U.S. patent application number 16/799707 was filed with the patent office on 2021-01-14 for cardiovascular related uses of il-1beta antibodies and binding fragments thereof.
This patent application is currently assigned to XOMA (US) LLC. The applicant listed for this patent is XOMA (US) LLC. Invention is credited to Jeffrey D. Feldstein, Patrick J. Scannon, Alan M. Solinger.
Application Number | 20210009679 16/799707 |
Document ID | / |
Family ID | 1000005120471 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210009679 |
Kind Code |
A1 |
Scannon; Patrick J. ; et
al. |
January 14, 2021 |
CARDIOVASCULAR RELATED USES OF IL-1BETA ANTIBODIES AND BINDING
FRAGMENTS THEREOF
Abstract
Disclosed are methods for the reduction, prevention or treatment
of cardiovascular events and/or cardiovascular diseases, including
acute cardiovascular disease or chronic cardiovascular disease
using anti-IL-1.beta. binding molecules (e.g., IL-1.beta. binding
antibodies and fragments thereof). The present disclosure also
relates to methods for prevention or treatment of cardiovascular
events and/or cardiovascular diseases, including by reducing a
cardiovascular event or disease.
Inventors: |
Scannon; Patrick J.; (San
Francisco, CA) ; Solinger; Alan M.; (US) ;
Feldstein; Jeffrey D.; (Livingston, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
XOMA (US) LLC |
Emeryville |
CA |
US |
|
|
Assignee: |
XOMA (US) LLC
Emeryville
CA
|
Family ID: |
1000005120471 |
Appl. No.: |
16/799707 |
Filed: |
February 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15647203 |
Jul 11, 2017 |
10611832 |
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16799707 |
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12790738 |
May 28, 2010 |
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15647203 |
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61313001 |
Mar 11, 2010 |
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61252571 |
Oct 16, 2009 |
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61182679 |
May 29, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/245 20130101;
A61K 2039/505 20130101; A61P 9/04 20180101; A61K 39/395
20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1. A method of reducing a cardiovascular event in a subject,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof, wherein the subject is a subject with a history
of a previous cardiovascular event or a history of at least one
risk factor for cardiovascular disease, and wherein the
cardiovascular event is myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina, or a revascularization
procedure.
2-17. (canceled)
18. A method of reducing mortality following a cardiovascular event
in a subject, comprising administering to said subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof.
19-38. (canceled)
39. A method of reducing a cardiovascular event in a subject with a
history of at least one risk factor for cardiovascular disease,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof, and wherein said risk factor is not Type 2
diabetes, obesity, hyperglycemia, dyslipidemia, hyperlipidemia,
chronic renal failure, high blood glucose, chronic kidney disease,
hypertension, atherosclerosis or metabolic syndrome.
40-57. (canceled)
58. A method of treating a cardiovascular event in a subject,
wherein the cardiovascular event is myocardial infarction, stroke,
congestive heart failure, acute coronary syndrome or angina,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof and at least one other pharmaceutical composition
comprising an active agent other than an IL-1.beta. antibody or
fragment.
59. (canceled)
60. A method for treating a cardiovascular event in a subject,
wherein the cardiovascular event is myocardial infarction, stroke,
congestive heart failure, acute coronary syndrome or angina,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof and a revascularization procedure.
61. (canceled)
62. A method of reducing restenosis in a subject following a
revascularization procedure, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof.
63-64. (canceled)
65. A method of treating acute hypertension in a subject comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
one or more antihypertensive agents.
66-107. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/313,001, filed Mar. 11, 2010, U.S. Provisional
Application No. 61/252,571 filed Oct. 16, 2009, and U.S.
Provisional Application No. 61/182,679 filed May 29, 2009, the
disclosures of which are incorporated by reference herein in their
entirety.
FIELD OF INVENTION
[0002] The present disclosure relates generally to IL-113 binding
molecules (e.g., IL-113 binding antibodies and fragments thereof)
for the reduction, prevention or treatment of cardiovascular events
and/or cardiovascular diseases (e.g., acute cardiovascular disease
or chronic cardiovascular disease).
BACKGROUND OF THE INVENTION
[0003] Inflammation has become a central theme in the pathogenesis
of cardiovascular disease over the past decade, and a wide range of
cardiac diseases has been associated with inflammation and cytokine
modulation (Mehra et al., 2005, J. Leukocyte Biol. 78:805-818).
Proinflammatory cytokines may be secreted by every nucleated cell
type in the myocardium, including the cardiac myocyte, in response
to various forms of stress/injury. They are elevated in conditions
as diverse as inflammatory myocarditis, allograft rejection,
cardiac ischemic states, congestive heart failure (CHF), and
reperfusion injury.
[0004] IL-113 is a pro-inflammatory cytokine secreted by a number
of different cell types including monocytes and macrophages. When
released as part of an inflammatory reaction, IL-1.beta. produces a
range of biological effects, mainly mediated through induction of
other inflammatory mediators such as corticotrophin, platelet
factor-4, prostaglandin E2 (PGE2), IL-6, and IL-8. IL-1.beta.
induces both local and systemic inflammatory effects through the
activation of the IL-1 receptor found on almost all cell types. The
interleukin-1 (IL-1) family of cytokines has been implicated in a
number of disease states. IL-1 family members include IL-1.alpha.,
IL-1.beta., and IL-1Ra. Although related by their ability to bind
to IL-1 receptors (IL-1R1 and IL-1R2), each of these cytokines is
different, being expressed by a different gene and having a
different primary amino acid sequence. Furthermore, the
physiological activities of these cytokines can be distinguished
from each other.
SUMMARY OF THE INVENTION
[0005] The present disclosure relates generally to IL-1.beta.
binding molecules (e.g., IL-1.beta. binding antibodies and
fragments thereof) for the reduction, prevention or treatment of
cardiovascular events and/or cardiovascular diseases, including
acute cardiovascular disease or chronic cardiovascular disease. The
present disclosure also relates to methods for prevention or
treatment of cardiovascular events and/or cardiovascular diseases,
including by reducing a cardiovascular event or disease.
[0006] The present disclosure provides methods of reducing a
cardiovascular event in a subject, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof, wherein the subject
is a subject with a history of a previous cardiovascular event or a
history of at least one risk factor for cardiovascular disease, and
wherein the cardiovascular event is myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina, or a revascularization
procedure.
[0007] The present disclosure provides methods of reducing a
cardiovascular event (e.g., delaying time to event, reducing
likelihood or risk of event, preventing an event, reducing severity
of event, reducing time to recovery) in a subject with a history of
at least one risk factor for cardiovascular disease, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof,
and wherein the cardiovascular event is myocardial infarction,
stroke, cardiovascular death, congestive heart failure, cardiac
arrest, acute coronary syndrome, angina, or a revascularization
procedure.
[0008] The present disclosure also provides methods of reducing a
cardiovascular event (e.g., delaying time to event, reducing
likelihood or risk of event, preventing an event, reducing severity
of event, reducing time to recovery) in a subject with a history of
a previous cardiovascular event, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof, and wherein said
cardiovascular event is myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina or a revascularization procedure.
In some embodiments, the previous cardiovascular event is a first
cardiovascular event. In some embodiments, the previous or first
cardiovascular event is selected from the group consisting of
myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome, angina and a revascularization procedure. In
some embodiments, the previous or first cardiovascular event is
myocardial infarction or acute coronary syndrome. In some
embodiments, the myocardial infarction is myocardial infarction
with ST elevation (e.g., ST-segment elevation myocardial
infarction, STEMI). In some embodiments, the myocardial infarction
is myocardial infarction without ST elevation (e.g., non-ST-segment
elevation myocardial infarction, NSTEMI). In some embodiments the
presence or absence of ST elevation is determined by
electrocardiogram (e.g., ECG, EKG). In some embodiments, the method
of reducing a cardiovascular event is a method of reducing a second
or subsequent cardiovascular event. In some embodiments, the
cardiovascular event (e.g., second or subsequent cardiovascular
event) is selected from the group consisting of myocardial
infarction, stroke, cardiovascular death, congestive heart failure,
cardiac arrest, acute coronary syndrome, angina and a
revascularization procedure. In some embodiments, the first
cardiovascular event and second cardiovascular event are the same
types of cardiovascular events. In some embodiments, the first
cardiovascular event and second cardiovascular event are different
types of cardiovascular events.
[0009] In some embodiments, the revascularization procedure is a
coronary, carotid or peripheral arterial revascularization
procedure. In some embodiments, the coronary, carotid or peripheral
arterial revascularization procedure is a percutaneous coronary
intervention (PCI), a stent implant, coronary artery bypass graft
(CABG), carotid endarterectomy, peripheral vascular disease bypass
surgery, or peripheral angioplasty surgery.
[0010] In some embodiments, said subject also has a history of at
least one risk factor for cardiovascular disease. In some
embodiments, the risk factor is manifest coronary heart disease,
coronary artery disease, thrombosis, transient ischaemic attack,
left ventricular hypertrophy, arteriosclerosis, restenosis, tobacco
smoking or peripheral vascular disease. In some embodiments, the
risk factor is elevated triglycerides, systemic inflammation, high
blood phosphorus levels, high parathyroid hormone levels,
microalbuminuria, or high homocysteine levels. In some embodiments,
the risk factor is obesity, hyperglycemia, chronic renal failure,
high blood glucose, chronic kidney disease, or metabolic syndrome.
In some embodiments, the risk factor is end stage renal disease. In
some embodiments, the risk factor is hypertension, dyslipidemia,
hyperlipidemia, elevated total cholesterol, elevated LDL
cholesterol, or low HDL cholesterol or atherosclerosis. In some
embodiments, the hypertension is manifested as a blood pressure of
greater than or equal to 180/110 mm Hg. In some other embodiments,
the hypertension is mild-to-moderate, with systolic blood pressure
(SBP) of 140 to 180 mm Hg and/or diastolic blood pressure (DBP) of
90 to 110 mm Hg.
[0011] In some embodiments, the subject has elevated levels of
C-reactive protein (CRP).
[0012] In some embodiments, the subject is older than 55 years.
[0013] In some embodiments, the subject is older than 65 years.
[0014] In some embodiments, the subject is non-hypertensive.
[0015] In some embodiments, the subject has poorly controlled
hypertension.
[0016] In some embodiments, the subject has an arrhythmia.
[0017] In some embodiments, the subject has a "Type A"
personality.
[0018] In some embodiments, the subject has a sedentary
lifestyle.
[0019] In some embodiments, the subject has diabetes mellitus. In
some embodiments, said diabetes mellitus is Type 2 diabetes.
[0020] In some embodiments, the subject has a history of two or
more said risk factors.
[0021] In some embodiments, the subject has a history of three or
more said risk factors.
[0022] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP
levels.
[0023] The present disclosure also provides methods of reducing
mortality following a cardiovascular event in a subject, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment
thereof.
[0024] In some embodiments, the cardiovascular event is myocardial
infarction, stroke, congestive heart failure, acute coronary
syndrome, angina or a revascularization procedure. In some
embodiments, the cardiovascular event is myocardial infarction or
acute coronary syndrome. In some embodiments, the myocardial
infarction is myocardial infarction with ST elevation (e.g.,
ST-segment elevation myocardial infarction, STEMI). In some
embodiments, the myocardial infarction is myocardial infarction
without ST elevation (e.g., non-ST-segment elevation myocardial
infarction, NSTEMI). In some embodiments the presence or absence of
ST elevation is determined by electrocardiogram (e.g., ECG, EKG).
In some embodiments, the revascularization procedure is a coronary,
carotid or peripheral arterial revascularization procedure. In some
embodiments, the coronary, carotid or peripheral arterial
revascularization procedure is a percutaneous coronary intervention
(PCI), a stent implant, coronary artery bypass graft (CABG),
carotid endarterectomy, peripheral vascular disease bypass surgery,
or peripheral angioplasty surgery.
[0025] In some embodiments, the subject does not have Type 2
diabetes.
[0026] In some embodiments, the subject has survived a previous
cardiovascular event of myocardial infarction or stroke.
[0027] In some embodiments, the occurrence of said cardiovascular
event is a reoccurrence of a cardiovascular event of myocardial
infarction or stroke.
[0028] In some embodiments, the subject has a history of one or
more risk factors for cardiovascular disease. In some embodiments,
the risk factor is manifest coronary heart disease, coronary artery
disease, thrombosis, transient ischaemic attack, left ventricular
hypertrophy, arteriosclerosis, restenosis, tobacco smoking or
peripheral vascular disease. In some embodiments, the risk factor
is elevated triglycerides, systemic inflammation, high blood
phosphorus levels, high parathyroid hormone levels,
microalbuminuria, or high homocysteine levels. In some embodiments,
the risk factor is obesity, hyperglycemia, chronic renal failure,
high blood glucose, chronic kidney disease, or metabolic syndrome.
In some embodiments, the risk factor is end stage renal disease. In
some embodiments, the risk factor is hypertension, dyslipidemia,
hyperlipidemia, elevated total cholesterol, elevated LDL
cholesterol, or low HDL cholesterol or atherosclerosis. In some
embodiments, the hypertension is manifested as a blood pressure of
greater than or equal to 180/110 mm Hg. In some other embodiments,
the hypertension is mild-to-moderate, with systolic blood pressure
(SBP) of 140 to 180 mm Hg and/or diastolic blood pressure (DBP) of
90 to 110 mm Hg.
[0029] In some embodiments, the subject is non-hypertensive.
[0030] In some embodiments, the subject has poorly controlled
hypertension.
[0031] In some embodiments, the subject has an arrhythmia.
[0032] In some embodiments, the subject has a "Type A"
personality.
[0033] In some embodiments, the subject has a sedentary
lifestyle.
[0034] In some embodiments, the subject has a history of two or
more said risk factors.
[0035] In some embodiments, the subject has a history of three or
more said risk factors.
[0036] In some embodiments, the subject is a patient with
cardiovascular disease, including acute cardiovascular disease
(e.g., not associated with congestive heart failure) or chronic
cardiovascular disease (e.g., associated with multiple risk factors
for atherosclerotic cardiovascular disease).
[0037] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP
levels.
[0038] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of at least one
risk factor for cardiovascular disease, comprising administering to
said subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof, and
wherein said risk factor is not Type 2 diabetes, obesity,
hyperglycemia, dyslipidemia, hyperlipidemia, chronic renal failure,
high blood glucose, chronic kidney disease, hypertension,
atherosclerosis or metabolic syndrome.
[0039] In some embodiments, the cardiovascular event is myocardial
infarction, stroke, cardiac arrest, congestive heart failure,
cardiovascular death, acute coronary syndrome (e.g., diagnosed),
angina or a revascularization procedure. In some embodiments, the
revascularization procedure is a coronary, carotid or peripheral
arterial revascularization procedure. In some embodiments, the
coronary, carotid or peripheral arterial revascularization
procedure is a percutaneous coronary intervention (PCI), a stent
implant, coronary artery bypass graft (CABG), carotid
endarterectomy, peripheral vascular disease bypass surgery, or
peripheral angioplasty surgery.
[0040] In some embodiments, the risk factor is manifest coronary
heart disease, coronary artery disease, thrombosis, transient
ischaemic attack, left ventricular hypertrophy, arteriosclerosis,
restenosis, tobacco smoking or peripheral vascular disease. In some
embodiments, the risk factor is elevated triglycerides, systemic
inflammation, high blood phosphorus levels, high parathyroid
hormone levels, microalbuminuria, or high homocysteine levels.
[0041] In some embodiments, the subject has elevated levels of
C-reactive protein (CRP).
[0042] In some embodiments, the subject is older than 55 years.
[0043] In some embodiments, the subject is older than 65 years.
[0044] In some embodiments, the subject has a history of two or
more said risk factors.
[0045] In some embodiments, the subject has a history of three or
more said risk factors.
[0046] In some embodiments, the subject is a patient with
cardiovascular disease, including acute cardiovascular disease
(e.g., not associated with congestive heart failure) or chronic
cardiovascular disease (e.g., associated with multiple risk factors
for atherosclerotic cardiovascular disease).
[0047] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP
levels.
[0048] The present disclosure also provides methods of treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof and at least one other
pharmaceutical composition comprising an active agent other than an
IL-1.beta. antibody or fragment.
