070) 5.3% 19.0% 33.1% 26.7% 15.9% 100.0% Overall
(100%; 110,739) 8.3% 24.0% 30.9% 22.6% 14.1% 100.0%
[0141] Conclusions: In this large, contemporary population of
patients at high CV risk, few achieved LDL-C goals of <70 mg/dL
(optional goal for very high CV risk) or <100 mg/dL. Further,
many other patients were not at LDL-C goal despite receiving a
high-potency statin. These data show that there is a need for
ongoing efforts to improve adherence to LLT as well as new
therapies to improve goal attainment and CV outcomes.
Example 4
Monoclonal Antibody to PCSK-9, MAb316P Dose-Dependently Decreases
Atherosclerosis, Induces a More Stable Plaque Phenotype and
Enhances the Effects of Atorvastatin in APOE*3Leiden.CETP
Transgenic Mice
Background
[0142] The aim of this study was to investigate the effects of two
dosages of mAb316P, alone and in combination with atorvastatin on
plasma lipids, atherosclerosis development, and lesion composition
in APOE*3Leiden.CETP mice. This is a well-established model for
hyperlipidemia and atherosclerosis with all features of familial
dysbetalipoproteinemia (FD) in humans, which is characterized by
accumulation of remnant lipoproteins and an increased very LDL
(VLDL) cholesterol to LDL-C ratio. APOE*3Leiden mice have an
impaired clearance of (V)LDL and increased TG levels, and are
thereby mimicking the slow clearance observed in humans, in
contrast to normal wild-type mice, which have a very rapid
clearance of apoB-containing lipoproteins. The lipoprotein profile
in APOE*3Leiden.CETP mice reflects that of FD patients with a
similar response to lipid-modifying therapies, including statins,
fibrates, niacin, and cholesteryl ester transfer protein (CETP)
inhibitors. This is illustrated by a comparable reduction in
cholesterol in all apoB-containing lipoprotein subfractions with
statin treatment. It was hypothesized that mAb316P alone could
reduce progression of atherosclerosis and add to the
atheroprotective effects of atorvastatin. Inhibition of
atherosclerosis by atorvastatin in APOE*3Leiden.CETP mice has been
observed previously.
Methods
i) Animals
[0143] Ninety female APOE*3Leiden.CETP transgenic mice (9 to 13
weeks of age), expressing human CETP under control of its natural
flanking regions, were used. During the study, mice were housed
under standard conditions with a 12-hour light-dark cycle and had
free access to food and water. Animal experiments were approved by
the Institutional Animal Care and Use Committee of The Netherlands
Organization for Applied Research.
ii) Experimental Design
[0144] Mice received a semi-synthetic cholesterol-rich diet,
containing 15% (w/w) cacao butter and 0.15% cholesterol
(Western-type diet [WTD]; Hope Farms, Woerden, The Netherlands) for
a run-in period of 3 weeks to increase plasma total cholesterol
(TC) levels up to .about.15 mmol/L. Body weight (BW) and food
intake were monitored regularly during the study. After matching
based on BW, TC, plasma TG, and age, mice (n=15 per group) received
a WTD alone or were treated with two dosages of mAb316P (3 or 10
mg/kg) alone or in combination with atorvastatin (3.6 mg/kg/d) for
18 weeks, and an arm with atorvastatin alone was added. MAb316P was
administered via weekly subcutaneous injections and atorvastatin
was added to the diet. The dose of atorvastatin was calculated in
order to attempt a TC reduction of about 20% to 30%. At the end of
the treatment period, all animals were sacrificed by CO.sub.2
inhalation. Livers and hearts were isolated to assess hepatic LDLR
protein levels, lipid content, atherosclerosis development, and
plaque composition.
iii) Plasma Lipids, Lipoprotein Analysis, and Measurement of
MAb316P Levels
[0145] Plasma was isolated from blood collected in
ethylenediaminetetraacetic acid (EDTA)-coated cups via tail vein
bleeding after a 4-hour fast every 2 to 4 weeks. Plasma TC and TG
were determined using enzymatic kits according to the
manufacturer's protocols (cat. no. 1458216 and cat. no. 1488872,
respectively; Roche/Hitachi) and average plasma TC and TG levels
were calculated. Lipoprotein profiles for TC were measured after
lipoprotein separation by fast protein liquid chromatography (FPLC)
after 4, 12, and 18 weeks of treatment. MAb316P levels were
measured by a human Fc enzyme-linked immunosorbent assay.
iv) Hepatic LDLR Protein Levels
[0146] Liver tissues were homogenized in lysis buffer (50 mM
Tris-HCL [pH=7.4], 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40
[Igepal], 1 mM EDTA, protease inhibitor cocktail [complete, Roche],
1 mM PMSF, 1 mM Na3VO4) and then centrifuged at 6500 rpm at
4.degree. C. for 30 minutes. Protein concentration in cell lysates
was determined by bicinchonic acid protein assay (Thermo
Scientific) according to manufacturer's instructions. 50 .mu.g of
protein lysates was separated by SDS-PAGE and then transferred to
polyvinylidene fluoride membranes (Millipore). Blots were subjected
to goat anti-mouse LDLR from R&D Systems and rabbit anti-goat
horseradish peroxidase (HRP) from AbD Serotec or mouse
anti-.alpha.-Tubulin from Sigma and horse anti-mouse HRP from Cell
Signaling Technologies (according to the manufacturer's
instructions); blots were developed with West Femto Super Signal
ECL (Thermo Scientific) and subjected to the Chemi-Doc-it imaging
system. Intensities of protein bands were quantified using Image J
software.
v) Histological Assessment of Atherosclerosis
[0147] Hearts were isolated, fixed in formalin, and embedded in
paraffin. Cross-sections (5 .mu.m each) of the entire aortic root
area were stained with hematoxylin-phloxin-saffron. For each mouse,
four sections at intervals of 50 .mu.m were used for quantitative
and qualitative assessment of the atherosclerotic lesions. To
determine atherosclerotic lesion size and severity, the lesions
were classified into five categories according to the American
Heart Association classification: I) early fatty streak, II)
regular fatty streak, III) mild plaque, IV) moderate plaque, and V)
severe plaque. Total lesion area and number of lesions per
cross-section, as well as the percentage undiseased segments, were
calculated. To assess lesion severity as a percentage of all
lesions, type I-III lesions were classified as mild lesions, and
type IV-V lesions were classified as severe lesions. To determine
the total plaque load in the thoracic aorta, perfusion-fixed aortas
(from the aortic origin to the diaphragm) were cleaned of
extravascular fat, opened longitudinally, pinned en face, and
stained for lipids with oil-red O as described previously by
Verschuren et al. (Arterioscler Thromb Vasc Biol. 2005;
25:161-167). Data were normalized for analyzed surface area and
expressed as percentage of the stained area. Photos/images were
taken with the Olympus BX51 microscope and lesion areas were
measured using Cell D imaging software (Olympus Soft Imaging
Solutions).
[0148] In the aortic root, lesion composition was determined for
the severe lesions (type IV-V) as a percentage of lesion area after
immunostaining with mouse anti-human alpha actin (1:800; Monosan,
Uden, The Netherlands) for smooth muscle cells (SMC), and rat
anti-mouse Mac-3 (1:25; BD Pharmingen, the Netherlands) for
macrophages followed by sirius red staining for collagen. Necrotic
area and cholesterol clefts, monocyte adhesion to the endothelium,
T-cells abundance in the aortic root area and the calculation of
plaque stability index (defined as the ratio of collagen and SMC
area as stabilization factors to macrophage and necrotic area as
destabilization factors) were determined as previously described by
Kuhnast et al. (J Hypertens. 2012; 30:107-116), Stary et al.
(Arterioscler Thromb Vasc Biol. 1995; 15:1512-1531), and Kuhnast et
al. (PLoS One. 2013; 8: e66467). Rat anti-mouse CD54 antibody,
GTX76543 (GeneTex, Inc., San Antonio, Tex., USA) was used for
immunostaining of intercellular adhesion molecule 1 (ICAM-1).