[0049] In some embodiments, the cardiovascular event is myocardial
infarction or acute coronary syndrome. In some embodiments, the
myocardial infarction is myocardial infarction with ST elevation
(e.g., ST-segment elevation myocardial infarction, STEMI). In some
embodiments, the myocardial infarction is myocardial infarction
without ST elevation (e.g., non-ST-segment elevation myocardial
infarction, NSTEMI). In some embodiments the presence or absence of
ST elevation is determined by electrocardiogram (e.g., ECG,
EKG).
[0050] In some embodiments, the active agent of said at least one
other pharmaceutical composition is a cholesterol lowering agent, a
statin, an HMG-CoA reductase inhibitor, a calcium channel blocker,
a beta blocker, an antihypertensive, a diuretic, aspirin, niacin,
an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II
receptor blocker, a vasodilator, an anticoagulant, a inhibitor of
platelet aggregation, a thrombolytic or digitalis.
[0051] The present disclosure also provides methods of treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof and (e.g., in
conjunction with) a revascularization procedure.
[0052] In some embodiments, the cardiovascular event is myocardial
infarction or acute coronary syndrome. In some embodiments, the
myocardial infarction is myocardial infarction with ST elevation
(e.g., ST-segment elevation myocardial infarction, STEMI). In some
embodiments, the myocardial infarction is myocardial infarction
without ST elevation (e.g., non-ST-segment elevation myocardial
infarction, NSTEMI). In some embodiments the presence or absence of
ST elevation is determined by electrocardiogram (e.g., ECG,
EKG).
[0053] In some embodiments, the revascularization procedure is a
coronary, carotid or peripheral arterial revascularization
procedure.
[0054] The present disclosure also provides methods of treating
cardiovascular disease, including, for example, acute
cardiovascular disease or chronic cardiovascular disease, in a
subject, comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof and (e.g., in conjunction with) a
revascularization procedure.
[0055] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a reduction in the
relative risk (e.g., lower risk, frequency, incidence, severity) of
MACE (major adverse cardiac event, e.g., myocardial infarction,
stroke, death, such as CV death, and/or composite thereof),
including, for example, in patients with cardiovascular disease,
such as acute cardiovascular disease or chronic cardiovascular
disease, or in patients with multiple risk factors for
atherosclerotic cardiovascular disease (e.g., age 55, age 65, plus
one or more of: CABG, NSTEMI, hypertension, elevated cholesterol or
on statins, elevated CRP, prior history of Myocardial
infarction/stroke no less than 6 months, prior history of ACS or
TIA, smoking, history of PCI, type 2 diabetes).
[0056] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve an in time to first MACE
event, revascularization procedures (e.g., CABG), all cause
mortality, peripheral vascular disease, first documented angina
endpoint, hospitalization for congestive heart failure (CHF),
decrease in number of hospital visits, duration of hospital stay,
rehospitalization for ischemic events (e.g., angina and/or CHF),
infarct size, diastolic volume, ejection fraction or use of
diuretics.
[0057] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve plaque regression, plaque
stabilization and/or inhibition of plaque rupture.
[0058] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP levels,
BNP levels, troponin levels, C-peptide levels, LDL levels, blood
pressure or blood sugar (HbA1c).
[0059] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease or no increase
in SAE, malignancy, hypoglycemia, serious infection rate, infection
rate, immunogenicity or heart failure.
[0060] The present disclosure also provides methods of reducing
restenosis in a subject following a revascularization procedure,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof.
[0061] In some embodiments, the revascularization procedure is a
coronary, carotid or peripheral arterial revascularization
procedure.
[0062] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP
levels.
[0063] The present disclosure also provides methods of treating
acute hypertension in a subject comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof and one or more
antihypertensive agents. In some embodiments, the subject has a
blood pressure of greater than or equal to 180/110 mm Hg. In some
other embodiments, the subject has mild-to-moderate hypertension,
with systolic blood pressure (SBP) of 140 to 180 mm Hg and/or
diastolic blood pressure (DBP) of 90 to 110 mm Hg. In some
embodiments, the antihypertensive agent is administered
intravenously. In some embodiments, the antihypertensive agent is
selected from the group consisting of alpha/beta-adrenergic
blocking agents, angiotensin-converting enzyme inhibitors,
angiotensin II receptor antagonists, antiadrenergic agents,
beta-adrenergic blocking agents, calcium-channel blocking agents,
diuretics, and vasodilators. In some embodiments, the
antihypertensive agent is carvedilol, labetalol, benazepril,
captopril, enalapril, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, trandolapril, candesartan,
eprosartan, irbesartan, losartan, telmisartan, valsartan,
clonidine, doxazosin, guanabenz, guanadrel, guanethidine,
guanfacine, mecamylamine, methyldopa, prazosin, reserpine,
terazosin, acebutolol, atenolol, betaxolol, bisoprolol, carteolol,
metoprolol, nadolol, penbutolol, pindolol, propranolol, timolol,
amlodipine, diltiazem, felodipine, isradipine, nicardipine,
nifedipine, nisoldipine, verapamil, amiloride, benzthiazide,
chlorothiazide, chlorthalidone, furosemide, hydrochlorothiazide,
indapamide, metolazone, polythiazide, spironolactone, torsemide,
trichlormethiazide, hydralazine, nitroglycerin, sodium
nitroprusside, clevidipine or minoxidil. In some embodiments, the
antihypertensive agent is labetalol, metoprolol, hydralazine,
nitroglycerin, nicardipine, sodium nitroprusside or
clevidipine.
[0064] The present disclosure also provides methods of reducing,
preventing or treating a cardiovascular event or disease (e.g.,
acute cardiovascular disease or chronic cardiovascular disease) in
a subject comprising administering to the subject an
anti-IL-1.beta. binding antibody or binding fragment thereof in
combination with (e.g., in conjunction with) (e.g., before, during
or after) a medical or surgical intervention. Such antibodies may
be administered in therapeutically effective amounts. Such
interventions may be therapeutically effective. In some
embodiments, a medical intervention is an active agent, such as a
drug or a biologic, including, for example, any one or more of the
active agents described herein. In some embodiments, a medical
intervention is an out-patient medical treatment or procedure. In
some embodiments, a medical intervention is an in-patient
hospitalization. In some embodiments, a surgical intervention is a
revascularization procedure, including, for example, any one or
more of the revascularization procedures described herein. In some
embodiments, a surgical intervention involves a heart valve repair
or replacement, coronary bypass surgery, heart transplant or heart
pump. In some embodiments, a surgical intervention involves a
biventricular cardiac pacemaker, internal cardiac defibrillator
(ICD) or myectomy. In some embodiments, a medical intervention is
smoking cessation medication or smoking cessation counseling.
[0065] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of at least one
risk factor for cardiovascular disease, comprising (a) identifying,
diagnosing or selecting the subject with the history of at least
one risk factor for cardiovascular disease and (b) administering to
the subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof, and
wherein the cardiovascular event is myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina, or a revascularization
procedure.
[0066] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of a previous
cardiovascular event, comprising (a) identifying, diagnosing or
selecting the subject with the history of the previous
cardiovascular event and (b) administering to the subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof, and wherein the
cardiovascular event is myocardial infarction, stroke, acute
coronary syndrome, angina or a revascularization procedure. In some
embodiments, the previous cardiovascular event is a first
cardiovascular event. In some embodiments, the previous or first
cardiovascular event is selected from the group consisting of
myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome, angina and a revascularization procedure. In
some embodiments, the previous or first cardiovascular event is
myocardial infarction or acute coronary syndrome. In some
embodiments, the myocardial infarction is myocardial infarction
with ST elevation (e.g., ST-segment elevation myocardial
infarction, STEMI). In some embodiments, the myocardial infarction
is myocardial infarction without ST elevation (e.g., non-ST-segment
elevation myocardial infarction, NSTEMI). In some embodiments the
presence or absence of ST elevation is determined by
electrocardiogram (e.g., ECG, EKG). In some embodiments, the method
of reducing a cardiovascular event is a method of reducing a second
or subsequent cardiovascular event. In some embodiments, the
cardiovascular event (e.g., second or subsequent cardiovascular
event) is selected from the group consisting of myocardial
infarction, stroke, cardiovascular death, congestive heart failure,
cardiac arrest, acute coronary syndrome, angina and a
revascularization procedure. In some embodiments, the first
cardiovascular event and second cardiovascular event are the same
types of cardiovascular events. In some embodiments, the first
cardiovascular event and second cardiovascular event are different
types of cardiovascular events.
[0067] The present disclosure also provides methods of reducing
mortality following a cardiovascular event in a subject, comprising
(a) identifying, diagnosing or selecting the subject having the
cardiovascular event and (b) administering to the subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof. In some embodiments, the
cardiovascular event is selected from the group consisting of
myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome, angina and a revascularization procedure. In
some embodiments, the cardiovascular event is myocardial infarction
or acute coronary syndrome. In some embodiments, the myocardial
infarction is myocardial infarction with ST elevation (e.g.,
ST-segment elevation myocardial infarction, STEMI). In some
embodiments, the myocardial infarction is myocardial infarction
without ST elevation (e.g., non-ST-segment elevation myocardial
infarction, NSTEMI). In some embodiments the presence or absence of
ST elevation is determined by electrocardiogram (e.g., ECG,
EKG).
[0068] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of at least one
risk factor for cardiovascular disease, comprising (a) identifying,
diagnosing or selecting the subject with the history of at least
one risk factor for cardiovascular disease and (b) administering to
the subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof, and
wherein the risk factor is not Type 2 diabetes, obesity,
hyperglycemia, dyslipidemia, hyperlipidemia, chronic renal failure,
high blood glucose, chronic kidney disease, hypertension,
atherosclerosis or metabolic syndrome. In some embodiments, the
cardiovascular event is selected from the group consisting of
myocardial infarction, stroke, cardiovascular death, congestive
heart failure, cardiac arrest, acute coronary syndrome, angina and
a revascularization procedure.
[0069] The present disclosure also provides methods of treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising (a) identifying, diagnosing
or selecting the subject with the cardiovascular event and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
at least one other pharmaceutical composition comprising an active
agent other than an IL-1.beta. antibody or fragment. In some
embodiments, the previous or first cardiovascular event is
myocardial infarction or acute coronary syndrome. In some
embodiments, the myocardial infarction is myocardial infarction
with ST elevation (e.g., ST-segment elevation myocardial
infarction, STEMI). In some embodiments, the myocardial infarction
is myocardial infarction without ST elevation (e.g., non-ST-segment
elevation myocardial infarction, NSTEMI). In some embodiments the
presence or absence of ST elevation is determined by
electrocardiogram (e.g., ECG, EKG).
[0070] The present disclosure also provides methods for treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising (a) identifying, diagnosing
or selecting the subject with the cardiovascular event and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
(e.g., in conjunction with) a revascularization procedure. In some
embodiments, the previous or first cardiovascular event is
myocardial infarction or acute coronary syndrome. In some
embodiments, the myocardial infarction is myocardial infarction
with ST elevation (e.g., ST-segment elevation myocardial
infarction, STEMI). In some embodiments, the myocardial infarction
is myocardial infarction without ST elevation (e.g., non-ST-segment
elevation myocardial infarction, NSTEMI). In some embodiments the
presence or absence of ST elevation is determined by
electrocardiogram (e.g., ECG, EKG).
[0071] The present disclosure also provides methods of reducing
restenosis in a subject following a revascularization procedure,
comprising (a) identifying, diagnosing or selecting the subject
with the revascularization procedure and (b) administering to the
subject a therapeutically effective amount of an anti-IL-1p binding
antibody or binding fragment thereof.
[0072] The present disclosure also provides methods of treating
acute hypertension in a subject comprising (a) identifying,
diagnosing or selecting the subject with acute hypertension and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
one or more antihypertensive agents. In some embodiments, the
hypertension is manifested as a blood pressure of greater than or
equal to 180/110 mm Hg. In some other embodiments, the hypertension
is mild-to-moderate, with systolic blood pressure (SBP) of 140 to
180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mm
Hg.
[0073] In any and/or all of the aforementioned embodiments,
administering said therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof may be
sufficient to achieve a decrease in CRP levels.
[0074] The present disclosure also provides pharmaceutical
compositions for use in any and/or all of the aforementioned
methods, including for example, for the reduction, prevention or
treatment of cardiovascular events and/or cardiovascular diseases,
including acute cardiovascular disease or chronic cardiovascular
disease, by administering a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof.
[0075] Various methods and pharmaceutical compositions are provided
herein, including for example, those described above. The present
disclosure further provides IL-1.beta. binding antibodies and
binding fragments thereof, as well as suitable dose amounts and
dosing regimens that may be used in or with any and/or all of the
aforementioned methods and pharmaceutical compositions.
[0076] In some embodiments of any and/or all of the methods and
pharmaceutical compositions described above, the antibody or
fragment binds to human IL-1.beta. with a dissociation constant of
about 1 nM or less. In some embodiments, the antibody or fragment
binds to human IL-1.beta. with a dissociation constant of about 500
pM or less. In some embodiments, the anti-IL-1.beta. binding
antibody or binding fragment thereof binds to human IL-1.beta. with
a dissociation constant of about 250 pM or less. In some
embodiments, the anti-IL-1.beta. binding antibody or binding
fragment thereof binds to human IL-1.beta. with a dissociation
constant of about 100 pM or less. In some embodiments of any of the
methods described above, the anti-IL-1.beta. binding antibody or
binding fragment thereof binds to human IL-1.beta. with a
dissociation constant of about 50 pM or less. In some embodiments
of any of the methods described above, the anti-IL-1.beta. binding
antibody or binding fragment thereof binds to human IL-1.beta. with
a dissociation constant of about 5 pM or less. In some embodiments,
the anti-IL-1.beta. binding antibody or binding fragment thereof
binds to human IL-1.beta. with a dissociation constant of about 1
pM or less. In some embodiments, the anti-IL-1.beta. binding
antibody or binding fragment thereof binds to human IL-1.beta. with
a dissociation constant of about 0.3 pM or less.
[0077] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof is a neutralizing antibody.
[0078] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof binds to an IL-1.beta. epitope such that the bound
antibody or fragment substantially permits the binding of
IL-1.beta. to IL-1 receptor I (IL-1RI).
[0079] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof does not detectably bind to IL-1.alpha., IL-1R or
IL-1Ra.
[0080] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof competes with the binding of an antibody having
the light chain variable region of SEQ ID NO:1 and the heavy chain
variable region of SEQ ID NO:2. In some embodiments of any and/or
all of the methods described above, the anti-IL-1.beta. binding
antibody or binding fragment thereof binds to an epitope that is
the same or substantially the same as an epitope that is bound by
an antibody having the light chain variable region of SEQ ID NO:1
and the heavy chain variable region of SEQ ID NO:2. In some
embodiments of any and/or all of the methods described above, the
anti-IL-1.beta. binding antibody or binding fragment thereof
comprises a light chain variable region of SEQ ID NO:1 and a heavy
chain variable region of SEQ ID NO:2.
[0081] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof binds to an epitope incorporating Glu64 of
IL-1.beta..
[0082] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof binds to amino acids 1-34 of the N terminus of
IL-1.beta..
[0083] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof is humanized or human.
[0084] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof is administered in one or more doses of 3 mg/kg of
antibody or fragment. In some embodiments, the anti-IL-1.beta.
binding antibody or binding fragment thereof is administered in one
or more doses of 1 mg/kg or less of antibody or fragment. In some
embodiments, the anti-IL-1.beta. binding antibody or binding
fragment thereof is administered in one or more doses of 0.3 mg/kg
or less of antibody or fragment. In some embodiments, the
anti-IL-1.beta. binding antibody or binding fragment thereof is
administered in one or more doses of 0.1 mg/kg or less of antibody
or fragment. In some embodiments, the anti-IL-1.beta. binding
antibody or binding fragment thereof is administered in one or more
doses of 0.03 mg/kg or less of antibody or fragment. In some
embodiments, the one or more doses are at least 0.01 mg/kg of
antibody or fragment. In some embodiments of any of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof is administered in one or more doses of 0.03 mg/kg
to 1 mg/kg.
[0085] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof is administered as a fixed dose, independent of a
dose per subject weight ratio. In some embodiments, the
anti-IL-1.beta. binding antibody or binding fragment thereof is
administered in one or more doses of 100 mg or less of antibody or
fragment. In some embodiments, the anti-IL-1.beta. binding antibody
or binding fragment thereof is administered in one or more doses of
25 mg or less of antibody or fragment In some embodiments, the
anti-IL-1.beta. binding antibody or binding fragment thereof is
administered in one or more doses of 10 mg or less of antibody or
fragment. In some embodiments, the anti-IL-1.beta. binding antibody
or binding fragment thereof is administered in one or more doses of
at least 0.5 mg of antibody or fragment. In some embodiments, the
anti-IL-1.beta. binding antibody or binding fragment thereof is
administered in one or more doses of 1 mg to 100 mg of antibody or
fragment. In some embodiments, said fixed dose of anti-IL-1.beta.
binding antibody or binding fragment thereof is administered using
a pre-filled syringe or delivery device.
[0086] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof is administered by subcutaneous, intravenous or
intramuscular injection.
[0087] In some embodiments of any and/or all of the methods
described above, administration of an initial dose of
anti-IL-1.beta. binding antibody or binding fragment thereof is
followed by the administration of one or more subsequent doses. In
some embodiments, said initial dose and one or more subsequent
doses are administered at an interval of about once every week to
about once every 12 months. In some embodiments, said initial dose
and one or more subsequent doses are administered at an interval of
about once every two weeks to about once every 6 months. In some
embodiments, said initial dose and one or more subsequent doses are
administered at an interval of about once every month to about once
every 6 months. In some embodiments, said initial dose and one or
more subsequent doses are administered at an interval of about once
every month to about once every 3 months. In some embodiments, said
initial dose and one or more subsequent doses are administered at
an interval of about once every 3 months to about once every 6
months.
[0088] In some embodiments of any and/or all of the aforementioned
methods dosing regimens are provided, wherein the dosing regimen
comprises more than one dosing interval for administration of an
IL-1.beta. binding antibody or binding fragment thereof. In some
embodiments, the dosage regimen comprises at least two (e.g., two,
three, four, five, six) different dosing intervals for
administration of the IL-1.beta. antibody or fragment thereof. In
some embodiments, the dosage regimen comprises two different dosing
intervals for administration of the IL-1.beta. antibody or fragment
thereof. In some embodiments, the dosing regimen comprises two
different dosing intervals for administration of the IL-1.beta.
binding antibody or binding fragment thereof, wherein a first
dosing interval comprises administration of one or more doses of
the IL-1.beta. antibody or fragment thereof and a second dosing
interval comprises administration of one or more doses of the
IL-1.beta. antibody or fragment thereof, and wherein the first
dosing interval is shorter in time than the second dosing interval.
For example, the first dosing interval may be days or weeks, and
the second dosing interval may be months. In some embodiments, the
first dosing interval is about 5 days to about 28 days, about 7
days to about 21 days, about 12 days to about 16 days, or about 14
days. In some embodiments, the second dosing interval is about 1
month to about 3 months, about 1 month to about 2 months, or about
1 month. In some embodiments, the first dosing interval is about 7
days and the second dosing interval is about 1 month.
[0089] In some embodiments, administration of an initial dose of
anti-IL-1.beta. binding antibody or binding fragment thereof is
followed by administration of one or more subsequent doses, and
wherein the dosing intervals between administration of the initial
dose and a second dose, and the second dose and a third dose are
about 7 days to about 21 days, and wherein the dosing intervals
between administration of subsequent doses is about 1 month to
about 3 months. In some embodiments, the dosing intervals between
administration of the initial dose and a second dose, and the
second dose and a third dose are about 12 to 16 days, and the
dosing intervals between administration of subsequent doses is
about 1 month to about 2 months. In some embodiments, the dosing
intervals between administration of the initial dose and a second
dose, and the second dose and a third dose are about 14 days, and
the dosing intervals between administration of subsequent doses is
about 1 month.
[0090] In some preferred embodiments of any and/or all of the
aforementioned methods, dose amounts and/or dosing regimens, the
IL-1.beta. binding antibody or binding fragment thereof (e.g.,
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof) is first administered within
1 week of the cardiovascular event, within 96 hours of the
cardiovascular event, within 72 hours of the cardiovascular event,
within 48 hours of the cardiovascular event, within 24 hours of the
cardiovascular event, or within 12 hours of the cardiovascular
event.
[0091] In some embodiments of any and/or all of the methods
described above, administration of an initial dose of the
anti-IL-1.beta. binding antibody or binding fragment thereof is
followed by the administration of one or more subsequent doses, and
wherein said one or more subsequent doses are in an amount that is
approximately the same or less than the initial dose.
[0092] In some embodiments of any and/or all of the methods
described above, administration of an initial dose of the
anti-IL-1.beta. binding antibody or binding fragment thereof is
followed by the administration of one or more subsequent doses, and
wherein at least one of the subsequent doses is in an amount that
is more than the initial dose.
[0093] In some embodiments of any and/or all of the methods
described above, the anti-IL-1.beta. binding antibody or binding
fragment thereof thereof has a lower IC.sub.50 than an IL-1.beta.
receptor antagonist in a human whole blood IL-1.beta. inhibition
assay that measures IL-1.beta. induced production of IL-8. In some
embodiments, the IL-1.beta. receptor antagonist is anakinra.
[0094] In some embodiments, any and/or all of the methods described
above may further comprise administering at least one other
pharmaceutical composition comprising an active agent other than an
anti-IL-1.beta. binding antibody or binding fragment thereof. In
some embodiments, the active agent of said at least one other
pharmaceutical composition is a cholesterol lowering agent. In some
embodiments, the active agent of said at least one other
pharmaceutical composition is a statin or an HMG-CoA reductase
inhibitor (e.g., lovastatin, pravastatin, simvastatin, fluvastatin,
atorvastatin, cerivastatin, mevastatin, pitavastatin, rosuvastatin
or mixtures thereof or mixtures with Ezetimibe, niacin, Amlodipine
Besylate). In some embodiments, the active agent of said at least
one other pharmaceutical composition is a calcium channel blocker
(e.g., amlodipine, diltiazem, nifedipine, nicardipine, verapamil)
or a beta blocker (e.g., esmolol, metoprolol, nadolol, penbutolol).
In some embodiments, the active agent of said at least one other
pharmaceutical composition is an antihypertensive (e.g., labetalol,
metoprolol, hydralazine, nitroglycerin, nicardipine, sodium
nitroprusside, clevidipine), a diuretic (e.g., a thiazide diuretic,
chlorthalidone, furosemide, hydrochlorothiazide, indapamide,
metolazone, amiloride hydrochloride, spironolactone, triamterene)
or aspirin. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an angiotensin-converting
enzyme (ACE) inhibitor (e.g. ramipril, ramiprilat, captopril,
lisinopril) or an angiotensin II receptor blocker (e.g., losartan,
olmesartan, valsartan). In some embodiments, the active agent of
said at least one other pharmaceutical composition is a
vasodilator. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an anticoagulant (e.g.,
acenocoumarol, phenprocoumon, warfarin heparin, low molecular
weight heparin) or inhibitor of platelet aggregation (e.g.,
clopidogrel, ticlopidine, cilostazol, dipyridamole, eptifibatide,
aspirin, abciximab, eptifibatide, tirofiban). In some embodiments,
the active agent of said at least one other pharmaceutical
composition is a thrombolytic (e.g., streptokinase, urokinase,
alteplase, reteplase, tenecteplase). In some embodiments, the
active agent of said at least one other pharmaceutical composition
is digitalis. In some embodiments, the active agent of said at
least one other pharmaceutical composition is digoxin or
nesiritide. In some embodiments, the active agent of said at least
one other pharmaceutical composition is oxygen. In some
embodiments, the active agent of said at least one other
pharmaceutical composition is a thrombin inhibitor (e.g., hirudin,
bivalirudin). In some embodiments, the active agent of said at
least one other pharmaceutical composition is a nitrate (e.g.,
glyceryl trinitrate (GTN)/nitroglycerin, isosorbide dinitrate,
isosorbide mononitrate). In some embodiments, the active agent of
said at least one other pharmaceutical composition is an analgesic
(e.g., morphine sulfate). In some embodiments, the active agent of
said at least one other pharmaceutical composition is a renin
inhibitor. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an endothelin A receptor
inhibitor. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an aldosterone
inhibitor.
[0095] The present disclosure also provides uses of an
anti-IL-1.beta. binding antibody or binding fragment thereof which
has a lower IC.sub.50 than an IL-1.beta. receptor antagonist in a
human whole blood IL-1.beta. inhibition assay that measures
IL-1.beta. induced production of IL-8, in the manufacture of a
composition for use in the reduction, prevention or treatment of a
cardiac event or a cardiovascular disease.
[0096] These IL-1.beta. binding antibodies and binding fragments
thereof, as well as suitable dose amounts and dosing regimens
and/or other pharmaceutical compositions comprising an active agent
other than an anti-IL-1.beta. antibody or fragment thereof, as
provided herein, may be used in or with any of the aforementioned
methods and/or pharmaceutical compositions, including for
example:
[0097] Methods and/or pharmaceutical compositions for use in
reducing a cardiovascular event (e.g., delaying time to event,
reducing likelihood or risk of event, preventing an event, reducing
severity of event, reducing time to recovery) in a subject with a
history of at least one risk factor for cardiovascular disease,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof, and wherein the cardiovascular event is
myocardial infarction, stroke, cardiovascular death, congestive
heart failure, cardiac arrest, acute coronary syndrome, angina, or
a revascularization procedure;
[0098] Methods and/or pharmaceutical compositions for use in
reducing a cardiovascular event (e.g., delaying time to event,
reducing likelihood or risk of event, preventing an event, reducing
severity of event, reducing time to recovery) in a subject with a
history of a previous cardiovascular event, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof,
and wherein said cardiovascular event is myocardial infarction,
stroke, cardiovascular death, congestive heart failure, cardiac
arrest, acute coronary syndrome, angina or a revascularization
procedure;
[0099] Methods and/or pharmaceutical compositions for use in
reducing mortality following a cardiovascular event in a subject,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof;
[0100] Methods and/or pharmaceutical compositions for use in
reducing a cardiovascular event in a subject with a history of at
least one risk factor for cardiovascular disease, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof,
and wherein said risk factor is not Type 2 diabetes, obesity,
hyperglycemia, dyslipidemia, hyperlipidemia, chronic renal failure,
high blood glucose, chronic kidney disease, hypertension,
atherosclerosis or metabolic syndrome;
[0101] Methods and/or pharmaceutical compositions for use in
treating a cardiovascular event in a subject, wherein the
cardiovascular event is myocardial infarction, stroke, congestive
heart failure, acute coronary syndrome or angina, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
at least one other pharmaceutical composition comprising an active
agent other than an IL-1.beta. antibody or fragment;
[0102] Methods and/or pharmaceutical compositions for use in
treating a cardiovascular event in a subject, wherein the
cardiovascular event is myocardial infarction, stroke, congestive
heart failure, acute coronary syndrome or angina, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
a revascularization procedure;
[0103] Methods and/or pharmaceutical compositions for use in
treating cardiovascular disease, including, for example, acute
cardiovascular disease or chronic cardiovascular disease, in a
subject, comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof and a revascularization procedure;
[0104] Methods and/or pharmaceutical compositions for use in
reducing restenosis in a subject following a revascularization
procedure, comprising administering to said subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof;
[0105] Methods and/or pharmaceutical compositions for use in
treating acute hypertension in a subject, comprising administering
to said subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof and
one or more antihypertensive agents;
[0106] Methods and/or pharmaceutical compositions for use in
reducing, preventing or treating a cardiovascular event or disease
(e.g., acute cardiovascular disease or chronic cardiovascular
disease) in a subject, comprising administering to the subject an
anti-IL-1.beta. binding antibody or binding fragment thereof in
combination with a medical or surgical intervention;
[0107] Methods and/or pharmaceutical compositions for use in
inhibiting platelet activity in a subject, comprising administering
to said subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof;
[0108] Methods and/or pharmaceutical compositions for use in
reducing a cardiovascular event in a subject with a history of at
least one risk factor for cardiovascular disease, comprising (a)
identifying, diagnosing or selecting the subject with the history
of at least one risk factor for cardiovascular disease and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof,
and wherein the cardiovascular event is myocardial infarction,
stroke, cardiovascular death, congestive heart failure, cardiac
arrest, acute coronary syndrome, angina, or a revascularization
procedure;
[0109] Methods and/or pharmaceutical compositions for use in
reducing a cardiovascular event in a subject with a history of a
previous cardiovascular event, comprising (a) identifying,
diagnosing or selecting the subject with the history of the
previous cardiovascular event and (b) administering to the subject
a therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof, and wherein the
cardiovascular event is myocardial infarction, stroke, acute
coronary syndrome, angina or a revascularization procedure;
[0110] Methods and/or pharmaceutical compositions for use in
reducing mortality following a cardiovascular event in a subject,
comprising (a) identifying, diagnosing or selecting the subject
having the cardiovascular event and (b) administering to the
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof;
[0111] Methods and/or pharmaceutical compositions for use in
reducing a cardiovascular event in a subject with a history of at
least one risk factor for cardiovascular disease, comprising (a)
identifying, diagnosing or selecting the subject with the history
of at least one risk factor for cardiovascular disease and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof,
and wherein the risk factor is not Type 2 diabetes, obesity,
hyperglycemia, dyslipidemia, hyperlipidemia, chronic renal failure,
high blood glucose, chronic kidney disease, hypertension,
atherosclerosis or metabolic syndrome;
[0112] Methods and/or pharmaceutical compositions for use in
treating a cardiovascular event in a subject, wherein the
cardiovascular event is myocardial infarction, stroke, congestive
heart failure, acute coronary syndrome or angina, comprising (a)
identifying, diagnosing or selecting the subject with the
cardiovascular event and (b) administering to the subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof and at least one other
pharmaceutical composition comprising an active agent other than an
IL-1.beta. antibody or fragment;
[0113] Methods and/or pharmaceutical compositions for use in
treating a cardiovascular event in a subject, wherein the
cardiovascular event is myocardial infarction, stroke, congestive
heart failure, acute coronary syndrome or angina, comprising (a)
identifying, diagnosing or selecting the subject with the
cardiovascular event and (b) administering to the subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof and (e.g., in conjunction
with) a revascularization procedure;
[0114] Methods and/or pharmaceutical compositions for use in
reducing restenosis in a subject following a revascularization
procedure, comprising (a) identifying, diagnosing or selecting the
subject with the revascularization procedure and (b) administering
to the subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof;
[0115] Methods and/or pharmaceutical compositions for use in
treating acute hypertension in a subject comprising (a)
identifying, diagnosing or selecting the subject with acute
hypertension and (b) administering to the subject a therapeutically
effective amount of an anti-IL-43 binding antibody or binding
fragment thereof and one or more antihypertensive agents.
[0116] It should be understood that where the present specification
provides methods of using IL-1.beta. antibodies or binding
fragments thereof with certain properties (such as Kd values or
IC.sub.50 values), such as for example, for the reduction,
prevention or treatment of cardiovascular events and/or
cardiovascular diseases, including acute cardiovascular disease or
chronic cardiovascular disease, this also means to embody the use
of such antibodies or fragments thereof in the manufacture of a
medicament for use in these methods. Further, the disclosure also
encompasses IL-1.beta. antibodies or binding fragments thereof
having these properties as well as pharmaceutical compositions
comprising these antibodies or fragments thereof for use in the
methods provided herein, such as for example, for the reduction,
prevention or treatment of cardiovascular events and/or
cardiovascular diseases, including acute cardiovascular disease or
chronic cardiovascular disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0117] FIG. 1 is a graph showing serum concentrations following IV
administration of 0.01, 0.03, 0.1, 0.3, or 1.0 mg/kg of an
anti-IL-1.beta. antibody in human subjects.
[0118] FIG. 2 is a graph showing serum concentrations following SC
administration of 0.03, 0.1 and 0.3 mg/kg of an anti-IL-1p antibody
in human subjects
[0119] FIG. 3 is a graph showing median percent change in CRP at
day 28 following administration of 0.01, 0.03, 0.1, 0.3, or 1.0
mg/kg of an anti-IL-1.beta. antibody in human subjects.