Photos/images of the lesions were taken with the Olympus BX40
microscope with Nuance 2 multispectral imaging system, and stained
areas were quantified using Image J software.
vi) Hepatic Lipid Analysis and Fecal Excretion of Bile Acids and
Neutral Sterols
[0149] Liver tissue samples were homogenized in phosphate-buffered
saline, and the protein content was measured. Lipids were
extracted, separated by high-performance thin-layer chromatography
on silica gel plates, and analyzed with TINA2.09 software (Raytest
Isotopen Messgerate Straubenhardt, Germany), as previously
described by Post et al. (Hepatology. 1999; 30:491-500).
[0150] Mice were housed at five mice per cage, and feces were
collected during two consecutive periods of 72 hours and 48 hours,
respectively. Aliquots of lyophilized feces were used for
determination of neutral and acidic sterol content by
gas-liquid-chromatography, as previously described by Post et al.
(Arterioscler Thromb Vasc Biol. 2003; 23:892-897).
vii) Flow Cytometric Analysis
[0151] After 8 weeks of treatment, peripheral blood mononuclear
cells (PBMCs) were isolated from fresh blood samples and were
sorted into GR-1+ (neutrophils/granulocytes), GR-1-
(lymphocytes/monocytes), CD3+ (T-cells), CD19+ (B-cells) and
CD11b+/Ly6C.sup.low and CD11b+/Ly6C.sup.hi (monocytes) cells using
flow cytometric (FACS) analysis. The following conjugated
monoclonal antibodies were used from Becton Dickinson: GR-1 FITC,
CD3 PerCpCy5-5, CD19 V450, CD11 b APC and Ly6C PE-Cy7.
viii) Statistical Analysis
[0152] Significance of differences between the groups was
calculated non-parametrically using a Kruskal-Wallis test for
independent samples, followed by a Mann-Whitney U-test for
independent samples. Linear regression analyses were used to assess
correlations between variables. Since the atherosclerotic lesion
area showed a quadratic dependence on plasma cholesterol exposure,
it was transformed using square root transformation. IBM SPSS
Statistics 20 for Windows (SPSS, Chicago, USA) was used for
statistical analyses. All groups were compared to the control group
and to the atorvastatin group, and 3 mg/kg mAb316P was compared to
10 mg/kg mAb316P either with or without atorvastatin. Values are
presented as means.+-.SD. P-values <0.05 were considered
statistically significant for single comparison. Bonferroni's
method was used to determine the level of significance in the case
of multiple comparisons. In the figures, significant effects after
correction for multiple comparisons are indicated by *** to compare
to the control group, .dagger..dagger..dagger. to compare to the
atorvastatin group, and .dagger-dbl..dagger-dbl..dagger-dbl. to
compare 3 mg/kg mAb316P to 10 mg/kg mAb316P.
Results
[0153] i) MAb316P and Atorvastatin Monotreatment and their
Combination Decrease Plasma Total Cholesterol and Triglycerides in
APOE*3Leiden.CETP Mice
[0154] Circulating mAb316P levels were detected in all groups
administered mAb316P and ranged between 5 to 12 .mu.g/mL (3 mg/kg
dose) and 12 to 30 .mu.g/mL (10 mg/kg dose) during the 18-week
study. The APOE*3Leiden.CETP mice on a cholesterol-containing WTD
(control group) reached average plasma TC and TG levels of
16.2.+-.1.8 mmol/L and 2.9.+-.0.6 mmol/L, respectively (FIGS. 1A
and 1B). Compared to the control, mAb316P decreased average plasma
TC (-37%, P<0.001; -46%, P<0.001) and TG (-33%, P<0.001;
-39%, P<0.001), and further decreased TC in combination with
atorvastatin (-48%, P<0.001; -58%, P<0.001). Compared to
atorvastatin, both combination treatments decreased TC (-36%,
P<0.001; -48%, P<0.001) and TG (-40%, P<0.001; -51%,
P<0.001) to a greater extent than atorvastatin alone. The
reductions in TC after mAb316P alone (-14%, P<0.01; 3 mg mAb316P
versus 10 mg mAb316P) and in combination with atorvastatin (-19%,
P<0.001; 3 mg mAb316P+atorvastatin versus 10 mg
mAb316P+atorvastatin) were dose-dependent and sustained during the
study. TC reductions after mAb316P (FIG. 1C), atorvastatin, and
their combination (FIG. 1D) were confined to apoB-containing
lipoproteins. No effects on BW (gain) and food intake were noted in
any treatment groups compared with the control.
ii) MAb316P, Without and with Atorvastatin, Decreases Plasma Lipids
by Reducing Low-Density Lipoprotein Receptor Degradation, and
Reduces Non-HDL-Cholesterol
[0155] Hepatic LDLR protein levels were measured to verify whether
PCSK9 inhibition by mAb316P decreases plasma lipids by rescuing
LDLR degradation (FIG. 2A). Hepatic LDLR protein levels were
increased after mAb316P treatment alone (+80%, P<0.01; +133%,
P<0.01) and together with atorvastatin (+98%, P<0.001; +178%,
P<0.05). Compared to atorvastatin alone, both the combination
treatments increased LDLR protein levels to a greater extent (+71%,
P<0.0045; +140%, P<0.01). An inverse correlation between LDLR
protein levels and plasma TC confirms the involvement of the LDLR
in lowering of TC by mAb316P (R.sup.2=0.50, P<0.001).
[0156] Non-HDL-Cholesterol levels were also measured to verify
whether PCSK9 inhibition by mAb316P decreases such levels by
up-regulation of the LDLR (FIG. 2B).
iii) MAb316P Does Not Affect Liver Lipids and Fecal Bile Acid and
Neutral Sterol Excretion
[0157] To evaluate the consequences of mAb316P-induced alterations
in lipoprotein metabolism on hepatic lipid metabolism and excretion
into feces, liver lipids and excretion of bile acids and neutral
sterols in stool were determined. MAb316P did not affect liver
weight nor the hepatic content of cholesterol and TG, whereas
atorvastatin and the combination treatments led to significant
reductions in liver weight (-15%, P=0.067; -17%, P<0.05, N.S.
after correction for multiple comparisons; -20%, P<0.0045,
respectively) and hepatic cholesteryl esters (-48%, P<0.0045;
-41%, P<0.0045 and -44%, P<0.05, N.S. after correction for
multiple comparisons, respectively) as compared to the control
group, without a change in hepatic triglycerides (Table 3). Fecal
output of bile acids and neutral sterols was not changed by the
treatments (Table 4). These data indicate that despite the greater
influx of cholesterol from the plasma compartment, hepatic
cholesterol homeostasis is maintained during mAb316P and statin
treatment.
TABLE-US-00003 TABLE 3 Effect of mAb316P, atorvastatin and their
combination on liver lipids. Liver lipids (.mu.g/mg protein) FC CE
TG Control 11.6 .+-. 1.6 50.6 .+-. 14.0 119.2 .+-. 33.3 3 mg
mAb316P 11.2 .+-. 1.4.dagger. 48.2 .+-. 8.2.dagger..dagger..dagger.
117.7 .+-. 21.6 10 mg mAb316P 11.4 .+-. 2.0.dagger. 53.9 .+-.
10.4.dagger..dagger..dagger. 142.1 .+-. 43.0.dagger. Atorvastatin
9.5 .+-. 0.9* 26.2 .+-. 4.8*** 90.6 .+-. 28.5 3 mg mAb316P + 10.4
.+-. 1.8 29.6 .+-. 5.8*** 103.5 .+-. 36.8 atorvastatin 10 mg
mAb316P + 10.7 .+-. 1.2 28.3 .+-. 9.0* 109.8 .+-. 28.8 atorvastatin
FC: free cholesterol; CE: cholesterol esters; TG: triglycerides. *P
< 0.05, ***P < 0.0045 as compared to control; .dagger.P <
0.05, .dagger..dagger..dagger.P < 0.0045 as compared to
atorvastatin.
TABLE-US-00004 TABLE 4 Effect of mAb316P, atorvastatin and their
combination on neutral sterol and bile acid excretion. Neutral
sterol excretion (.mu.mol/100 g Bile acid excretion mouse/day)
(.mu.mol/100 g mouse/day) Control 25.8 .+-. 5.5 13.5 .+-. 3.3 3 mg
mAb316P 20.4 .+-. 6.2.dagger. 14.3 .+-. 2.7.dagger. 10 mg mAb316P
21.6 .+-. 5.6.dagger. 12.4 .+-. 3.2 Atorvastatin 30.3 .+-. 6.5 10.7
.+-. 2.4 3 mg mAb316P + atorvastatin 28.6 .+-. 6.0 11.4 .+-. 2.2 10
mg mAb316P + 27.5 .+-. 4.4 12.7 .+-. 1.6 atorvastatin .dagger.P
< 0.05 as compared to atorvastatin.
iv) MAb316P Dose-Dependently Reduces Atherosclerosis Development
and Enhances the Atheroprotective Effects of Atorvastatin
[0158] Effects of mAb316P on atherosclerosis development, in the
absence and presence of atorvastatin, were assessed in the aortic
root and arch after 18 weeks of treatment. Representative images of
atherosclerotic lesions, as illustrated in FIG. 3, show that
mAb316P, atorvastatin, and their combination reduced lesion
progression. To confirm a reduction in atherosclerosis development,
the lesion area and the number of lesions per cross-section were
evaluated (FIG. 4A and FIG. 4B), along with lesion severity (FIG.
4C). For the control group, total lesion area was
278.+-.89.times.103 .mu.m2 per cross-section, which consisted of
4.0.+-.0.7 lesions per cross section. MAb316P dose-dependently
decreased atherosclerotic lesion size (-71%, P<0.001; -88%,
P<0.001) and dose-dependently enhanced the effects of
atorvastatin (-89%, P<0.001; -98%, P<0.001) as compared to
the control. In addition, mAb316P, with and without atorvastatin,
also decreased the number of lesions (-17%, P<0.05, N.S. after
correction for multiple comparisons; -30%, P<0.0045 and -41%,
P<0.001; -77%, P<0.001, respectively). Mice treated with
mAb316P, alone and in combination with atorvastatin, had more
lesion-free sections and fewer severe (type IV-V) lesions compared
with the control. Atorvastatin alone decreased lesion size (-35%,
P<0.05, N.S. after correction for multiple comparisons) and
reduced severity to a lesser extent with no effect on the number of
lesions or undiseased segments. When compared to atorvastatin
monotreatment, the combinations further decreased lesion size
(-82%, P<0.001; -97%, P<0.001) and number of lesions (-38%,
P<0.001; -76%, P<0.001) and increased the amount of
undiseased segments.
[0159] To evaluate the effect of mAb316P treatment on lesion
development at another spot along the aorta prone to development of
atherosclerosis, plaque surface in the aortic arch was measured
(FIG. 4D). At this site, lesion development is delayed as compared
with the aortic root. In line with the effects on atherogenesis in
the aortic origin, 10 mg/kg mAb316P alone (-67%, P<0.05, N.S.
after correction for multiple comparisons) and both doses together
with atorvastatin (-73%, P<0.0045; -73%, P<0.0045) reduced
the total plaque area.
[0160] The anti-atherogenic effect of mAb316P and atorvastatin were
evaluated (FIG. 5) and a strong correlation between plasma TC
levels and atherosclerotic lesion area in the aortic root was
observed (R.sup.2=0.84, P<0.001; FIG. 5), indicating an
important role of cholesterol in the development of
atherosclerosis.
v) MAb316P Reduces Monocyte and T-Cell Recruitment and Improves
Lesion Stability Index
[0161] As a functional marker of vessel wall inflammation, the
number of monocytes adhering to the activated endothelium (FIG. 8A)
and the number of T-cells in the aortic root area (FIG. 8B) was
counted and calculated per cross section (FIG. 7A). In the control
group, 5.7.+-.4.2 adhering monocytes and 16.7.+-.7.7 T-cells were
present. When administered alone and together with atorvastatin,
the higher dose of mAb316P (10 mg/kg) decreased the adhering
monocytes (-57%, P<0.01, N.S. after correction for multiple
comparisons, and -75%, P<0.001) and the abundance of T-cells
(-37%, P<0.05, N.S. after correction for multiple comparisons,
and -62%, P<0.001). To further underline the mechanism by which
mAb316P reduced monocyte adherence, endothelial ICAM-1 expression
by immunohistochemistry (FIG. 8C) was assessed. For the control,
39% of the endothelium was positive for ICAM-1 compared to 19%
(P<0.001) after 10 mg/kg mAb316P monotreatment and 16%
(P<0.001) when given in combination with atorvastatin. The
reduction in monocyte adherence was, therefore, corroborated by a
reduction in adhesion molecule expression in endothelial cells
after mAb316P treatment alone and in combination with
atorvastatin.
[0162] After investigating lesion morphology, treatment effects on
plaque composition were analyzed in the severe lesions (type IV-V),
which are considered to be the most vulnerable lesions as shown by
representative images in FIG. 6. To illustrate that plaque
stability is not always dependent on the size of the lesions,
included are representative images of similar size lesions for the
control group and the mAb316P group. Lesion macrophage area (FIG.
7A left) plus lesion necrotic core area (including cholesterol
clefts) (FIG. 7A right), were quantified as destabilization factors
whereas SMC in the fibrotic cap (FIG. 7B left) and collagen area
(FIG. 7B right) were quantified as stabilization factors. All were
expressed as a percentage of total lesion area. Lesions in the
control group consisted of 10.3% macrophages, 4.8% necrotic core
and cholesterol clefts, 3.1% SMC in the cap and 48.4% collagen.
Lesion stability index (FIG. 7C) for the control group was
3.5.+-.0.8. When administered in combination with atorvastatin, the
lower dose of mAb316P (3 mg/kg) decreased the destabilization
factors (-26%, P<0.05, N.S. after correction for multiple
comparisons) and increased the stabilization factors (+19%,
P<0.05, N.S. after correction for multiple comparisons), whereas
the higher dose (10 mg/kg) alone and in combination with
atorvastatin decreased the destabilization factors (-37%,
P<0.001; +73%, P<0.001) and increased the stabilization
factors (+19%, P<0.05, N.S. after correction for multiple
comparisons; +29%, P<0.001). MAb316P, therefore, improved lesion
stability as shown by an increase in lesion stability index alone
(+24%, N.S.; +113%, P<0.0045) and together with atorvastatin
(+116%, P<0.05, N.S. after correction for multiple comparisons;
556%, P<0.001).
vi) MAb316P Tends to Reduce the Circulating Monocytes
[0163] The effects of mAb316P alone and in combination with
atorvastatin on white blood cell count was assessed by flow
cytometry. Interestingly, mAb316P alone and together with
atorvastatin reduced granulocytes/neutrophils (-20%, P<0.05,
N.S. after correction for multiple comparisons; -34%, P<0.001)
and monocytes (-28%, P<0.05, N.S. after correction for multiple
comparisons; -39%, P<0.001) when expressed as a percentage of
the PBMC population. More specifically, mAb316P alone and in
combination with atorvastatin tended to decrease pro-inflammatory
Ly6C.sup.low (-8%, P=0.061; -19%, P<0.001) and increase
anti-inflammatory Ly6C.sup.low (+12%, P=0.089; +35%, P<0.001)
monocytes. Therefore, the effect of mAb316P on vascular recruitment
and adhesion of monocytes may be augmented by a reduction in
circulating monocytes.
TABLE-US-00005 TABLE 5 Effect of mAb316P, atorvastatin, and their
combination on white blood cell count as assessed by flow
cytometric analysis after 8 weeks of treatment. 10 mg/kg 10 mg/kg
MAb316P + Control MAb316P Atorvastatin Atorvastatin
Neutrophils/Granulocytes 8.9 .+-. 2.4 7.1 .+-. 2.0*.dagger..dagger.