[0120] FIG. 4 is graphs showing changes in echocardiographic values
in a myocardial infarction animal model.
[0121] FIG. 5 is graphs showing measurements of akinetic segments
(surrogate for infarct size), anterior wall (infarct) thickness,
MPI or Tei index (marker of combined systolic and diastolic
dysfunction and a surrogate marker for heart failure related
mortality), and TAPSE (marker of right ventricular function and a
surrogate marker for AMI related mortality in a myocardial
infarction animal model.
[0122] FIG. 6 is graphs showing inhibition of the release of
macrophage-induced pro-inflammatory cytokines from endothelial
cells.
[0123] FIG. 7 is graphs showing inhibition of the release of
macrophage-induced cytokines and degradative enzymes from smooth
muscle cells.
[0124] FIG. 8 is graphs showing reduction in the formation of
atherosclerotic lesions in the aortas of ApoE knockout mice.
[0125] FIG. 9 is photographs of en face analysis showing reduction
in the formation of atherosclerotic lesions in the aortas of ApoE
knockout mice.
DETAILED DESCRIPTION
[0126] The present disclosure relates to methods and related
articles of manufacture for the treatment and/or prevention of
cardiovascular disease, including, for example, acute
cardiovascular disease or chronic cardiovascular disease. The
methods may be used for reducing, treating or preventing a
cardiovascular event, such as myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina, or a revascularization procedure
in a subject, including in a subject with a history of a risk
factor for cardiovascular disease. The methods may also be used to
reduce mortality following a cardiovascular event in a subject. Use
of anti-IL-1.beta. binding antibodies or binding fragments as
disclosed herein, offers potential advantages over previously
available options, such as for example greater safety (e.g.,
reduced side effects), greater efficacy, targeting of the
inflammatory component of disease, and/or less frequent dosing.
[0127] The interleukin-1 (IL-1) family of cytokines has been
implicated in several disease states such as rheumatoid arthritis
(RA), osteoarthritis, Crohn's disease, ulcerative colitis (UC),
septic shock, chronic obstructive pulmonary disease (COPD), asthma,
graft versus host disease, atherosclerosis, adult T-cell leukemia,
multiple myeloma, multiple sclerosis, stroke, and Alzheimer's
disease. IL-1 family members include IL-1.alpha., IL-1.beta., and
IL-1Ra. Although related by their ability to bind to IL-1 receptors
(IL-1R1, IL-1R2), each of these cytokines is expressed by a
different gene and has a different primary amino acid sequence.
Furthermore, the physiological activities of these cytokines can be
distinguished from each other.
[0128] Compounds that disrupt IL-1 receptor signaling have been
investigated as therapeutic agents to treat IL-1 mediated diseases,
such as for example some of the aforementioned diseases. These
compounds include recombinant IL-1Ra (Amgen Inc., Thousand Oaks,
Calif.), IL-1 receptor "trap" peptide (Regeneron Inc., Tarrytown,
N.Y.), as well as animal-derived IL-1.beta. antibodies and
recombinant IL-1.beta. antibodies and fragments thereof. Compounds
that directly target the IL-1.beta. ligand are believed to provide
a superior strategy, particularly when administering an IL-1.beta.
antibody with high affinity.
Antibodies, Humanized Antibodies, and Human Engineered
Antibodies
[0129] IL-1 (e.g., IL-1.beta.) binding antibodies may be provided
as polyclonal antibodies, monoclonal antibodies (mAbs), recombinant
antibodies, chimeric antibodies, CDR-grafted antibodies, fully
human antibodies, single chain antibodies, and/or bispecific
antibodies, as well as fragments, including variants and
derivatives thereof, provided by known techniques, including, but
not limited to enzymatic cleavage, peptide synthesis or recombinant
techniques.
[0130] Antibodies generally comprise two heavy chain polypeptides
and two light chain polypeptides, though single domain antibodies
having one heavy chain and one light chain, and heavy chain
antibodies devoid of light chains are also contemplated. There are
five types of heavy chains, called alpha, delta, epsilon, gamma and
mu, based on the amino acid sequence of the heavy chain constant
domain. These different types of heavy chains give rise to five
classes of antibodies, IgA (including IgA.sub.1 and IgA.sub.2),
IgD, IgE, IgG and IgM, respectively, including four subclasses of
IgG, namely IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4. There
are also two types of light chains, called kappa (.kappa.) or
lambda (.lamda.) based on the amino acid sequence of the constant
domains. A full-length antibody includes a constant domain and a
variable domain. The constant region need not be present in an
antigen binding fragment of an antibody. Antigen binding fragments
of an antibody disclosed herein can include Fab, Fab',
F(ab').sub.2, and F(v) antibody fragments. As discussed in more
detail below, IL-1.beta. binding fragments encompass antibody
fragments and antigen-binding polypeptides that will bind
IL-1.beta..
[0131] Each of the heavy chain and light chain sequences of an
antibody, or antigen binding fragment thereof, includes a variable
region with three complementarity determining regions (CDRs) as
well as non-CDR framework regions (FRs). The terms "heavy chain"
and "light chain," as used herein, mean the heavy chain variable
region and the light chain variable region, respectively, unless
otherwise noted. Heavy chain CDRs are referred to herein as CDR-H1,
CDR-H2, and CDR-H3. Light chain CDRs are referred to herein as
CDR-L1, CDR-L2, and CDR-L3. Variable regions and CDRs in an
antibody sequence can be identified (i) according to general rules
that have been developed in the art or (ii) by aligning the
sequences against a database of known variable regions. Methods for
identifying these regions are described in Kontermann and Dubel,
eds., Antibody Engineering, Springer, New York, N.Y., 2001, and
Dinarello et al., Current Protocols in Immunology, John Wiley and
Sons Inc., Hoboken, N.J., 2000. Databases of antibody sequences are
described in and can be accessed through "The Kabatman" database at
www.bioinf.org.uk/abs (maintained by A. C. Martin in the Department
of Biochemistry & Molecular Biology University College London,
London, England) and VBASE2 at www.vbase2.org, as described in
Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674
(2005). The "Kabatman" database web site also includes general
rules of thumb for identifying CDRs. The term "CDR," as used
herein, is as defined in Kabat et al., Sequences of Immunological
Interest, 5.sup.th ed., U.S. Department of Health and Human
Services, 1991, unless otherwise indicated.
[0132] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) immunizing
injections of the relevant antigen and an adjuvant, using standard
techniques known in the art. An improved antibody response may be
obtained by conjugating the relevant antigen to a protein that is
immunogenic in the species to be immunized, e.g., keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin
inhibitor using a bifunctional or derivatizing agent, for example,
maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride or other agents known in the
art.
[0133] Monoclonal antibody refers to an antibody obtained from a
population of substantially homogeneous antibodies. Monoclonal
antibodies are generally highly specific, and may be directed
against a single antigenic site, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes). In
addition to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the homogeneous
culture, uncontaminated by other immunoglobulins with different
specificities and characteristics.
[0134] Monoclonal antibodies to be used in accordance with the
present disclosure may be made by the hybridoma method first
described by Kohler et al., (Nature, 256:495-7, 1975), or may be
made by recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567). The monoclonal antibodies may also be isolated from
phage antibody libraries using the techniques described in, for
example, Clackson et al., (Nature 352:624-628, 1991) and Marks et
al., (J. Mol. Biol. 222:581-597, 1991).
[0135] It is further contemplated that antibodies of the present
disclosure may be used as smaller antigen binding fragments of the
antibody well-known in the art and described herein. The present
disclosure encompasses IL-1 (e.g., IL-1.beta.) binding antibodies
that include two full length heavy chains and two full length light
chains. Alternatively, the IL-1.beta. binding antibodies can be
constructs such as single chain antibodies or "mini" antibodies
that retain binding activity to IL-1.beta.. Such constructs can be
prepared by methods known in the art such as, for example, the PCR
mediated cloning and assembly of single chain antibodies for
expression in E. coli (as described in Antibody Engineering, The
practical approach series, J. McCafferty, H. R. Hoogenboom, and D.
J. Chiswell, editors, Oxford University Press, 1996). In this type
of construct, the variable portions of the heavy and light chains
of an antibody molecule are PCR amplified from cDNA. The resulting
amplicons are then assembled, for example, in a second PCR step,
through a linker DNA that encodes a flexible protein linker
composed of the amino acids Gly and Ser. This linker allows the
variable heavy and light chain portions to fold in such a way that
the antigen binding pocket is regenerated and antigen is bound with
affinities often comparable to the parent full-length dimeric
immunoglobulin molecule.
[0136] The IL-1 (e.g., IL-1.beta.) binding antibodies and fragments
encompass variants of the exemplary antibodies, fragments and
sequences disclosed herein. Variants include peptides and
polypeptides comprising one or more amino acid sequence
substitutions, deletions, and/or additions that have the same or
substantially the same affinity and specificity of epitope binding
as one or more of the exemplary antibodies, fragments and sequences
disclosed herein. Thus, variants include peptides and polypeptides
comprising one or more amino acid sequence substitutions,
deletions, and/or additions to the exemplary antibodies, fragments
and sequences disclosed herein where such substitutions, deletions
and/or additions do not cause substantial changes in affinity and
specificity of epitope binding. For example, a variant of an
antibody or fragment may result from one or more changes to an
antibody or fragment, where the changed antibody or fragment has
the same or substantially the same affinity and specificity of
epitope binding as the starting sequence. Variants may be naturally
occurring, such as allelic or splice variants, or may be
artificially constructed. Variants may be prepared from the
corresponding nucleic acid molecules encoding said variants.
Variants of the present antibodies and IL-1.beta. binding fragments
may have changes in light and/or heavy chain amino acid sequences
that are naturally occurring or are introduced by in vitro
engineering of native sequences using recombinant DNA techniques.
Naturally occurring variants include "somatic" variants which are
generated in vivo in the corresponding germ line nucleotide
sequences during the generation of an antibody response to a
foreign antigen.
[0137] Variants of IL-1 (e.g., IL-1.beta.) binding antibodies and
binding fragments may also be prepared by mutagenesis techniques.
For example, amino acid changes may be introduced at random
throughout an antibody coding region and the resulting variants may
be screened for binding affinity for IL-1.beta. or for another
property. Alternatively, amino acid changes may be introduced in
selected regions of an IL-1.beta. antibody, such as in the light
and/or heavy chain CDRs, and/or in the framework regions, and the
resulting antibodies may be screened for binding to IL-1.beta. or
some other activity. Amino acid changes encompass one or more amino
acid substitutions in a CDR, ranging from a single amino acid
difference to the introduction of multiple permutations of amino
acids within a given CDR, such as CDR3. In another method, the
contribution of each residue within a CDR to IL-1.beta. binding may
be assessed by substituting at least one residue within the CDR
with alanine. Lewis et al. (1995), Mol. Immunol. 32: 1065-72.
Residues which are not optimal for binding to IL-1.beta. may then
be changed in order to determine a more optimum sequence. Also
encompassed are variants generated by insertion of amino acids to
increase the size of a CDR, such as CDR3. For example, most light
chain CDR3 sequences are nine amino acids in length. Light chain
sequences in an antibody which are shorter than nine residues may
be optimized for binding to IL-1.beta. by insertion of appropriate
amino acids to increase the length of the CDR.
[0138] Variants may also be prepared by "chain shuffling" of light
or heavy chains. Marks et al. (1992), Biotechnology 10: 779-83. A
single light (or heavy) chain can be combined with a library having
a repertoire of heavy (or light) chains and the resulting
population is screened for a desired activity, such as binding to
IL-1.beta.. This permits screening of a greater sample of different
heavy (or light) chains in combination with a single light (or
heavy) chain than is possible with libraries comprising repertoires
of both heavy and light chains.
[0139] The IL-1 (e.g., IL-1.beta.) binding antibodies and fragments
of the present disclosure encompass derivatives of the exemplary
antibodies, fragments and sequences disclosed herein. Derivatives
include polypeptides or peptides, or variants, fragments or
derivatives thereof, which have been chemically modified. Examples
include covalent attachment of one or more polymers, such as water
soluble polymers, N-linked, or O-linked carbohydrates, sugars,
phosphates, and/or other such molecules. The derivatives are
modified in a manner that is different from naturally occurring or
starting peptide or polypeptides, either in the type or location of
the molecules attached. Derivatives further include deletion of one
or more chemical groups which are naturally present on the peptide
or polypeptide.
[0140] The IL-1.beta. binding antibodies and fragments can be
bispecific. Bispecific antibodies or fragments can be of several
configurations. For example, bispecific antibodies may resemble
single antibodies (or antibody fragments) but have two different
antigen binding sites (variable regions). Bispecific antibodies can
be produced by chemical techniques (Kranz et al. (1981), Proc.
Natl. Acad. Sci. USA, 78: 5807), by "polydoma" techniques (U.S.
Pat. No. 4,474,893) or by recombinant DNA techniques. Bispecific
antibodies of the present disclosure can have binding specificities
for at least two different epitopes, at least one of which is an
epitope of IL-1.beta.. The IL-1.beta. binding antibodies and
fragments can also be heteroantibodies. Heteroantibodies are two or
more antibodies, or antibody binding fragments (Fab) linked
together, each antibody or fragment having a different
specificity.
[0141] Techniques for creating recombinant DNA versions of the
antigen-binding regions of antibody molecules which bypass the
generation of monoclonal antibodies are contemplated for the
present IL-1 (e.g., IL-1.beta.) binding antibodies and fragments.
DNA is cloned into a bacterial expression system. One example of
such a technique suitable for the practice of the present
disclosure uses a bacteriophage lambda vector system having a
leader sequence that causes the expressed Fab protein to migrate to
the periplasmic space (between the bacterial cell membrane and the
cell wall) or to be secreted. One can rapidly generate and screen
great numbers of functional Fab fragments for those which bind
IL-1.beta.. Such IL-1.beta. binding agents (Fab fragments with
specificity for an IL-1.beta. polypeptide) are specifically
encompassed within the IL-1.beta. binding antibodies and fragments
of the present disclosure.
[0142] The present IL-1 (e.g., IL-1.beta.) binding antibodies and
fragments can be humanized or human engineered antibodies. As used
herein, a humanized antibody, or antigen binding fragment thereof,
is a recombinant polypeptide that comprises a portion of an antigen
binding site from a non-human antibody and a portion of the
framework and/or constant regions of a human antibody. A human
engineered antibody or antibody fragment is a non-human (e.g.,
mouse) antibody that has been engineered by modifying (e.g.,
deleting, inserting, or substituting) amino acids at specific
positions so as to reduce or eliminate any detectable
immunogenicity of the modified antibody in a human.
[0143] Humanized antibodies include chimeric antibodies and
CDR-grafted antibodies. Chimeric antibodies are antibodies that
include a non-human antibody variable region linked to a human
constant region. Thus, in chimeric antibodies, the variable region
is mostly non-human, and the constant region is human. Chimeric
antibodies and methods for making them are described in Morrison,
et al., Proc. Natl. Acad Sci. USA, 81: 6841-6855 (1984), Boulianne,
et al., Nature, 312: 643-646 (1984), and PCT Application
Publication WO 86/01533. Although, they can be less immunogenic
than a mouse monoclonal antibody, administrations of chimeric
antibodies have been associated with human anti-mouse antibody
responses (HAMA) to the non-human portion of the antibodies.
Chimeric antibodies can also be produced by splicing the genes from
a mouse antibody molecule of appropriate antigen-binding
specificity together with genes from a human antibody molecule of
appropriate biological activity, such as the ability to activate
human complement and mediate ADCC. Morrison et al. (1984), Proc.
Natl. Acad. Sci., 81: 6851; Neuberger et al. (1984), Nature, 312:
604. One example is the replacement of a Fc region with that of a
different isotype.