5.1 .+-. 1.6*** 5.9 .+-. 2.0*** (% of PBMC population)
Lymphocytes/Monocytes 91.1 .+-. 2.4 92.9 .+-. 2.0*.dagger..dagger.
94.9 .+-. 1.6 *** 94.1 .+-. 2.0*** (% of PBMC population) T-cells
22.9 .+-. 4.6 21.4 .+-. 5.0.dagger. 17.0 .+-. 4.8*** 18.5 .+-. 4.5*
(% of PBMC population) B-cells 63.9 .+-. 8.5 66.3 .+-. 15.0 69.5
.+-. 17.4*** 66.9 .+-. 2.0*** (% of PBMC population) Monocytes 12.3
.+-. 5.0 8.9 .+-. 2.5*.dagger..dagger..dagger. 5.3 .+-. 2.4*** 7.5
.+-. 2.8***.dagger. (% of PBMC population) CD11b + Ly6C.sup.hi 62.2
.+-. 8.5 57.5 .+-. 8.4 51.2 .+-. 6.4*** 50.2 .+-. 4.1*** (% of
monocytes) P = 0.061.dagger. CD11b + Ly6C.sup.low 35.6 .+-. 7.7
40.0 .+-. 8.0 47.8 .+-. 6.3*** 48.0 .+-. 3.5*** (% of monocytes) P
= 0.089.dagger. *P < 0.05, ***P < 0.0125 as compared to
control; .dagger.P < 0.05, .dagger..dagger.P < 0.01,
.dagger..dagger..dagger.P < 0.0125 as compared to atorvastatin
(n = 15 per group)
SUMMARY
[0164] The present study was designed to investigate the effects of
mAb316P per se on atherosclerosis development and in combination
with atorvastatin. Taken together, these data demonstrate that
mAb316P dose-dependently decreases plasma cholesterol, progression
of atherosclerosis and plaque vulnerability, and enhances the
beneficial effects of atorvastatin in APOE*3Leiden.CETP mice. This
is the first study to show that a monoclonal antibody to PCSK9
reduces atherosclerosis development.
Sequence CWU 1
1
1981118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ser Thr Ile Ser Gly
Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Ile Ile Ser Arg Asp Ser Ser Lys His Thr Leu Tyr 65 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 Ser Asn Trp Gly Asn Phe Asp Leu Trp Gly Arg Gly Thr
100 105 110 Leu Val Thr Val Ser Ser 115 28PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Gly
Phe Thr Phe Asn Asn Tyr Ala 1 5 38PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 3Ile Ser Gly Ser Gly Gly
Thr Thr 1 5 411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 4Ala Lys Asp Ser Asn Trp Gly Asn Phe Asp
Leu 1 5 10 5447PRTArtificial SequenceDescription of Artificial
Sequence Synthetic REGN727 heavy chain polypeptide 5Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35
40 45 Ser Thr Ile Ser Gly Ser Gly Gly Thr Thr Asn Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Ser Ser Lys His
Thr Leu Tyr 65 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 Ser Asn Trp Gly Asn Phe
Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165
170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 445 6113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 6Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys
Lys Ser Ser Gln Ser Val Leu Tyr Arg 20 25 30 Ser Asn Asn Arg Asn
Phe Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Asn
Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70
75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln
Gln 85 90 95 Tyr Tyr Thr Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile 100 105 110 Lys 712PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Gln Ser Val Leu Tyr Arg Ser
Asn Asn Arg Asn Phe 1 5 10 83PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Trp Ala Ser 1
9220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic REGN727 light chain polypeptide 9Asp Ile Val Met Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr
Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Arg 20 25 30 Ser Asn
Asn Arg Asn Phe Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45
Pro Pro Asn Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50
55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys Gln Gln 85 90 95 Tyr Tyr Thr Thr Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175
Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180
185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 220 109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Gln Gln Tyr Tyr Thr Thr Pro Tyr Thr 1 5
11127PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Glu Met Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser His 20 25 30 Trp Met Lys Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Ile Val Leu Met Val Tyr Asp Met Asp Tyr Tyr Tyr Tyr
100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 125 128PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 12Gly Phe Thr Phe Ser Ser His Trp 1 5
138PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Ile Asn Gln Asp Gly Ser Glu Lys 1 5
1420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Ala Arg Asp Ile Val Leu Met Val Tyr Asp Met Asp
Tyr Tyr Tyr Tyr 1 5 10 15 Gly Met Asp Val 20 15112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1
5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30 Asn Gly Asn 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 Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Met Gln Thr 85 90 95 Leu Gln Thr Pro Leu
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110
1611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gln Ser Leu Leu His Ser Asn Gly Asn Asn Tyr 1 5
10 173PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Leu Gly Ser 1 189PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Met
Gln Thr Leu Gln Thr Pro Leu Thr 1 5 198PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Gly
Phe Thr Phe Ser Ser His Trp 1 5 208PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Ile
Asn Gln Asp Gly Ser Glu Lys 1 5 2120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Ala
Arg Asp Ile Val Leu Met Val Tyr Asp Met Asp Tyr Tyr Tyr Tyr 1 5 10
15 Gly Met Asp Val 20 2211PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Gln Ser Leu His His Ser Asn
Gly Asn Asn Tyr 1 5 10 233PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 23Leu Gly Ser 1
249PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Met Gln Thr Leu Gln Thr Pro Leu Thr 1 5
258PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Gly Phe Thr Phe Ser Ser His Trp 1 5
268PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Ile Asn Gln Asp Gly Ser Glu Lys 1 5
2720PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Ala Arg Asp Ile Val Leu Met Val Tyr His Met Asp
Tyr Tyr Tyr Tyr 1 5 10 15 Gly Met Asp Val 20 2811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Gln
Ser Leu Leu His Ser Asn Gly Asn Asn Tyr 1 5 10 293PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Leu
Gly Ser 1 309PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 30Met Gln Thr Leu Gln Thr Pro Leu Thr 1
5 318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Gly Phe Thr Phe Ser Ser His Trp 1 5
328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 32Ile Asn Gln Asp Gly Ser Glu Lys 1 5
3320PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Ala Arg Asp Ile Val Leu Met Val Tyr His Met Asp
Tyr Tyr Tyr Tyr 1 5 10 15 Gly Met Asp Val 20 3411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Gln
Ser Leu His His Ser Asn Gly Asn Asn Tyr 1 5 10 353PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Leu
Gly Ser 1 369PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 36Met Gln Thr Leu Gln Thr Pro Leu Thr 1
5 37131PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VH; m2CX1D05 polypeptide 37Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Asn Ser His 20 25 30 Ala Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Gly Ile Asn Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg His Tyr Glu Ile Gln Ile Gly Arg Tyr Gly
Met Asn Val Tyr 100 105 110 Tyr Leu Met Tyr Arg Phe Ala Ser Trp Gly
Gln Gly Thr Leu Val Thr 115 120 125 Val Ser Ser 130
3810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VH CDR1; m2CX1D05 peptide 38Gly Gly Thr Phe Asn Ser His
Ala Ile Ser 1 5 10 3920PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VH CDR2; m2CX1D05 peptide 39Trp Met
Gly Gly Ile Asn Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln 1 5 10 15
Lys Phe Gln Gly 20 4022PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VH CDR3; m2CX1D05 peptide 40His Tyr
Glu Ile Gln Ile Gly Arg Tyr Gly Met Asn Val Tyr Tyr Leu 1 5 10 15
Met Tyr Arg Phe Ala Ser 20 41213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic LC; m2CX1D05 polypeptide 41Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser Ala 20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Asn Gly 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 Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Phe Asp Gly Asp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150
155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr
Lys Ser Phe 195 200 205 Asn Arg Gly Glu Ala 210 4211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic VL CDR 1;
m2CX1D05 peptide 42Arg Ala Ser Gln Gly Ile Arg Ser Ala Leu Asn 1 5
10 4311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VL CDR2; m2CX1D05 peptide 43Leu Leu Ile Tyr Asn Gly Ser
Thr Leu Gln Ser 1 5 10 447PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VL CDR3; m2CX1D05 peptide 44Gln Gln
Phe Asp Gly Asp Pro 1 5 45119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VH; 1B20 polypeptide 45Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser
Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25
30 Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro
Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Trp Tyr Lys Pro
Leu Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 4610PRTArtificial SequenceDescription of Artificial
Sequence Synthetic VH CDR1; 1B20 peptide 46Gly Tyr Ser Phe Thr Asn
Tyr Trp Ile Ser 1 5 10 4720PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VH CDR2; 1B20 peptide 47Trp Met Gly
Ile Ile Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro 1 5 10 15 Ser
Phe Gln Gly 20 4810PRTArtificial SequenceDescription of Artificial
Sequence Synthetic VH CDR3; 1B20 peptide 48Asp Tyr Trp Tyr Lys Pro
Leu Phe Asp Ile 1 5 10 49220PRTArtificial SequenceDescription of
Artificial Sequence Synthetic LC; 1B20 polypeptide 49Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser 20 25
30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Ser Ser Phe Pro Ile Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155
160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Ala 210 215 220 5017PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VL CDR1; 1B20 peptide 50Arg Ser Ser
Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu 1 5 10 15 Ala
5111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic VL CDR2; 1B20 peptide 51Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser 1 5 10 528PRTArtificial SequenceDescription of
Artificial Sequence Synthetic VL CDR3; 1B20 peptide 52Gln Gln Tyr
Ser Ser Phe Pro Ile 1 5 53120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic variable heavy antibody region
polypeptide 53Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Ser Ser Tyr 20 25 30 Gly Met Tyr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Gly Ser
Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Glu Arg Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110 Leu Val Thr Val Ser Ser Ala Ser 115 120 5416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX132 heavy
chain CDR1 antibody region peptide 54Lys Ala Ser Gly Tyr Thr Phe
Ser Ser Tyr Gly Met Tyr Trp Val Arg 1 5 10 15 5523PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX132 heavy
chain CDR2 antibody region peptide 55Trp Ile Gly Trp Ile Asp Pro
Gly Ser Gly Gly Thr Lys Tyr Asn Glu 1 5 10 15 Lys Phe Lys Gly Lys
Ala Thr 20 5615PRTArtificial SequenceDescription of Artificial
Sequence Synthetic AX132 heavy chain CDR3 antibody region peptide
56Cys Ala Arg Glu Arg Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln 1 5
10 15 57108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic variable light antibody region polypeptide 57Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Tyr 20 25
30 Leu Asn 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 Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Val
Trp Asp Ser Ser Pro Pro 85 90 95 Val Val Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 5817PRTArtificial SequenceDescription of
Artificial Sequence Synthetic AX213 and AX132 light chain CDR1
antibody region peptide 58Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly
Ser Tyr Leu Asn Trp Tyr 1 5 10 15 Gln 5913PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX213 and
AX132 light chain CDR2 antibody region peptide 59Leu Ile Tyr Asp
Ala Ser Asn Arg Ala Thr Gly Ile Pro 1 5 10 6016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX132 &
AX213 light chain CDR3 antibody region peptide 60Tyr Tyr Cys Gln
Val Trp Asp Ser Ser Pro Pro Val Val Phe Gly Gly 1 5 10 15
61120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic variable heavy antibody region polypeptide 61Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr 20 25
30 Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45 Gly Arg Ile Asp Pro Gly Asn Gly Gly Thr Arg Tyr Asn Glu
Lys Phe 50 55 60 Lys Gly Lys Ala Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Asn Asp Gly Tyr Ser
Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser
Ala Ser 115 120 6216PRTArtificial SequenceDescription of Artificial
Sequence Synthetic AX213 heavy chain CDR1 antibody region peptide
62Lys Ala Ser Gly Tyr Thr Phe Ser Arg Tyr Gly Ile Asn Trp Val Arg 1
5 10 15 6323PRTArtificial SequenceDescription of Artificial
Sequence Synthetic AX213 heavy chain CDR2 antibody region peptide
63Trp Ile Gly Arg Ile Asp Pro Gly Asn Gly Gly Thr Arg Tyr Asn Glu 1
5 10 15 Lys Phe Lys Gly Lys Ala Thr 20 6415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX213 heavy
chain CDR3 antibody region peptide 64Cys Ala Arg Ala Asn Asp Gly
Tyr Ser Phe Asp Tyr Trp Gly Gln 1 5 10 15 65108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic variable light
antibody region polypeptide 65Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Ile Thr Cys
Arg Ala Ser Gln Tyr Val Gly Ser Tyr 20 25 30 Leu Asn 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 Pro 65 70
75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Val Trp Asp Ser Ser Pro
Pro 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105 6617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic AX213 and AX132 light chain CDR1 antibody region peptide
66Ile Thr Cys Arg Ala Ser Gln Tyr Val Gly Ser Tyr Leu Asn Trp Tyr 1
5 10 15 Gln 6713PRTArtificial SequenceDescription of Artificial
Sequence Synthetic AX213 and AX132 light chain CDR2 antibody region
peptide 67Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro 1 5
10 6816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic AX132 & AX213 light chain CDR3 antibody region
peptide 68Tyr Tyr Cys Gln Val Trp Asp Ser Ser Pro Pro Val Val Phe
Gly Gly 1 5 10 15 69119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic AX1 VH antibody sequence polypeptide
69Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser
Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Arg Ile Asn Pro Asp Ser Gly Ser Thr Lys
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Arg
Leu Ser Trp Asp Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 7016PRTArtificial SequenceDescription of
Artificial Sequence Synthetic AX1 VH CDR1 antibody sequence peptide
70Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg 1
5 10 15 7123PRTArtificial SequenceDescription of Artificial
Sequence Synthetic AX1 VH CDR2 antibody sequence peptide 71Trp Ile
Gly Arg Ile Asn Pro Asp Ser Gly Ser Thr Lys Tyr Asn Glu 1 5 10 15
Lys Phe Lys Gly Arg Ala Thr 20 7216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX1 VH CDR3
antibody sequence peptide 72Cys Ala Arg Gly Gly Arg Leu Ser Trp Asp
Phe Asp Val Trp Gly Gln 1 5 10 15 73109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX1 VL
antibody sequence polypeptide 73Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Arg Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu 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 Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Ala Tyr Asp Tyr Ser Leu
Gly 85 90 95 Gly Tyr Val Phe Gly Asp Gly Thr Lys Val Glu Ile Lys
100 105 7411PRTArtificial SequenceDescription of Artificial
Sequence Synthetic AX1 VL CDR1 antibody sequence peptide 74Arg Ala
Ser Gln Asp Ile Ser Arg Tyr Leu Ala 1 5 10 757PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX1 AX9 AX189
VL CDR2 antibody sequence peptide 75Ala Ala Ser Ser Leu Gln Ser 1 5
7611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic AX1 VL CDR3 antibody sequence peptide 76Ala Ala Tyr Asp
Tyr Ser Leu Gly Gly Tyr Val 1 5 10 77121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX9 AX189 VH
antibody sequence polypeptide 77Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Trp Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile
Asp Pro Tyr Asn Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys
Gly Lys Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Tyr Gly Tyr Tyr Leu Gly Ser Tyr Ala Met Asp
Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
7816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic AX9 AX189 VH CDR1 antibody sequence peptide 78Lys Ala Ser
Gly Tyr Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg 1 5 10 15
7923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic AX9 AX189 VH CDR2 antibody sequence peptide 79Trp Ile Gly
Arg Ile Asp Pro Tyr Asn Gly Gly Thr Lys Tyr Asn Glu 1 5 10 15 Lys
Phe Lys Gly Lys Ala Thr 20 8018PRTArtificial SequenceDescription of
Artificial Sequence Synthetic AX9 AX189 VH CDR3 antibody sequence
peptide 80Cys Ala Arg Tyr Gly Tyr Tyr Leu Gly Ser Tyr Ala Met Asp
Tyr Trp 1 5 10 15 Gly Gln 81109PRTArtificial SequenceDescription of
Artificial Sequence Synthetic AX189 VL antibody sequence
polypeptide 81Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Arg Tyr 20 25 30 Leu Thr Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu 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 Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Ala Tyr Asp Tyr Ser Leu
Ser 85 90 95 Gly Tyr Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 8211PRTArtificial SequenceDescription of Artificial
Sequence Synthetic AX189 VL CDR1 antibody sequence peptide 82Arg
Ala Ser Gln Asp Val Ser Arg Tyr Leu Thr 1 5 10 837PRTArtificial
SequenceDescription of Artificial Sequence Synthetic AX1 AX9 AX189
VL CDR2 antibody sequence peptide 83Ala Ala Ser Ser Leu Gln Ser 1 5
8411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic AX189 VL CDR3 antibody sequence peptide 84Gln Ala Tyr Asp
Tyr Ser Leu Ser Gly Tyr Val 1 5 10 85115PRTHomo sapiens 85Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Ser Tyr 20
25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala
Gln Lys Leu 50 55 60 Gln Gly Arg Gly Thr Met Thr Thr Asp Pro Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Gly Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser Ser 115
865PRTHomo sapiens 86Ser Tyr Gly Ile Ser 1 5 8717PRTHomo sapiens
87Trp Val Ser Phe Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln 1
5 10 15 Gly 886PRTHomo sapiens 88Gly Tyr Gly Met Asp Val 1 5
89109PRTHomo sapiens 89Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Ser Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val
Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Tyr Thr Ser Thr 85 90
95 Ser Met Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
9014PRTHomo sapiens 90Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn
Ser Val Ser 1 5 10 917PRTHomo sapiens 91Glu Val Ser Asn Arg Pro Ser
1 5 929PRTHomo sapiens 92Asn Ser Tyr Thr Ser Thr Ser Met Val 1 5
93123PRTHomo sapiens 93Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser
Ser Ser Ser Tyr Ile Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Arg Asp Tyr Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val
100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
9410PRTHomo sapiens 94Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn 1 5
10 9517PRTHomo sapiens 95Ser Ile Ser Ser Ser Ser Ser Tyr Ile Ser
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 9614PRTHomo sapiens 96Asp Tyr
Asp Phe Trp Ser Ala Tyr Tyr Asp Ala Phe Asp Val 1 5 10 97111PRTHomo
sapiens 97Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro
Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn
Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro
Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Ser Gly Asn Ser Asn Arg
Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser
Gly Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
9814PRTHomo sapiens 98Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly Tyr
Asp Val His 1 5 10 997PRTHomo sapiens 99Gly Asn Ser Asn Arg Pro Ser
1 5 10011PRTHomo sapiens 100Gln Ser Tyr Asp Ser Ser Leu Ser Gly Ser
Val 1 5 10 101114PRTHomo sapiens 101Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Ala Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser 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 Tyr Tyr Asp Gly Ile Asn Lys His Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Leu Asp Trp Gly Gln Gly Thr
Leu Val Thr Val 100 105 110 Ser Ser 10210PRTHomo sapiens 102Gly Phe
Thr Phe Ser Ser Tyr Gly Met His 1 5 10 10317PRTHomo sapiens 103Val
Ile Tyr Tyr Asp Gly Ile Asn Lys His Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 1045PRTHomo sapiens 104Asp Arg Gly Leu Asp 1 5 105113PRTHomo
sapiens 105Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser
Val Leu Tyr Ser 20 25 30 Ser Asn Ser Lys Asn Tyr Leu Val Trp Tyr
Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr
Ser Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110
Lys 10617PRTHomo sapiens 106Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser
Asn Ser Lys Asn Tyr Leu 1 5 10 15 Val 1077PRTHomo sapiens 107Trp
Ala Ser Thr Arg Glu Ser 1 5 1089PRTHomo sapiens 108Gln Gln Tyr Tyr
Ser Thr Pro Trp Thr 1 5 109118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic anti-PCSK9 monoclonal antibody pJG04
(clones LGT-209 and LGT-210) Vh heavy chain variable region
(FR1-FR4) polypeptide 109Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Ser Thr Met 20 25 30 Tyr Met Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp
Pro Ala Asn Glu His Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Tyr Tyr Tyr Tyr Asn Met Asp Tyr Trp Gly Gln Gly
Thr 100 105 110 Leu Val Thr Val Ser Ser 115 1105PRTArtificial
SequenceDescription of Artificial Sequence Synthetic anti-PCSK9
monoclonal antibody clones LGT-209, LGT-210 and LGT-211 heavy chain
CDR1 peptide 110Thr Met Tyr Met Ser 1 5 11117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic anti-PCSK9
monoclonal antibody clones LGT-209, LGT-210 and LGT-211 heavy chain
CDR2 peptide 111Arg Ile Asp Pro Ala Asn Glu His Thr Asn Tyr Ala Gln
Lys Phe Gln 1 5 10 15 Gly 1129PRTArtificial SequenceDescription of
Artificial Sequence Synthetic anti-PCSK9 monoclonal antibody
pJG04(clones LGT-209 and LGT-210) Vh heavy chain complementarity
determining region 3 (CDR3) peptide 112Ser Tyr Tyr Tyr Tyr Asn Met
Asp Tyr 1 5 113106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic anti-PCSK9 monoclonal antibody pJG10(clones
LGT-209 and LGT-211) Vk light chain variable region (FR1-FR4)
polypeptide 113Gln Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val
Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Gly Val Phe Arg Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Gly Arg Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala
Val Tyr Tyr Cys Leu Gln Trp Ser Ser Asp Pro Pro Thr 85 90 95 Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 11410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic anti-PCSK9
monoclonal antibody clones LGT-209, LGT-210 and LGT-211 light chain
CDR1 peptide 114Arg Ala Ser Gln Ser Val Ser Tyr Met His 1 5 10
1157PRTArtificial SequenceDescription of Artificial Sequence
Synthetic anti-PCSK9 monoclonal antibody clones LGT-209, LGT-210
and LGT-211 light chain CDR1 peptide 115Gly Val Phe Arg Arg Ala Thr
1 5 1169PRTArtificial SequenceDescription of Artificial Sequence
Synthetic mouse anti-PCSK9 monoclonal antibody LFU720 and
anti-PCSK9 monoclonal antibody clones LGT-209, LGT-210 and LGT-211
light chain CDR3 peptide 116Leu Gln Trp Ser Ser Asp Pro Pro Thr 1 5
117118PRTHomo sapiens 117Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Ser
Pro Phe Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Arg Pro Leu Tyr Ala Ser Asp Leu Trp Gly Gln Gly
Thr 100 105 110 Thr Val Thr Val Ser Ser 115 1187PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Gly
Tyr Thr Phe Thr Ser Tyr 1 5 1196PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 119Ser Pro Phe Gly Gly Arg
1 5 1209PRTArtificial SequenceDescription of Artificial Sequence
Synthetic variable heavy chain CDR peptide 120Glu Arg Pro Leu Tyr
Ala Ser Asp Leu 1 5 121107PRTHomo sapiens 121Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg Tyr
Ser Leu Trp Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 12211PRTArtificial SequenceDescription of Artificial
Sequence Synthetic variable light chain CDR peptide 122Arg Ala Ser
Gln Gly Ile Ser Ser Ala Leu Ala 1 5 10 1237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic variable light
chain CDR peptide 123Ser Ala Ser Tyr Arg Tyr Thr 1 5
1249PRTArtificial SequenceDescription of Artificial Sequence
Synthetic variable light chain CDR peptide 124Gln Gln Arg Tyr Ser
Leu Trp Arg Thr 1 5 125118PRTHomo sapiens 125Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr
Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
12610PRTHomo sapiens 126Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His 1 5
10 12717PRTHomo sapiens 127Glu Ile His Pro Ser Gly Gly Arg Thr Asn
Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser 1289PRTHomo sapiens 128Glu
Arg Pro Leu Tyr Ala Met Asp Tyr 1 5 129107PRTHomo sapiens 129Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr His Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Arg Tyr Ser Leu Trp Arg 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 13011PRTHomo sapiens 130Lys Ala Ser Gln
Asp Val His Thr Ala Val Ala 1 5 10 1317PRTHomo sapiens 131His Ala
Ser Tyr Arg Tyr Thr 1 5 1329PRTHomo sapiens 132Gln Gln Arg Tyr Ser
Leu Trp Arg Thr 1 5 133118PRTHomo sapiens 133Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Glu Ile His Pro Ser Gly Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60 Lys Ser Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr
Val Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Arg Pro Leu Tyr Ala Ser Asp Leu Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
13410PRTHomo sapiens 134Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His 1 5
10 13517PRTHomo sapiens 135Glu Ile His Pro Ser Gly Gly Arg Thr Asn
Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ser 1369PRTHomo sapiens 136Glu
Arg Pro Leu Tyr Ala Ser Asp Leu 1 5 137107PRTHomo sapiens 137Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val His Thr Ala
20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr His Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Arg Tyr Ser Leu Trp Arg 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 13811PRTHomo sapiens 138Lys Ala Ser Gln
Asp Val His Thr Ala Val Ala 1 5 10 1397PRTHomo sapiens 139His Ala
Ser Tyr Arg Tyr Thr 1 5 1409PRTHomo sapiens 140Gln Gln Arg Tyr Ser
Leu Trp Arg Thr 1 5 141118PRTMus musculus 141Gln Val Gln Leu Gln
Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Glu Ile Asn Pro Ser Asn Gly Arg Thr Asn Tyr Asn Glu Lys Phe
50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Pro Leu Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115
1427PRTMus musculus 142Gly Tyr Thr Phe Thr Ser Tyr 1 5 1436PRTMus
musculus 143Asn Pro Ser Asn Gly Arg 1 5 1449PRTMus musculus 144Glu
Arg Pro Leu Tyr Ala Met Asp Tyr 1 5 145108PRTMus musculus 145Asp
Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10
15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala
20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp
Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr
Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys
Gln Gln Arg Tyr Ser Thr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 14611PRTMus musculus 146Lys Ala Ser
Gln Asp Val Ser Thr Ala Val Ala 1 5 10 1477PRTArtificial
SequenceDescription of Artificial Sequence Synthetic variable light
chain CDR peptide 147Ser Ala Ser Tyr Arg Tyr Thr 1 5 1489PRTMus
musculus 148Gln Gln Arg Tyr Ser Thr Pro Arg Thr 1 5 149115PRTMus
musculus 149Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly
Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly
Gly Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu
Thr Val Asp Lys Ser Tyr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Trp Leu Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110
Val Ser Ala 115 1507PRTMus musculus 150Gly Tyr Thr Phe Thr Asp Tyr
1 5 1516PRTMus musculus 151Asn Pro Asn Asn Gly Gly 1 5 1526PRTMus
musculus 152Trp Leu Leu Phe Ala Tyr 1 5 153108PRTMus musculus
153Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly
Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Lys Ala Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val
Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Asn Val Leu Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr
Phe Cys Gln Gln Phe Tyr Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Arg 100 105 15411PRTMus musculus 154Lys
Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 1557PRTMus musculus
155Ser Ala Ser Tyr Arg Tyr Ser 1 5 1569PRTMus musculus 156Gln Gln
Phe Tyr Ser Tyr Pro Tyr Thr 1 5 157123PRTMus musculus 157Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20
25 30 Tyr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp
Ile 35 40 45 Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Gly Ile Tyr Tyr
Arg Tyr Asp Arg Asn Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser 115 120 1587PRTMus musculus 158Gly Tyr Thr
Phe Thr Asp Tyr 1 5 1596PRTMus musculus 159Asn Pro Asn Asn Gly Gly
1 5 16014PRTMus musculus 160Gly Gly Ile Tyr Tyr Arg Tyr Asp Arg Asn
Tyr Phe Asp Tyr 1 5 10 161107PRTMus musculus 161Asp Ile Gln Met Thr
Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val
Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40
45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu
Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser
Lys Leu Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
Lys 100 105 16211PRTMus musculus 162Ser Ala Ser Gln Gly Ile Ser Asn
Tyr Leu Asn 1 5 10 1637PRTMus musculus 163Tyr Thr Ser Ser Leu His
Ser 1 5 1649PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 164Gln Gln Tyr Ser Lys Leu Pro Phe Thr 1
5 165117PRTMus musculus 165Glu Val Lys Leu Val Glu Ser Glu Gly Gly
Leu Val Gln Pro Gly Ser 1 5 10 15 Ser Met Lys Leu Ser Cys Thr Ala
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ala Trp Val Arg
Gln Val Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Asn
Tyr Asp Gly Ser Asn Thr Ser Tyr Leu Asp Ser Leu 50 55 60 Lys Ser
Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Ile Leu Tyr 65 70 75 80
Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85
90 95 Ala Arg Glu Lys Phe Ala Ala Met Asp Tyr Trp Gly Gln Gly Thr
Ser 100 105 110 Val Thr Val Ser Ser 115 1667PRTMus musculus 166Gly
Phe Thr Phe Ser Asp Tyr 1 5 1676PRTMus musculus 167Asn Tyr Asp Gly
Ser Asn 1 5 1688PRTMus musculus 168Glu Lys Phe Ala Ala Met Asp Tyr
1 5 169108PRTMus musculus 169Asp Ile Val Met Thr Gln Ser His Lys
Phe Met Ser Thr Ser Phe Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys
Lys Ala Ser Gln Asp Val Ser Asn Ala 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly His Ser Pro Lys Leu Leu Ile 35 40 45 Phe Ser Ala
Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 65 70
75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro
Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100
105 17011PRTMus musculus 170Lys Ala Ser Gln Asp Val Ser Asn Ala Leu
Ala 1 5 10 1717PRTArtificial SequenceDescription of Artificial
Sequence Synthetic variable light chain CDR peptide 171Ser Ala Ser
Tyr Arg Tyr Thr 1 5 1729PRTMus musculus 172Gln Gln His Tyr Ser Thr
Pro Trp Thr 1 5 173121PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 173Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg His 20 25 30 Thr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Ile Gly Ser Arg Glu Leu Tyr
Ile Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 17410PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 174Gly Phe Thr Phe Thr Arg His Thr Ile
His 1 5 10 17517PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 175Arg Ile Ser Pro Ala Asn Gly Asn Thr
Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 17612PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 176Trp
Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr 1 5 10
177108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 177Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser
Phe Leu Tyr 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 Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Ile Gln Pro 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
17811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 178Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1
5 10 1797PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 179Ser Ala Ser Phe Leu Tyr Ser 1 5
1809PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 180Gln Gln Ser Tyr Arg Ile Gln Pro Thr 1 5
181121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 181Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Thr 20 25 30 Ala Ile His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Ser
Pro Ala Asn Gly Asn Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Trp Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr Trp
Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