[0144] CDR-grafted antibodies are antibodies that include the CDRs
from a non-human "donor" antibody linked to the framework region
from a human "recipient" antibody. Generally, CDR-grafted
antibodies include more human antibody sequences than chimeric
antibodies because they include both constant region sequences and
variable region (framework) sequences from human antibodies. Thus,
for example, a CDR-grafted humanized antibody of the present
disclosure can comprise a heavy chain that comprises a contiguous
amino acid sequence (e.g., about 5 or more, 10 or more, or even 15
or more contiguous amino acid residues) from the framework region
of a human antibody (e.g., FR-1, FR-2, or FR-3 of a human antibody)
or, optionally, most or all of the entire framework region of a
human antibody. CDR-grafted antibodies and methods for making them
are described in, Jones et al., Nature, 321: 522-525 (1986),
Riechmann et al., Nature, 332: 323-327 (1988), and Verhoeyen et
al., Science, 239: 1534-1536 (1988)). Methods that can be used to
produce humanized antibodies also are described in U.S. Pat. Nos.
4,816,567, 5,721,367, 5,837,243, and 6,180,377. CDR-grafted
antibodies are considered less likely than chimeric antibodies to
induce an immune reaction against non-human antibody portions.
However, it has been reported that framework sequences from the
donor antibodies are required for the binding affinity and/or
specificity of the donor antibody, presumably because these
framework sequences affect the folding of the antigen-binding
portion of the donor antibody. Therefore, when donor, non-human CDR
sequences are grafted onto unaltered human framework sequences, the
resulting CDR-grafted antibody can exhibit, in some cases, loss of
binding avidity relative to the original non-human donor antibody.
See, e.g., Riechmann et al., Nature, 332: 323-327 (1988), and
Verhoeyen et al., Science, 239: 1534-1536 (1988).
[0145] Human engineered antibodies include for example "veneered"
antibodies and antibodies prepared using HUMAN ENGINEERING.TM.
technology (U.S. Pat. No. 5,869,619). HUMAN ENGINEERING.TM.
technology is commercially available, and involves altering an
non-human antibody or antibody fragment, such as a mouse or
chimeric antibody or antibody fragment, by making specific changes
to the amino acid sequence of the antibody so as to produce a
modified antibody with reduced immunogenicity in a human that
nonetheless retains the desirable binding properties of the
original non-human antibodies. Generally, the technique involves
classifying amino acid residues of a non-human (e.g., mouse)
antibody as "low risk", "moderate risk", or "high risk" residues.
The classification is performed using a global risk/reward
calculation that evaluates the predicted benefits of making
particular substitution (e.g., for immunogenicity in humans)
against the risk that the substitution will affect the resulting
antibody's folding and/or antigen-binding properties. Thus, a low
risk position is one for which a substitution is predicted to be
beneficial because it is predicted to reduce immunogenicity without
significantly affecting antigen binding properties. A moderate risk
position is one for which a substitution is predicted to reduce
immunogenicity, but is more likely to affect protein folding and/or
antigen binding. High risk positions contain residues most likely
to be involved in proper folding or antigen binding. Generally, low
risk positions in a non-human antibody are substituted with human
residues, high risk positions are rarely substituted, and
humanizing substitutions at moderate risk positions are sometimes
made, although not indiscriminately. Positions with prolines in the
non-human antibody variable region sequence are usually classified
as at least moderate risk positions.
[0146] The particular human amino acid residue to be substituted at
a given low or moderate risk position of a non-human (e.g., mouse)
antibody sequence can be selected by aligning an amino acid
sequence from the non-human antibody's variable regions with the
corresponding region of a specific or consensus human antibody
sequence. The amino acid residues at low or moderate risk positions
in the non-human sequence can be substituted for the corresponding
residues in the human antibody sequence according to the alignment.
Techniques for making human engineered proteins are described in
greater detail in Studnicka et al., Protein Engineering, 7: 805-814
(1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and
5,869,619, and PCT Application Publication WO 93/11794.
[0147] "Veneered" antibodies are non-human or humanized (e.g.,
chimeric or CDR-grafted antibodies) antibodies that have been
engineered to replace certain solvent-exposed amino acid residues
so as to further reduce their immunogenicity or enhance their
function. As surface residues of a chimeric antibody are presumed
to be less likely to affect proper antibody folding and more likely
to elicit an immune reaction, veneering of a chimeric antibody can
include, for instance, identifying solvent-exposed residues in the
non-human framework region of a chimeric antibody and replacing at
least one of them with the corresponding surface residues from a
human framework region. Veneering can be accomplished by any
suitable engineering technique, including the use of the
above-described HUMAN ENGINEERING.TM. technology.
[0148] In a different approach, a recovery of binding avidity can
be achieved by "de-humanizing" a CDR-grafted antibody.
De-humanizing can include restoring residues from the donor
antibody's framework regions to the CDR grafted antibody, thereby
restoring proper folding. Similar "de-humanization" can be achieved
by (i) including portions of the "donor" framework region in the
"recipient" antibody or (ii) grafting portions of the "donor"
antibody framework region into the recipient antibody (along with
the grafted donor CDRs).
[0149] For a further discussion of antibodies, humanized
antibodies, human engineered, and methods for their preparation,
see Kontermann and Dubel, eds., Antibody Engineering, Springer, New
York, N.Y., 2001.
[0150] Exemplary humanized or human engineered antibodies include
IgG, IgM, IgE, IgA, and IgD antibodies. The present antibodies can
be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can
comprise a kappa or lambda light chain. For example, a human
antibody can comprise an IgG heavy chain or defined fragment, such
as at least one of isotypes, IgG1, IgG2, IgG3 or IgG4. As a further
example, the present antibodies or fragments can comprise an IgG1
heavy chain and an IgG1 light chain.
[0151] The present antibodies and fragments can be human
antibodies, such as antibodies which bind IL-1.beta. polypeptides
and are encoded by nucleic acid sequences which are naturally
occurring somatic variants of human germline immunoglobulin nucleic
acid sequence, and fragments, synthetic variants, derivatives and
fusions thereof. Such antibodies may be produced by any method
known in the art, such as through the use of transgenic mammals
(such as transgenic mice) in which the native immunoglobulin
repertoire has been replaced with human V-genes in the mammal
chromosome. Such mammals appear to carry out VDJ recombination and
somatic hypermutation of the human germline antibody genes in a
normal fashion, thus producing high affinity antibodies with
completely human sequences.
[0152] Human antibodies to target protein can also be produced
using transgenic animals that have no endogenous immunoglobulin
production and are engineered to contain human immunoglobulin loci.
For example, WO 98/24893 discloses transgenic animals having a
human Ig locus wherein the animals do not produce functional
endogenous immunoglobulins due to the inactivation of endogenous
heavy and light chain loci. WO 91/00906 also discloses transgenic
non-primate mammalian hosts capable of mounting an immune response
to an immunogen, wherein the antibodies have primate constant
and/or variable regions, and wherein the endogenous immunoglobulin
encoding loci are substituted or inactivated. WO 96/30498 and U.S.
Pat. No. 6,091,001 disclose the use of the Cre/Lox system to modify
the immunoglobulin locus in a mammal, such as to replace all or a
portion of the constant or variable region to form a modified
antibody molecule. WO 94/02602 discloses non-human mammalian hosts
having inactivated endogenous Ig loci and functional human Ig loci.
U.S. Pat. No. 5,939,598 discloses methods of making transgenic mice
in which the mice lack endogenous heavy chains, and express an
exogenous immunoglobulin locus comprising one or more xenogeneic
constant regions. See also, U.S. Pat. Nos. 6,114,598 6,657,103 and
6,833,268.
[0153] Using a transgenic animal described above, an immune
response can be produced to a selected antigenic molecule, and
antibody producing cells can be removed from the animal and used to
produce hybridomas that secrete human monoclonal antibodies.
Immunization protocols, adjuvants, and the like are known in the
art, and are used in immunization of, for example, a transgenic
mouse as described in WO 96/33735. This publication discloses
monoclonal antibodies against a variety of antigenic molecules
including IL-6, IL-8, TNFa, human CD4, L selectin, gp39, and
tetanus toxin. The monoclonal antibodies can be tested for the
ability to inhibit or neutralize the biological activity or
physiological effect of the corresponding protein. WO 96/33735
discloses that monoclonal antibodies against IL-8, derived from
immune cells of transgenic mice immunized with IL-8, blocked IL-8
induced functions of neutrophils. Human monoclonal antibodies with
specificity for the antigen used to immunize transgenic animals are
also disclosed in WO 96/34096 and U.S. patent application no.
20030194404; and U.S. patent application no. 20030031667.
[0154] Additional transgenic animals useful to make monoclonal
antibodies include the Medarex HuMAb-MOUSE.RTM., described in U.S.
Pat. No. 5,770,429 and Fishwild, et al. (Nat. Biotechnol.
14:845-851, 1996), which contains gene sequences from unrearranged
human antibody genes that code for the heavy and light chains of
human antibodies. Immunization of a HuMAb-MOUSE.RTM. enables the
production of fully human monoclonal antibodies to the target
protein.
[0155] Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002)
describes the TransChromo Mouse (TCMOUSE.TM.) which comprises
megabase-sized segments of human DNA and which incorporates the
entire human immunoglobulin (hIg) loci. The TCMOUSE.TM. has a fully
diverse repertoire of hIgs, including all the subclasses of IgGs
(IgG1-G4). Immunization of the TC MOUSE.TM. with various human
antigens produces antibody responses comprising human
antibodies.
[0156] See also Jakobovits et al., Proc. Natl. Acad. Sci. USA,
90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et al., Year in Immunol., 7:33 (1993); and U.S. Pat.
Nos. 5,591,669, 5,589,369, 5,545,807; and U.S Patent Publication
No. 20020199213. U.S. Patent Publication No. 20030092125 describes
methods for biasing the immune response of an animal to the desired
epitope. Human antibodies may also be generated by in vitro
activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
[0157] Human antibodies can also be generated through the in vitro
screening of antibody display libraries. See Hoogenboom et al.
(1991), J. Mol. Biol. 227: 381; and Marks et al. (1991), J. Mol.
Biol. 222: 581. Various antibody-containing phage display libraries
have been described and may be readily prepared. Libraries may
contain a diversity of human antibody sequences, such as human Fab,
Fv, and scFv fragments, that may be screened against an appropriate
target. Phage display libraries may comprise peptides or proteins
other than antibodies which may be screened to identify selective
binding agents of IL-1.beta..
[0158] The development of technologies for making repertoires of
recombinant human antibody genes, and the display of the encoded
antibody fragments on the surface of filamentous bacteriophage, has
provided a means for making human antibodies directly. The
antibodies produced by phage technology are produced as antigen
binding fragments-usually Fv or Fab fragments-in bacteria and thus
lack effector functions. Effector functions can be introduced by
one of two strategies: The fragments can be engineered either into
complete antibodies for expression in mammalian cells, or into
bispecific antibody fragments with a second binding site capable of
triggering an effector function.
[0159] The present disclosure contemplates a method for producing
target-specific antibody or antigen-binding portion thereof
comprising the steps of synthesizing a library of human antibodies
on phage, screening the library with target protein or a portion
thereof, isolating phage that bind target, and obtaining the
antibody from the phage. By way of example, one method for
preparing the library of antibodies for use in phage display
techniques comprises the steps of immunizing a non-human animal
comprising human immunoglobulin loci with target antigen or an
antigenic portion thereof to create an immune response, extracting
antibody producing cells from the immunized animal; isolating RNA
from the extracted cells, reverse transcribing the RNA to produce
cDNA, amplifying the cDNA using a primer, and inserting the cDNA
into a phage display vector such that antibodies are expressed on
the phage. Recombinant target-specific antibodies of the present
disclosure may be obtained in this way.
[0160] Phage-display processes mimic immune selection through the
display of antibody repertoires on the surface of filamentous
bacteriophage, and subsequent selection of phage by their binding
to an antigen of choice. One such technique is described in WO
99/10494, which describes the isolation of high affinity and
functional agonistic antibodies for MPL and msk receptors using
such an approach. Antibodies of the present disclosure can be
isolated by screening of a recombinant combinatorial antibody
library, preferably a scFv phage display library, prepared using
human V.sub.L and V.sub.H cDNAs prepared from mRNA derived from
human lymphocytes. Methodologies for preparing and screening such
libraries are known in the art. See e.g., U.S. Pat. No. 5,969,108.
There are commercially available kits for generating phage display
libraries (e.g., the Pharmacia Recombinant Phage Antibody System,
catalog no. 27-9400-01; and the Stratagene SurfZAP.TM. phage
display kit, catalog no. 240612). There are also other methods and
reagents that can be used in generating and screening antibody
display libraries (see, e.g., Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et
al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication
No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679;
Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al.
PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No.
WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; McCafferty et al., Nature (1990)
348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et
al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377;
Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et
al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.
[0161] In one embodiment, to isolate human antibodies specific for
the target antigen with the desired characteristics, a human
V.sub.H and V.sub.L library are screened to select for antibody
fragments having the desired specificity. The antibody libraries
used in this method are preferably scFv libraries prepared and
screened as described herein and in the art (McCafferty et al., PCT
Publication No. WO 92/01047, McCafferty et al., (Nature
348:552-554, 1990); and Griffiths et al., (EMBO J 12:725-734,
1993). The scFv antibody libraries preferably are screened using
target protein as the antigen.
[0162] Alternatively, the Fd fragment (V.sub.H-C.sub.H1) and light
chain (V.sub.L-C.sub.L) of antibodies are separately cloned by PCR
and recombined randomly in combinatorial phage display libraries,
which can then be selected for binding to a particular antigen. The
Fab fragments are expressed on the phage surface, i.e., physically
linked to the genes that encode them. Thus, selection of Fab by
antigen binding co-selects for the Fab encoding sequences, which
can be amplified subsequently. Through several rounds of antigen
binding and re-amplification, a procedure termed panning, Fab
specific for the antigen are enriched and finally isolated.
[0163] In 1994, an approach for the humanization of antibodies,
called "guided selection", was described. Guided selection utilizes
the power of the phage display technique for the humanization of
mouse monoclonal antibody (See Jespers, L. S., et al.,
Bio/Technology 12, 899-903 (1994)). For this, the Fd fragment of
the mouse monoclonal antibody can be displayed in combination with
a human light chain library, and the resulting hybrid Fab library
may then be selected with antigen. The mouse Fd fragment thereby
provides a template to guide the selection. Subsequently, the
selected human light chains are combined with a human Fd fragment
library. Selection of the resulting library yields entirely human
Fab.
[0164] A variety of procedures have been described for deriving
human antibodies from phage-display libraries (See, for example,
Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol, 222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and
5,573,905; Clackson, T., and Wells, J. A., TIBTECH 12, 173-184
(1994)). In particular, in vitro selection and evolution of
antibodies derived from phage display libraries has become a
powerful tool (See Burton, D. R., and Barbas III, C. F., Adv.
Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.
Immunol. 12, 433-455 (1994); U.S. patent publication no.
20020004215 and WO 92/01047; U.S. patent publication no.
20030190317; and U.S. Pat. Nos. 6,054,287 and 5,877,293.
[0165] Watkins, "Screening of Phage-Expressed Antibody Libraries by
Capture Lift," Methods in Molecular Biology, Antibody Phage
Display: Methods and Protocols 178: 187-193 (2002), and U.S. patent
publication no. 20030044772, published Mar. 6, 2003, describe
methods for screening phage-expressed antibody libraries or other
binding molecules by capture lift, a method involving
immobilization of the candidate binding molecules on a solid
support.
[0166] Fv fragments are displayed on the surface of phage, by the
association of one chain expressed as a phage protein fusion (e.g.,
with M13 gene III) with the complementary chain expressed as a
soluble fragment. It is contemplated that the phage may be a
filamentous phage such as one of the class I phages: fd, M13, f1,
If1, lke, ZJ/Z, Ff and one of the class II phages Xf, Pf1 and Pf3.
The phage may be M13, or fd or a derivative thereof.
[0167] Once initial human V.sub.L and V.sub.H segments are
selected, "mix and match" experiments, in which different pairs of
the initially selected V.sub.L and V.sub.H segments are screened
for target binding, are performed to select preferred
V.sub.LN.sub.H pair combinations. Additionally, to further improve
the quality of the antibody, the V.sub.L and V.sub.H segments of
the preferred V.sub.LN.sub.H pair(s) can be randomly mutated,
preferably within the any of the CDR1, CDR2 or CDR3 region of
V.sub.H and/or V.sub.L, in a process analogous to the in vivo
somatic mutation process responsible for affinity maturation of
antibodies during a natural immune response. This in vitro affinity
maturation can be accomplished by amplifying V.sub.L and V.sub.H
regions using PCR primers complimentary to the V.sub.H CDR1, CDR2,
and CDR3, or V.sub.L CDR1, CDR2, and CDR3, respectively, which
primers have been "spiked" with a random mixture of the four
nucleotide bases at certain positions such that the resultant PCR
products encode V.sub.L and V.sub.H segments into which random
mutations have been introduced into the V.sub.H and/or V.sub.L CDR3
regions. These randomly mutated V.sub.L and V.sub.H segments can be
rescreened for binding to target antigen.
[0168] Following screening and isolation of an target specific
antibody from a recombinant immunoglobulin display library, nucleic
acid encoding the selected antibody can be recovered from the
display package (e.g., from the phage genome) and subcloned into
other expression vectors by standard recombinant DNA techniques. If
desired, the nucleic acid can be further manipulated to create
other antibody forms of the present disclosure, as described below.
To express a recombinant human antibody isolated by screening of a
combinatorial library, the DNA encoding the antibody is cloned into
a recombinant expression vector and introduced into a mammalian
host cell, as described herein.
[0169] It is contemplated that the phage display method may be
carried out in a mutator strain of bacteria or host cell. A mutator
strain is a host cell which has a genetic defect which causes DNA
replicated within it to be mutated with respect to its parent DNA.
Example mutator strains are NR9046mutD5 and NR9046 mut T1.
[0170] It is also contemplated that the phage display method may be
carried out using a helper phage. This is a phage which is used to
infect cells containing a defective phage genome and which
functions to complement the defect. The defective phage genome can
be a phagemid or a phage with some function encoding gene sequences
removed. Examples of helper phages are M13K07, M13K07 gene III no.
3; and phage displaying or encoding a binding molecule fused to a
capsid protein.
[0171] Antibodies are also generated via phage display screening
methods using the hierarchical dual combinatorial approach as
disclosed in WO 92/01047 in which an individual colony containing
either an H or L chain clone is used to infect a complete library
of clones encoding the other chain (L or H) and the resulting
two-chain specific binding member is selected in accordance with
phage display techniques such as those described therein. This
technique is also disclosed in Marks et al, (Bio/Technology,
10:779-783, 1992).
[0172] Methods for display of peptides on the surface of yeast and
microbial cells have also been used to identify antigen specific
antibodies. See, for example, U.S. Pat. No. 6,699,658. Antibody
libraries may be attached to yeast proteins, such as agglutinin,
effectively mimicking the cell surface display of antibodies by B
cells in the immune system.
[0173] In addition to phage display methods, antibodies may be
isolated using ribosome mRNA display methods and microbial cell
display methods. Selection of polypeptide using ribosome display is
described in Hanes et al., (Proc. Natl Acad Sci USA, 94:4937-4942,
1997) and U.S. Pat. Nos. 5,643,768 and 5,658,754 issued to
Kawasaki. Ribosome display is also useful for rapid large scale
mutational analysis of antibodies. The selective mutagenesis
approach also provides a method of producing antibodies with
improved activities that can be selected using ribosomal display
techniques.
[0174] The IL-1 (e.g., IL-1.beta.) binding antibodies and fragments
may comprise one or more portions that do not bind IL-1.beta. but
instead are responsible for other functions, such as circulating
half-life, direct cytotoxic effect, detectable labeling, or
activation of the recipient's endogenous complement cascade or
endogenous cellular cytotoxicity. The antibodies or fragments may
comprise all or a portion of the constant region and may be of any
isotype, including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g.
IgG1, IgG2, IgG3 or IgG4), or IgM. In addition to, or instead of,
comprising a constant region, antigen-binding compounds of the
present disclosure may include an epitope tag, a salvage receptor
epitope, a label moiety for diagnostic or purification purposes, or
a cytotoxic moiety such as a radionuclide or toxin.
[0175] The constant region (when present) of the present antibodies
and fragments may be of the .gamma.1, .gamma.2, .gamma.3, .gamma.4,
.mu., .beta.2, or .delta. or c type, preferably of the .gamma.
type, more preferably of the y, type, whereas the constant part of
a human light chain may be of the .kappa. or .lamda. type (which
includes the .lamda..sub.1, .lamda..sub.2 and .lamda..sub.3
subtypes) but is preferably of the .kappa. type.
[0176] Variants also include antibodies or fragments comprising a
modified Fc region, wherein the modified Fc region comprises at
least one amino acid modification relative to a wild-type Fc
region. The variant Fc region may be designed, relative to a
comparable molecule comprising the wild-type Fc region, so as to
bind Fc receptors with a greater or lesser affinity.
[0177] For example, the present IL-1.beta. binding antibodies and
fragments may comprise a modified Fc region. Fe region refers to
naturally-occurring or synthetic polypeptides homologous to the IgG
C-terminal domain that is produced upon papain digestion of IgG.
IgG Fc has a molecular weight of approximately 50 kD. In the
present antibodies and fragments, an entire Fe region can be used,
or only a half-life enhancing portion. In addition, many
modifications in amino acid sequence are acceptable, as native
activity is not in all cases necessary or desired.
[0178] The Fc region can be mutated, if desired, to inhibit its
ability to fix complement and bind the Fc receptor with high
affinity. For murine IgG Fc, substitution of Ala residues for Glu
318, Lys 320, and Lys 322 renders the protein unable to direct
ADCC. Substitution of Glu for Leu 235 inhibits the ability of the
protein to bind the Fe receptor with high affinity. Various
mutations for human IgG also are known (see, e.g., Morrison et al.,
1994, The Immunologist 2: 119 124 and Brekke et al., 1994, The
Immunologist 2: 125).
[0179] In some embodiments, the present an antibodies or fragments
are provided with a modified Fc region where a naturally-occurring
Fc region is modified to increase the half-life of the antibody or
fragment in a biological environment, for example, the serum
half-life or a half-life measured by an in vitro assay. Methods for
altering the original form of a Fc region of an IgG also are
described in U.S. Pat. No. 6,998,253.
[0180] In certain embodiments, it may be desirable to modify the
antibody or fragment in order to increase its serum half-life, for
example, adding molecules such as PEG or other water soluble
polymers, including polysaccharide polymers, to antibody fragments
to increase the half-life. This may also be achieved, for example,
by incorporation of a salvage receptor binding epitope into the
antibody fragment (e.g., by mutation of the appropriate region in
the antibody fragment or by incorporating the epitope into a
peptide tag that is then fused to the antibody fragment at either
end or in the middle, e.g., by DNA or peptide synthesis) (see,
International Publication No. WO96/32478). Salvage receptor binding
epitope refers to an epitope of the Fe region of an IgG molecule
(e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is
responsible for increasing the in vivo serum half-life of the IgG
molecule.
[0181] A salvage receptor binding epitope can include a region
wherein any one or more amino acid residues from one or two loops
of a Fc domain are transferred to an analogous position of the
antibody fragment. Even more preferably, three or more residues
from one or two loops of the Fc domain are transferred. Still more
preferred, the epitope is taken from the CH2 domain of the Fc
region (e.g., of an IgG) and transferred to the CH1, CH3, or
V.sub.H region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fc
region and transferred to the C.sub.L region or V.sub.L region, or
both, of the antibody fragment. See also International applications
WO 97/34631 and WO 96/32478 which describe Fc variants and their
interaction with the salvage receptor.
[0182] Mutation of residues within Fc receptor binding sites can
result in altered effector function, such as altered ADCC or CDC
activity, or altered half-life. Potential mutations include
insertion, deletion or substitution of one or more residues,
including substitution with alanine, a conservative substitution, a
non-conservative substitution, or replacement with a corresponding
amino acid residue at the same position from a different IgG
subclass (e.g. replacing an IgG1 residue with a corresponding IgG2
residue at that position). For example it has been reported that
mutating the serine at amino acid position 241 in IgG4 to proline
(found at that position in IgG1 and IgG2) led to the production of
a homogeneous antibody, as well as extending serum half-life and
improving tissue distribution compared to the original chimeric
IgG4. (Angal et al., Mol Immunol. 30:105-8, 1993).
[0183] Antibody fragments are portions of an intact full length
antibody, such as an antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies;
single-chain antibody molecules (e.g., scFv); multispecific
antibody fragments such as bispecific, trispecific, and
multispecific antibodies (e.g., diabodies, triabodies,
tetrabodies); minibodies; chelating recombinant antibodies;
tribodies or bibodies; intrabodies; nanobodies; small modular
immunopharmaceuticals (SMIP), adnectins, binding-domain
immunoglobulin fusion proteins; camelized antibodies; V.sub.HH
containing antibodies; and any other polypeptides formed from
antibody fragments.
[0184] The present disclosure includes IL-1.beta. binding antibody
fragments comprising any of the foregoing heavy or light chain
sequences and which bind IL-1.beta.. The term fragments as used
herein refers to any 3 or more contiguous amino acids (e.g., 4 or
more, 5 or more 6 or more, 8 or more, or even 10 or more contiguous
amino acids) of the antibody and encompasses Fab, Fab',
F(ab').sub.2, and F(v) fragments, or the individual light or heavy
chain variable regions or portion thereof. IL-1.beta. binding
fragments include, for example, Fab, Fab', F(ab').sub.2, Fv and
scFv. These fragments lack the Fc fragment of an intact antibody,
clear more rapidly from the circulation, and can have less
non-specific tissue binding than an intact antibody. See Wahl et
al. (1983), J. Nucl. Med., 24: 316-25. These fragments can be
produced from intact antibodies using well known methods, for
example by proteolytic cleavage with enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab').sub.2
fragments).
[0185] In vitro and cell based assays are well described in the art
for use in determining binding of IL-1.beta. to IL-1 receptor type
I (IL-1R1), including assays that determining in the presence of
molecules (such as antibodies, antagonists, or other inhibitors)
that bind to IL-1.beta. or IL-1RI. (see for example Evans et al.,
(1995), J. Biol. Chem. 270:11477-11483; Vigers et al., (2000), J.
Biol. Chem. 275:36927-36933; Yanofsky et al., (1996), Proc. Natl.
Acad. Sci. USA 93:7381-7386; Fredericks et al., (2004), Protein
Eng. Des. Sel. 17:95-106; Slack et al., (1993), J. Biol. Chem.
268:2513-2524; Smith et al., (2003), Immunity 18:87-96; Vigers et
al., (1997), Nature 386:190-194; Ruggiero et al., (1997), J.
Immunol. 158:3881-3887; Guo et al., (1995), J. Biol. Chem.
270:27562-27568; Svenson et al., (1995), Eur. J. Immunol.
25:2842-2850; Arend et al., (1994), J. Immunol. 153:4766-4774).
Recombinant IL-1 receptor type I, including human IL-1 receptor
type I, for such assays is readily available from a variety of
commercial sources (see for example R&D Systems, SIGMA). IL-1
receptor type I also can be expressed from an expression construct
or vector introduced into an appropriate host cell using standard
molecular biology and transfection techniques known in the art. The
expressed IL-1 receptor type I may then be isolated and purified
for use in binding assays, or alternatively used directly in a cell
associated form.
[0186] For example, the binding of IL-1.beta. to IL-1 receptor type
I may be determined by immobilizing an IL-1.beta. binding antibody,
contacting IL-1.beta. with the immobilized antibody and determining
whether the IL-1.beta. was bound to the antibody, and contacting a
soluble form of IL-1RI with the bound IL-1.beta./antibody complex
and determining whether the soluble IL-1RI was bound to the
complex. The protocol may also include contacting the soluble
IL-1RI with the immobilized antibody before the contact with
IL-1.beta., to confirm that the soluble IL-1RI does not bind to the
immobilized antibody. This protocol can be performed using a
Biacore.RTM. instrument for kinetic analysis of binding
interactions. Such a protocol can also be employed to determine
whether an antibody or other molecule permits or blocks the binding
of IL-1.beta. to IL-1 receptor type I.
[0187] For other IL-1.beta./IL-1RI binding assays, the permitting
or blocking of IL-1.beta. binding to IL-1 receptor type I may be
determined by comparing the binding of IL-1.beta. to IL-1RI in the
presence or absence of IL-1.beta. antibodies or IL-1.beta. binding
fragments thereof. Blocking is identified in the assay readout as a
designated reduction of IL-1.beta. binding to IL-1 receptor type I
in the presence of anti-IL-1.beta. antibodies or IL-1.beta. binding
fragments thereof, as compared to a control sample that contains
the corresponding buffer or diluent but not an IL-1.beta. antibody
or IL-1.beta. binding fragment thereof. The assay readout may be
qualitatively viewed as indicating the presence or absence of
blocking, or may be quantitatively viewed as indicating a percent
or fold reduction in binding due to the presence of the antibody or
fragment.
[0188] Alternatively or additionally, when an IL-1.beta. binding
antibody or IL-1.beta. binding fragment substantially blocks
IL-1.beta. binding to IL-1R1, the IL-1.beta. binding to IL-1RI is
reduced by at least 10-fold, alternatively at least about 20-fold,
alternatively at least about 50-fold, alternatively at least about
100-fold, alternatively at least about 1000-fold, alternatively at
least about 10000-fold, or more, compared to binding of the same
concentrations of IL-1.beta. and IL-1RI in the absence of the
antibody or fragment. As another example, when an IL-1.beta.
binding antibody or IL-1.beta. binding fragment substantially
permits IL-1.beta. binding to IL-1R1, the IL-1.beta. binding to
IL-1RI is at least about 90%, alternatively at least about 95%,
alternatively at least about 99%, alternatively at least about
99.9%, alternatively at least about 99.99%, alternatively at least
about 99.999%, alternatively at least about 99.9999%, alternatively
substantially identical to binding of the same concentrations of
IL-1.beta. and IL-1R1 in the absence of the antibody or
fragment.
[0189] The present disclosure may in certain embodiments encompass
IL-1.beta. binding antibodies or IL-1.beta. binding fragments that
bind to the same epitope or substantially the same epitope as one
or more of the exemplary antibodies described herein. Alternatively
or additionally, the IL-1.beta. binding antibodies or IL-1.beta.
binding fragments compete with the binding of an antibody having
variable region sequences of AB7, described in U.S. application
Ser. No. 11/472,813 (sequences shown below). Alternatively or
additionally, the present disclosure encompasses IL-1.beta. binding
antibodies and fragments that bind to an epitope contained in the
amino acid sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 3). As
contemplated herein, one can readily determine if an IL-1.beta.
binding antibody or fragment binds to the same epitope or
substantially the same epitope as one or more of the exemplary
antibodies, such as for example the antibody designated AB7, using
any of several known methods in the art.
[0190] For example, the key amino acid residues (epitope) bound by
an IL-1.beta. binding antibody or fragment may be determined using
a peptide array, such as for example, a PepSpot.TM. peptide array
(JPT Peptide Technologies, Berlin, Germany), wherein a scan of
twelve amino-acid peptides, spanning the entire IL-1.beta. amino
acid sequence, each peptide overlapping by 11 amino acid to the
previous one, is synthesized directly on a membrane. The membrane
carrying the peptides is then probed with the antibody for which
epitope binding information is sought, for example at a
concentration of 2 mg/ml, for 2 hr at room temperature. Binding of
antibody to membrane bound peptides may be detected using a
secondary HRP-conjugated goat anti-human (or mouse, when
appropriate) antibody, followed by enhanced chemiluminescence
(ECL). The peptides spot(s) corresponding to particular amino acid
residues or sequences of the mature IL-1.beta. protein, and which
score positive for antibody binding, are indicative of the epitope
bound by the particular antibody.
[0191] Alternatively or in addition, antibody competition
experiments may be performed and such assays are well known in the
art. For example, to determine if an antibody or fragment binds to
an epitope contained in a peptide sequence comprising the amino
acids ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO: 3), which corresponds to
residues 83-105 of the mature IL-1.beta. protein, an antibody of
unknown specificity may be compared with any of the exemplary of
antibodies (e.g., AB7) of the present disclosure. Binding
competition assays may be performed, for example, using a
Biacore.RTM. instrument for kinetic analysis of binding
interactions or by ELISA. In such an assay, the antibody of unknown
epitope specificity is evaluated for its ability to compete for
binding against the known comparator antibody (e.g., AB7).