18210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 182Gly Phe Thr Phe Ser Ser Thr Ala Ile His 1 5 10
18317PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 183Arg Ile Ser Pro Ala Asn Gly Asn Thr Asn Tyr
Ala Asp Ser Val Lys 1 5 10 15 Gly 18412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 184Trp
Ile Gly Ser Arg Glu Leu Tyr Ile Met Asp Tyr 1 5 10
185108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 185Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser
Phe Leu Tyr 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 Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Ala Leu His 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
18611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 186Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1
5 10 1877PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 187Ser Ala Ser Phe Leu Tyr Ser 1 5
1889PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 188Gln Gln Ser Tyr Pro Ala Leu His Thr 1 5
189125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 189Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Pro Phe Ser Lys Leu 20 25 30 Gly Met Val Trp Val Arg
Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Ser Gly Gly
Gly Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Glu Gly Ile Ser Phe Gln Gly Gly Thr Tyr Thr Tyr Val Met 100 105
110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
19010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 190Gly Phe Pro Phe Ser Lys Leu Gly Met Val 1 5 10
19117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 191Thr Ile Ser Ser Gly Gly Gly Tyr Thr Tyr Tyr
Pro Asp Ser Val Lys 1 5 10 15 Gly 19216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 192Glu
Gly Ile Ser Phe Gln Gly Gly Thr Tyr Thr Tyr Val Met Asp Tyr 1 5 10
15 193112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 193Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Lys Ser Leu Leu His Arg 20 25 30 Asn Gly Ile Thr Tyr Ser
Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Gln Leu Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Tyr Gln Asn 85
90 95 Leu Glu Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110 19416PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 194Arg Ser Ser Lys Ser Leu
Leu His Arg Asn Gly Ile Thr Tyr Ser Tyr 1 5 10 15 1957PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 195Gln
Leu Ser Asn Leu Ala Ser 1 5 1969PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 196Tyr Gln Asn Leu Glu Leu
Pro Leu Thr 1 5 1972076DNAHomo sapiens 197atgggcaccg tcagctccag
gcggtcctgg tggccgctgc cactgctgct gctgctgctg 60ctgctcctgg gtcccgcggg
cgcccgtgcg caggaggacg aggacggcga ctacgaggag 120ctggtgctag
ccttgcgttc cgaggaggac ggcctggccg aagcacccga gcacggaacc
180acagccacct tccaccgctg cgccaaggat ccgtggaggt tgcctggcac
ctacgtggtg 240gtgctgaagg aggagaccca cctctcgcag tcagagcgca
ctgcccgccg cctgcaggcc 300caggctgccc gccggggata cctcaccaag
atcctgcatg tcttccatgg ccttcttcct 360ggcttcctgg tgaagatgag
tggcgacctg ctggagctgg ccttgaagtt gccccatgtc 420gactacatcg
aggaggactc ctctgtcttt gcccagagca tcccgtggaa cctggagcgg
480attacccctc cacggtaccg ggcggatgaa taccagcccc ccgacggagg
cagcctggtg 540gaggtgtatc tcctagacac cagcatacag agtgaccacc
gggaaatcga gggcagggtc 600atggtcaccg acttcgagaa tgtgcccgag
gaggacggga cccgcttcca cagacaggcc 660agcaagtgtg acagtcatgg
cacccacctg gcaggggtgg tcagcggccg ggatgccggc 720gtggccaagg
gtgccagcat gcgcagcctg cgcgtgctca actgccaagg gaagggcacg
780gttagcggca ccctcatagg cctggagttt attcggaaaa gccagctggt
ccagcctgtg 840gggccactgg tggtgctgct gcccctggcg ggtgggtaca
gccgcgtcct caacgccgcc 900tgccagcgcc tggcgagggc tggggtcgtg
ctggtcaccg ctgccggcaa cttccgggac 960gatgcctgcc tctactcccc
agcctcagct cccgaggtca tcacagttgg ggccaccaat 1020gcccaagacc
agccggtgac cctggggact ttggggacca actttggccg ctgtgtggac
1080ctctttgccc caggggagga catcattggt gcctccagcg actgcagcac
ctgctttgtg 1140tcacagagtg ggacatcaca ggctgctgcc cacgtggctg
gcattgcagc catgatgctg 1200tctgccgagc cggagctcac cctggccgag
ttgaggcaga gactgatcca cttctctgcc 1260aaagatgtca tcaatgaggc
ctggttccct gaggaccagc gggtactgac ccccaacctg 1320gtggccgccc
tgccccccag cacccatggg gcaggttggc agctgttttg caggactgta
1380tggtcagcac actcggggcc tacacggatg gccacagccg tcgcccgctg
cgccccagat 1440gaggagctgc tgagctgctc cagtttctcc aggagtggga
agcggcgggg cgagcgcatg 1500gaggcccaag ggggcaagct ggtctgccgg
gcccacaacg cttttggggg tgagggtgtc 1560tacgccattg ccaggtgctg
cctgctaccc caggccaact gcagcgtcca cacagctcca 1620ccagctgagg
ccagcatggg gacccgtgtc cactgccacc aacagggcca cgtcctcaca
1680ggctgcagct cccactggga ggtggaggac cttggcaccc acaagccgcc
tgtgctgagg 1740ccacgaggtc agcccaacca gtgcgtgggc cacagggagg
ccagcatcca cgcttcctgc 1800tgccatgccc caggtctgga atgcaaagtc
aaggagcatg gaatcccggc ccctcaggag 1860caggtgaccg tggcctgcga
ggagggctgg accctgactg gctgcagtgc cctccctggg 1920acctcccacg
tcctgggggc ctacgccgta gacaacacgt gtgtagtcag gagccgggac
1980gtcagcacta caggcagcac cagcgaaggg gccgtgacag ccgttgccat
ctgctgccgg 2040agccggcacc tggcgcaggc ctcccaggag ctccag
2076198692PRTHomo sapiens 198Met Gly Thr Val Ser Ser Arg Arg Ser
Trp Trp Pro Leu Pro Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu
Gly Pro Ala Gly Ala Arg Ala Gln Glu 20 25 30 Asp Glu Asp Gly Asp
Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu 35 40 45 Glu Asp Gly
Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe 50 55 60 His
Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val 65 70
75 80 Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala
Arg 85 90 95 Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr
Lys Ile Leu 100 105 110 His Val Phe His Gly Leu Leu Pro Gly Phe Leu
Val Lys Met Ser Gly 115 120 125 Asp Leu Leu Glu Leu Ala Leu Lys Leu
Pro His Val Asp Tyr Ile Glu 130 135 140 Glu Asp Ser Ser Val Phe Ala
Gln Ser Ile Pro Trp Asn Leu Glu Arg 145 150 155 160 Ile Thr Pro Pro
Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly 165 170 175 Gly Ser
Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp 180 185 190
His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val 195
200 205 Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys
Asp 210 215 220 Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg
Asp Ala Gly 225 230 235 240 Val Ala Lys Gly Ala Ser Met Arg Ser Leu
Arg Val Leu Asn Cys Gln 245 250 255 Gly Lys Gly Thr Val Ser Gly Thr
Leu Ile Gly Leu Glu Phe Ile Arg 260 265 270 Lys Ser Gln Leu Val Gln
Pro Val Gly Pro Leu Val Val Leu Leu Pro 275 280 285 Leu Ala Gly Gly
Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu 290 295 300 Ala Arg
Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp 305 310 315
320 Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val
325 330 335 Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr
Leu Gly 340 345 350 Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro
Gly Glu Asp Ile 355 360 365 Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys
Phe Val Ser Gln Ser Gly 370 375 380 Thr Ser Gln Ala Ala Ala His Val
Ala Gly Ile Ala Ala Met Met Leu 385 390 395 400 Ser Ala Glu Pro Glu
Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile 405 410 415 His Phe Ser
Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp 420 425 430 Gln
Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr 435 440
445 His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His
450 455 460 Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala
Pro Asp 465 470 475 480 Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg
Ser Gly Lys Arg Arg 485 490 495 Gly Glu Arg Met Glu Ala Gln Gly Gly
Lys Leu Val Cys Arg Ala His 500 505 510 Asn Ala Phe Gly Gly Glu Gly
Val Tyr Ala Ile Ala Arg Cys Cys Leu 515 520 525 Leu Pro Gln Ala Asn
Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala 530 535 540 Ser Met Gly
Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr 545 550 555 560
Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro 565
570 575 Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His
Arg 580 585 590 Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly
Leu Glu Cys 595 600 605 Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln
Glu Gln Val Thr Val 610 615 620 Ala Cys Glu Glu Gly Trp Thr Leu Thr
Gly Cys Ser Ala Leu Pro Gly 625 630 635 640 Thr Ser His Val Leu Gly
Ala Tyr Ala Val Asp Asn Thr Cys Val Val 645 650 655 Arg Ser Arg Asp
Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val 660 665 670 Thr Ala
Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser 675 680 685
Gln Glu Leu Gln 690
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