Competition for binding to a particular epitope is determined by a
reduction in binding to the IL-1.beta. epitope of at least about
50%, or at least about 70%, or at least about 80%, or at least
about 90%, or at least about 95%, or at least about 99% or about
100% for the known comparator antibody (e.g., AB7) and is
indicative of binding to substantially the same epitope.
[0192] In view of the identification in this disclosure of
IL-1.beta. binding regions in exemplary antibodies and/or epitopes
recognized by the disclosed antibodies, it is contemplated that
additional antibodies with similar binding characteristics and
therapeutic or diagnostic utility can be generated that parallel
the embodiments of this disclosure.
[0193] Antigen-binding fragments of an antibody include fragments
that retain the ability to specifically bind to an antigen,
generally by retaining the antigen-binding portion of the antibody.
It is well established that the antigen-binding function of an
antibody can be performed by fragments of a full-length antibody.
Examples of antigen-binding portions include (i) a Fab fragment,
which is a monovalent fragment consisting of the VL, VH, CL and CH1
domains; (ii) a F(ab').sup.2 fragment, which is a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment which is the VH and CH1 domains;
(iv) a Fv fragment which is the VL and VH domains of a single arm
of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which is a VH domain; and (vi) an isolated
complementarity determining region (CDR). Single chain antibodies
are also encompassed within the term antigen-binding portion of an
antibody. The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass monovalent or multivalent, or
monomeric or multimeric (e.g. tetrameric), CDR-derived binding
domains with or without a scaffold (for example, protein or
carbohydrate scaffolding).
[0194] The present IL-1.beta. binding antibodies or fragments may
be part of a larger immunoadhesion molecules, formed by covalent or
non-covalent association of the antibody or antibody portion with
one or more other proteins or peptides. Examples of such
immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibodies and
fragments comprising immunoadhesion molecules can be obtained using
standard recombinant DNA techniques, as described herein. Preferred
antigen binding portions are complete domains or pairs of complete
domains.
[0195] The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass domain antibody (dAb) fragments
(Ward et al., Nature 341:544-546, 1989) which consist of a V.sub.H
domain. The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass diabodies, which are bivalent
antibodies in which V.sub.H and V.sub.L domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see e.g., EP
404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123,
1994). Diabodies can be bispecific or monospecific.
[0196] The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass single-chain antibody fragments
(scFv) that bind to IL-1.beta.. An scFv comprises an antibody heavy
chain variable region (V.sub.H) operably linked to an antibody
light chain variable region (V.sub.L) wherein the heavy chain
variable region and the light chain variable region, together or
individually, form a binding site that binds IL-1.beta.. An scFv
may comprise a V.sub.H region at the amino-terminal end and a
V.sub.L region at the carboxy-terminal end. Alternatively, scFv may
comprise a V.sub.L region at the amino-terminal end and a V.sub.H
region at the carboxy-terminal end. Furthermore, although the two
domains of the Fv fragment, V.sub.L and V.sub.H, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883).
[0197] An scFv may optionally further comprise a polypeptide linker
between the heavy chain variable region and the light chain
variable region. Such polypeptide linkers generally comprise
between 1 and 50 amino acids, alternatively between 3 and 12 amino
acids, alternatively 2 amino acids. An example of a linker peptide
for linking heavy and light chains in an scFv comprises the 5 amino
acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 6). Other examples
comprise one or more tandem repeats of this sequence (for example,
a polypeptide comprising two to four repeats of Gly-Gly-Gly-Gly-Ser
(SEQ ID NO: 6) to create linkers.
[0198] The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass heavy chain antibodies (HCAb).
Exceptions to the H.sub.2L.sub.2 structure of conventional
antibodies occur in some isotypes of the immunoglobulins found in
camelids (camels, dromedaries and llamas; Hamers-Casterman et al.,
1993 Nature 363: 446; Nguyen et al., 1998 J. Mol. Biol. 275: 413),
wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001),
nurse sharks (Greenberg et al., Nature 374:168-73, 1995; Roux et
al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in the spotted
ratfish (Nguyen, et al., "Heavy-chain antibodies in Camelidae; a
case of evolutionary innovation," 2002 Immunogenetics 54(1):
39-47). These antibodies can apparently form antigen-binding
regions using only heavy chain variable regions, in that these
functional antibodies are dimers of heavy chains only (referred to
as "heavy-chain antibodies" or "HCAbs"). Accordingly, some
embodiments of the present IL-1.beta. binding antibodies and
fragments may be heavy chain antibodies that specifically bind to
IL-1.beta.. For example, heavy chain antibodies that are a class of
IgG and devoid of light chains are produced by animals of the genus
Camelidae which includes camels, dromedaries and llamas
(Hamers-Casterman et al., Nature 363:446-448 (1993)). HCAbs have a
molecular weight of about 95 kDa instead of the about 160 kDa
molecular weight of conventional IgG antibodies. Their binding
domains consist only of the heavy-chain variable domains, often
referred to as V.sub.HH to distinguish them from conventional
V.sub.H. Muyldermans et al., J. Mol. Recognit. 12:131-140 (1999).
The variable domain of the heavy-chain antibodies is sometimes
referred to as a nanobody (Cortez-Retamozo et al., Cancer Research
64:2853-57, 2004). A nanobody library may be generated from an
immunized dromedary as described in Conrath et al., (Antimicrob
Agents Chemother 45: 2807-12, 2001) or using recombinant
methods.
[0199] Since the first constant domain (C.sub.H1) is absent
(spliced out during mRNA processing due to loss of a splice
consensus signal), the variable domain (V.sub.HH) is immediately
followed by the hinge region, the C.sub.1-12 and the C.sub.H3
domains (Nguyen et al., Mol. Immunol. 36:515-524 (1999); Woolven et
al., Immunogenetics 50:98-101 (1999)). Camelid V.sub.HH reportedly
recombines with IgG2 and IgG3 constant regions that contain hinge,
CH2, and CH3 domains and lack a CHI domain (Hamers-Casterman et
al., supra). For example, llama IgG1 is a conventional
(H.sub.2L.sub.2) antibody isotype in which V.sub.H recombines with
a constant region that contains hinge, CH1, CH2 and CH3 domains,
whereas the llama IgG2 and IgG3 are heavy chain-only isotypes that
lack CH1 domains and that contain no light chains.
[0200] Although the HCAbs are devoid of light chains, they have an
antigen-binding repertoire. The genetic generation mechanism of
HCAbs is reviewed in Nguyen et al. Adv. Immunol 79:261-296 (2001)
and Nguyen et al., Immunogenetics 54:39-47 (2002). Sharks,
including the nurse shark, display similar antigen
receptor-containing single monomeric V-domains. Irving et al., J.
Immunol. Methods 248:31-45 (2001); Roux et al., Proc. Natl. Acad.
Sci. USA 95:11804 (1998).
[0201] V.sub.HHs comprise small intact antigen-binding fragments
(for example, fragments that are about 15 kDa, 118-136 residues).
Camelid V.sub.HH domains have been found to bind to antigen with
high affinity (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001),
with V.sub.HH affinities typically in the nanomolar range and
comparable with those of Fab and scFv fragments. V.sub.HHS are
highly soluble and more stable than the corresponding derivatives
of scFv and Fab fragments. V.sub.H fragments have been relatively
difficult to produce in soluble form, but improvements in
solubility and specific binding can be obtained when framework
residues are altered to be more V.sub.HH-like. (See, for example,
Reichman et al., J Immunol Methods 1999, 231:25-38.) V.sub.fflis
carry amino acid substitutions that make them more hydrophilic and
prevent prolonged interaction with BiP (immunoglobulin heavy-chain
binding protein), which normally binds to the H-chain in the
Endoplasmic Reticulum (ER) during folding and assembly, until it is
displaced by the L-chain. Because of the V.sub.HHs' increased
hydrophilicity, secretion from the ER is improved.
[0202] Functional V.sub.HHs may be obtained by proteolytic cleavage
of HCAb of an immunized camelid, by direct cloning of V.sub.HH
genes from B-cells of an immunized camelid resulting in recombinant
V.sub.HHs, or from naive or synthetic libraries. V.sub.HHs with
desired antigen specificity may also be obtained through phage
display methodology. Using V.sub.HHs in phage display is much
simpler and more efficient compared to Fabs or scFvs, since only
one domain needs to be cloned and expressed to obtain a functional
antigen-binding fragment. Muyldermans, Biotechnol. 74:277-302
(2001); Ghahroudi et al., FEBS Lett. 414:521-526 (1997); and van
der Linden et al., J. Biotechnol. 80:261-270 (2000). Methods for
generating antibodies having camelid heavy chains are also
described in U.S. Patent Publication Nos. 20050136049 and
20050037421.
[0203] Ribosome display methods may be used to identify and isolate
scFv and/or V.sub.HH molecules having the desired binding activity
and affinity. Irving et al., J. Immunol. Methods 248:31-45 (2001).
Ribosome display and selection has the potential to generate and
display large libraries (10.sup.14).
[0204] Other embodiments provide V.sub.HH-like molecules generated
through the process of camelisation, by modifying non-Camelidae
V.sub.Hs, such as human V.sub.HHs, to improve their solubility and
prevent non-specific binding. This is achieved by replacing
residues on the V.sub.Ls side of V.sub.Hs with V.sub.HH-like
residues, thereby mimicking the more soluble V.sub.HH fragments.
Camelised V.sub.H fragments, particularly those based on the human
framework, are expected to exhibit a greatly reduced immune
response when administered in vivo to a patient and, accordingly,
are expected to have significant advantages for therapeutic
applications. Davies et al., FEBS Lett. 339:285-290 (1994); Davies
et al., Protein Eng. 9:531-537 (1996); Tanha et al., J. Biol. Chem.
276:24774-24780 (2001); and Riechmann et al., Immunol. Methods
231:25-38 (1999).
[0205] A wide variety of expression systems are available for the
production of IL-1.beta. fragments including Fab fragments, scFv,
and V.sub.HHs. For example, expression systems of both prokaryotic
and eukaryotic origin may be used for the large-scale production of
antibody fragments and antibody fusion proteins. Particularly
advantageous are expression systems that permit the secretion of
large amounts of antibody fragments into the culture medium.
[0206] Production of bispecific Fab-scFv ("bibody") and trispecific
Fab-(scFv)(2) ("tribody") are described in Schoonjans et al. (J
Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B
Analyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies or
tribodies, a scFv molecule is fused to one or both of the VL-CL (L)
and VH-CH.sub.1 (Fd) chains, e.g., to produce a tribody two scFvs
are fused to C-term of Fab while in a bibody one scFv is fused to
C-term of Fab. A "minibody" consisting of scFv fused to CH3 via a
peptide linker (hingeless) or via an IgG hinge has been described
in Olafsen, et al., Protein Eng Des Sel. 2004 April;
17(4):315-23.
[0207] Intrabodies are single chain antibodies which demonstrate
intracellular expression and can manipulate intracellular protein
function (Biocca, et al., EMBO J 9:101-108, 1990; Colby et al.,
Proc Natl Acad Sci USA. 101:17616-21, 2004). Intrabodies, which
comprise cell signal sequences which retain the antibody construct
in intracellular regions, may be produced as described in
Mhashilkar et al (EMBO J14:1542-51, 1995) and Wheeler et al. (FASEB
J. 17:1733-5. 2003). Transbodies are cell-permeable antibodies in
which a protein transduction domains (PTD) is fused with single
chain variable fragment (scFv) antibodies Heng et al., (Med
Hypotheses. 64:1105-8, 2005).
[0208] The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass antibodies that are SMIPs or
binding domain immunoglobulin fusion proteins specific for target
protein. These constructs are single-chain polypeptides comprising
antigen binding domains fused to immunoglobulin domains necessary
to carry out antibody effector functions. See e.g., WO03/041600,
U.S. Patent publication 20030133939 and US Patent Publication
20030118592.
[0209] The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass immunoadhesins. One or more CDRs
may be incorporated into a molecule either covalently or
noncovalently to make it an immunoadhesin. An immunoadhesin may
incorporate the CDR(s) as part of a larger polypeptide chain, may
covalently link the CDR(s) to another polypeptide chain, or may
incorporate the CDR(s) noncovalently. The CDRs disclosed herein
permit the immunoadhesin to specifically bind to IL-1.beta..
[0210] The IL-1.beta. binding antibodies and fragments of the
present disclosure also encompass antibody mimics comprising one or
more IL-1.beta. binding portions built on an organic or molecular
scaffold (such as a protein or carbohydrate scaffold). Proteins
having relatively defined three-dimensional structures, commonly
referred to as protein scaffolds, may be used as reagents for the
design of antibody mimics. These scaffolds typically contain one or
more regions which are amenable to specific or random sequence
variation, and such sequence randomization is often carried out to
produce libraries of proteins from which desired products may be
selected. For example, an antibody mimic can comprise a chimeric
non-immunoglobulin binding polypeptide having an
immunoglobulin-like domain containing scaffold having two or more
solvent exposed loops containing a different CDR from a parent
antibody inserted into each of the loops and exhibiting selective
binding activity toward a ligand bound by the parent antibody.
Non-immunoglobulin protein scaffolds have been proposed for
obtaining proteins with novel binding properties. (Tramontano et
al., J. Mol. Recognit. 7:9, 1994; McConnell and Hoess, J. Mol.
Biol. 250:460, 1995). Other proteins have been tested as frameworks
and have been used to display randomized residues on alpha helical
surfaces (Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al.,
Protein Eng. 8:601, 1995), loops between alpha helices in alpha
helix bundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552,
1995), and loops constrained by disulfide bridges, such as those of
the small protease inhibitors (Markland et al., Biochemistry
35:8045, 1996; Markland et al., Biochemistry 35:8058, 1996; Rottgen
and Collins, Gene 164:243, 1995; Wang et al., J. Biol. Chem.
270:12250, 1995). Methods for employing scaffolds for antibody
mimics are disclosed in U.S. Pat. No. 5,770,380 and US Patent
Publications 2004/0171116, 2004/0266993, and 2005/0038229.
[0211] The anti-IL-1.beta. binding antibodies or binding fragments
thereof for use in the methods herein generally bind to IL-1.beta.
with high affinity (e.g., as determined with BIACORE). In preferred
embodiments, the antibody or fragment thereof binds to IL-1.beta.
with an equilibrium binding dissociation constant (K.sub.D) of
about 10 nM or less, about 5 nM or less, about 1 nM or less, about
500 pM or less, about 250 pM or less, about 100 pM or less, about
50 pM or less, or about 25 pM or less. In particularly preferred
embodiments, the antibody or antibody fragment binds to human
IL-1.beta. with a dissociation constant of about 100 pM or less,
about 50 pM or less, about 10 pM or less, about 5 pM or less, about
3 pM or less, about 1 pM or less, about 0.75 pM or less, about 0.5
pM or less, about 0.3 pM or less, about 0.2 pM or less, or about
0.1 pM or less. In particularly preferred embodiments, the antibody
or antibody fragment binds to human IL-1.beta. with a dissociation
constant of about 10 pM or less.
[0212] Antibodies or fragments of the present disclosure may, for
example, bind to IL-1.beta. with an IC.sub.50 of about 10 nM or
less, about 5 nM or less, about 2 nM or less, about 1 nM or less,
about 0.75 nM or less, about 0.5 nM or less, about 0.4 nM or less,
about 0.3 nM or less, or even about 0.2 nM or less, as determined
by enzyme linked immunosorbent assay (ELISA). Preferably, the
antibody or antibody fragment of the present disclosure does not
cross-react with any target other than IL-1. For example, the
present antibodies and fragments may bind to IL-1.beta., but do not
detectably bind to IL-1.alpha., or have at least about 100 times
(e.g., at least about 150 times, at least about 200 times, or even
at least about 250 times) greater selectivity in its binding of
IL-1.beta. relative to its binding of IL-1.alpha.. Antibodies or
fragments used according to the present disclosure may, in certain
embodiments, inhibit IL-1.beta. induced expression of serum IL-6 in
an animal by at least 50% (e.g., at least 60%, at least 70%, or
even at least 80%) as compared to the level of serum IL-6 in an
IL-1.beta. stimulated animal that has not been administered an
antibody or fragment of the present disclosure. Antibodies may bind
IL-1.beta. but permit or substantially permit the binding of the
bound IL-1.beta. ligand to IL-1 receptor type I (IL-1RI). In
contrast to many known IL-1.beta. binding antibodies that block or
substantially interfere with binding of IL-1.beta. to IL-1RI, the
antibodies designated ABS and AB7 (U.S. application Ser. No.
11/472,813) selectively bind to the IL-1.beta. ligand, but permit
the binding of the bound IL-1.beta. ligand to MARL For example, the
antibody designated AB7 binds to an IL-1.beta. epitope but still
permits the bound IL-1.beta. to bind to IL-1RI. In certain
embodiments, the antibody may decrease the affinity of interaction
of bound IL-1.beta. to bind to IL-1R1. Accordingly, the disclosure
provides, in a related aspect, use of an IL-1.beta. binding
antibody or IL-1.beta. binding antibody fragment that has at least
one of the aforementioned characteristics. Any of the foregoing
antibodies, antibody fragments, or polypeptides of the disclosure
can be humanized or human engineered, as described herein.
[0213] A variety of IL-1 (e.g., IL-1.beta.) antibodies and
fragments known in the art may be used according the methods
provided herein, including for example antibodies described in or
derived using methods described in the following patents and patent
applications: U.S. Pat. No. 4,935,343; US 2003/0026806; US
2003/0124617 (e.g., antibody AAL160); WO 2006/081139 (e.g.,
antibody 9.5.2); WO 03/034984; WO 95/01997 (e.g., antibody SK48-E26
VTKY); U.S. Pat. No. 7,446,175 (e.g., antibody ACZ 885); WO
03/010282 (e.g., antibody Hu007); WO 03/073982 (e.g., antibody
N55S), U.S. Pat. No. 7,541,033 (e.g., W17, U43, W13, W18, W20),
U.S. Pat. No. 7,491,392, WO 2004/072116, WO 2004/067568, EP 0 267
611 B1, EP 0 364 778 B1, and U.S. application Ser. No. 11/472,813.
As a non-limiting example, antibodies AB5 and AB7 (U.S. application
Ser. No. 11/472,813, WO2007/002261) may be used in accordance with
the present disclosure. Variable region sequences of AB5 and AB7
are as follows:
TABLE-US-00001 AB7 LIGHT CHAIN (SEQ ID NO: 1)
DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLIYY
TSKLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPWTFGQ GTKLEIK
The underlined sequences depict (from left to right) CDR1, 2 and
3.
TABLE-US-00002 HEAVY CHAIN (SEQ ID NO: 2)
QVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWL
AHIWWDGDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCARN
RYDPPWFVDWGQGTLVTVSS
The underlined sequences depict (from left to right) CDR1, 2 and
3.
TABLE-US-00003 AB5 LIGHT CHAIN (SEQ ID NO: 4)
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYY
TSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCLQGKMLPWTFGG GTKLEIK
The underlined sequences depict (from left to right) CDR1, 2 and
3.
TABLE-US-00004 HEAVY CHAIN (SEQ ID NO: 5)
QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWL
AHIWWDGDESYNPSLKTQLTISKDTSRNQVFLKITSVDTVDTATYFCARN
RYDPPWFVDWGQGTLVTVSS
The underlined sequences depict (from left to right) CDR1, 2 and
3.
[0214] In some embodiments, IL-1.beta. antibodies or fragments
thereof for use in any and/or all of the methods disclosed herein
may bind to human IL-1.beta. with a dissociation constant of about
1 nM or less. In some embodiments, the antibody or fragment binds
to human IL-1.beta. with a dissociation constant of about 500 pM or
less. In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof binds to human IL-1.beta. with a
dissociation constant of about 250 pM or less. In some embodiments,
the anti-IL-1.beta. binding antibody or binding fragment thereof
binds to human IL-1.beta. with a dissociation constant of about 100
pM or less. In some embodiments, the antibody or fragment binds to
human IL-1.beta. with a dissociation constant of about 50 pM or
less. In some embodiments, the antibody or fragment binds to human
IL-1.beta. with a dissociation constant of about 10 pM or less. In
some embodiments, the antibody or fragment binds to human
IL-1.beta. with a dissociation constant of about 5 pM or less. In
some embodiments, the antibody or fragment binds to human
IL-1.beta. with a dissociation constant of about 1 pM or less. In
some embodiments, the antibody or fragment binds to human
IL-1.beta. with a dissociation constant of about 0.3 pM or
less.
[0215] In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof is a neutralizing antibody.
[0216] In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof binds to an IL-1.beta. epitope such that
the bound antibody or fragment substantially permits the binding of
IL-1.beta. to IL-1 receptor I (IL-1RI).
[0217] In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof does not detectably bind to IL-1.alpha.,
IL-1R or IL-1Ra.
[0218] In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof competes with the binding of an antibody
having the light chain variable region of SEQ ID NO:1 and the heavy
chain variable region of SEQ ID NO:2. In some embodiments of any of
the methods described above, the anti-IL-1.beta. binding antibody
or binding fragment thereof binds to an epitope that is the same or
substantially the same as an epitope that is bound by an antibody
having the light chain variable region of SEQ ID NO:1 and the heavy
chain variable region of SEQ ID NO:2. In some embodiments of any of
the methods described above, the anti-IL-1.beta. binding antibody
or binding fragment thereof comprises a light chain variable region
of SEQ ID NO:1 and a heavy chain variable region of SEQ ID
NO:2.
[0219] In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof binds to an epitope incorporating Glu64 of
IL-1.beta..
[0220] In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof binds to amino acids 1-34 of the N
terminus of IL-1.beta..
[0221] In some embodiments, the anti-IL-1.beta. binding antibody or
binding fragment thereof is humanized or human.
[0222] The present disclosure also provides uses of an
anti-IL-1.beta. binding antibody or binding fragment thereof which
has a lower IC.sub.50 than an IL-1.beta. receptor antagonist in a
human whole blood IL-1.beta. inhibition assay that measures
IL-1.beta. induced production of IL-8, in the manufacture of a
composition for use in the reduction, prevention or treatment of a
cardiac event or a cardiovascular disease.
[0223] In another aspect, the methods comprise administering a
therapeutically effective amount of an anti-IL-1.beta. antibody or
fragment thereof, wherein the antibody or fragment thereof has a
lower IC.sub.50 than an IL-1.beta. receptor antagonist in a human
whole blood IL-1.beta. inhibition assay that measures IL-1.beta.
induced production of IL-8. In one embodiment, the antibody or
fragment has an IC.sub.50 that is less than about 90%, 80%, 70%,
60%, 50% of the IC.sub.50 of an IL-1.beta. receptor antagonist in a
human whole blood IL-1.beta. inhibition assay that measures
IL-1.beta. induced production of IL-8. In a further embodiment, the
antibody or fragment has an IC.sub.50 that is less than about 40%,
30%, 20%, 10% of the IC.sub.50 of an IL-1.beta. receptor antagonist
in a human whole blood IL-1.beta. inhibition assay that measures
IL-1.beta. induced production of IL-8. In a preferred embodiment,
the antibody or fragment has an IC.sub.50 that is less than about
8%, 5%, 4%, 3%, 2%, 1% of the IC.sub.50 of an IL-1.beta. receptor
antagonist in a human whole blood IL-1.beta. inhibition assay that
measures IL-1.beta. induced production of IL-8. In one embodiment,
the IL-1.beta. receptor antagonist is anakinra (i.e.,
Kineret.RTM.).
[0224] In another aspect, the method provided herein comprises
administering a therapeutically effective amount of an
anti-IL-1.beta. antibody or fragment thereof to the subject,
wherein the antibody or fragment thereof provides in vivo
inhibition of IL-1.beta. stimulated release of IL-6 in mice
compared to a control antibody using an assay that is described by
Economides et al., Nature Med., 9:47-52 (2003) which is
incorporated by reference. In one embodiment the antibody or
fragment provides in vivo inhibition of IL-1B stimulated release of
IL-6 in mice of at least about 10%, 20%, 30%, 40%, 50% compared to
the control antibody. In a further embodiment, the antibody or
fragment provides in vivo inhibition of IL-1 stimulated release of
IL-6 in mice of at least about 60%, 70%, 80%, 90%, 95% compared to
the control antibody. In one embodiment, the control antibody is an
isotype control antibody.
[0225] In another aspect, the disclosure provides a method
comprising administering a therapeutically effective amount of an
anti-IL-1.beta. antibody or fragment thereof to the human, wherein
the antibody or fragment thereof inhibits Staphylococcus
epidermidis induced cytokine production in human whole blood
compared to a control where no antibody is used. In one embodiment
the antibody or fragment provides a greater level of inhibition of
Staphylococcus epidermidis induced cytokine production in human
whole blood by at least about 10%, 20%, 30%, 40%, 50% compared to
the control. In a further embodiment, the antibody or fragment
provides a greater level of inhibition of Staphylococcus
epidermidis induced cytokine production in human whole blood by at
least about 60%, 70%, 80%, 90%, 95% compared to the control. In one
embodiment, the inhibited cytokines are IL-1.beta., IL-1.alpha.,
IL-6, IL-8, IL-1Ra, TNF.alpha. or IFN.gamma..
[0226] The antibodies and antibody fragments described herein can
be prepared by any suitable method. Suitable methods for preparing
such antibodies and antibody fragments are known in the art. Other
methods for preparing the antibodies and antibody fragments are as
described herein as part of the disclosure. The antibody, antibody
fragment, or polypeptide of the present disclosure, as described
herein, can be isolated or purified to any degree. As used herein,
an isolated compound is a compound that has been removed from its
natural environment. A purified compound is a compound that has
been increased in purity, such that the compound exists in a form
that is more pure than it exists (i) in its natural environment or
(ii) when initially synthesized and/or amplified under laboratory
conditions, wherein "purity" is a relative term and does not
necessarily mean "absolute purity."
Pharmaceutical Compositions
[0227] IL-1 (e.g., IL-1.beta.) binding antibodies and antibody
fragments for use according to the present disclosure can be
formulated in compositions, especially pharmaceutical compositions,
for use in the methods herein. Such compositions comprise a
therapeutically or prophylactically effective amount of an
IL-1.beta. binding antibody or antibody fragment of the disclosure
in admixture with a suitable carrier, e.g., a pharmaceutically
acceptable agent. Typically, IL-1.beta. binding antibodies and
antibody fragments of the disclosure are sufficiently purified for
administration to an animal before formulation in a pharmaceutical
composition.
[0228] Pharmaceutically acceptable agents include carriers,
excipients, diluents, antioxidants, preservatives, coloring,
flavoring and diluting agents, emulsifying agents, suspending
agents, solvents, fillers, bulking agents, buffers, delivery
vehicles, tonicity agents, cosolvents, wetting agents, complexing
agents, buffering agents, antimicrobials, and surfactants.
[0229] Neutral buffered saline or saline mixed with albumin are
exemplary appropriate carriers. The pharmaceutical compositions can
include antioxidants such as ascorbic acid; low molecular weight
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
Tween, pluronics, or polyethylene glycol (PEG). Also by way of
example, suitable tonicity enhancing agents include alkali metal
halides (preferably sodium or potassium chloride), mannitol,
sorbitol, and the like. Suitable preservatives include benzalkonium
chloride, thimerosal, phenethyl alcohol, methylparaben,
propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen
peroxide also can be used as preservative. Suitable cosolvents
include glycerin, propylene glycol, and PEG. Suitable complexing
agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting
agents include sorbitan esters, polysorbates such as polysorbate
80, tromethamine, lecithin, cholesterol, tyloxapal, and the like.
The buffers can be conventional buffers such as acetate, borate,
citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be
about pH 4-5.5, and Tris buffer can be about pH 7-8.5. Additional
pharmaceutical agents are set forth in Remington's Pharmaceutical
Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing
Company, 1990.
[0230] The composition can be in liquid form or in a lyophilized or
freeze-dried form and may include one or more lyoprotectants,
excipients, surfactants, high molecular weight structural additives
and/or bulking agents (see for example U.S. Pat. Nos. 6,685,940,
6,566,329, and 6,372,716). In one embodiment, a lyoprotectant is
included, which is a non-reducing sugar such as sucrose, lactose or
trehalose. The amount of lyoprotectant generally included is such
that, upon reconstitution, the resulting formulation will be
isotonic, although hypertonic or slightly hypotonic formulations
also may be suitable. In addition, the amount of lyoprotectant
should be sufficient to prevent an unacceptable amount of
degradation and/or aggregation of the protein upon lyophilization.
Exemplary lyoprotectant concentrations for sugars (e.g., sucrose,
lactose, trehalose) in the pre-lyophilized formulation are from
about 10 mM to about 400 mM. In another embodiment, a surfactant is
included, such as for example, nonionic surfactants and ionic
surfactants such as polysorbates (e.g. polysorbate 20, polysorbate
80); poloxamers (e.g. poloxamer 188); poly (ethylene glycol) phenyl
ethers (e.g. Triton); sodium dodecyl sulfate (SDS); sodium laurel
sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyk or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl ofeyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc). Exemplary amounts of surfactant that may be
present in the pre-lyophilized formulation are from about
0.001-0.5%. High molecular weight structural additives (e.g.
fillers, binders) may include for example, acacia, albumin, alginic
acid, calcium phosphate (dibasic), cellulose,
carboxymethylcellulose, carboxymethylcellulose sodium,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,
dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium
sulfate, amylose, glycine, bentonite, maltose, sorbitol,
ethylcellulose, disodium hydrogen phosphate, disodium phosphate,
disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar
gum, liquid glucose, compressible sugar, magnesium aluminum
silicate, maltodextrin, polyethylene oxide, polymethacrylates,
povidone, sodium alginate, tragacanth microcrystalline cellulose,
starch, and zein. Exemplary concentrations of high molecular weight
structural additives are from 0.1% to 10% by weight. In other
embodiments, a bulking agent (e.g., mannitol, glycine) may be
included.
[0231] Compositions can be suitable for parenteral administration.
Exemplary compositions are suitable for injection or infusion into
an animal by any route available to the skilled worker, such as
intraarticular, subcutaneous, intravenous, intramuscular,
intraperitoneal, intracerebral (intraparenchymal),
intracerebroventricular, intramuscular, intraocular, intraarterial,
intralesional, intrarectal, transdermal, oral, and inhaled routes.
A parenteral formulation typically will be a sterile, pyrogen-free,
isotonic aqueous solution, optionally containing pharmaceutically
acceptable preservatives.
[0232] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringers' dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers, such as those based on Ringer's dextrose,
and the like. Preservatives and other additives may also be
present, such as, for example, anti-microbials, anti-oxidants,
chelating agents, inert gases and the like. See generally,
Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980,
which is incorporated herein by reference.
[0233] Pharmaceutical compositions described herein can be
formulated for controlled or sustained delivery in a manner that
provides local concentration of the product (e.g., bolus, depot
effect) sustained release and/or increased stability or half-life
in a particular local environment. The present disclosure
contemplates that in certain embodiments such compositions may
include a significantly larger amount of antibody or fragment in
the initial deposit, while the effective amount of antibody or
fragment actually released and available at any point in time for
is in accordance with the disclosure herein an amount much lower
than the initial deposit. The compositions can include the
formulation of IL-1.beta. binding antibodies, antibody fragments,
nucleic acids, or vectors of the disclosure with particulate
preparations of polymeric compounds such as polylactic acid,
polyglycolic acid, etc., as well as agents such as a biodegradable
matrix, injectable microspheres, microcapsular particles,
microcapsules, bioerodible particles beads, liposomes, and
implantable delivery devices that provide for the controlled or
sustained release of the active agent which then can be delivered
as a depot injection. Techniques for formulating such sustained- or
controlled-delivery means are known and a variety of polymers have
been developed and used for the controlled release and delivery of
drugs. Such polymers are typically biodegradable and biocompatible.
Polymer hydrogels, including those formed by complexation of
enantiomeric polymer or polypeptide segments, and hydrogels with
temperature or pH sensitive properties, may be desirable for
providing drug depot effect because of the mild and aqueous
conditions involved in trapping bioactive protein agents (e.g.,
antibodies). See, for example, the description of controlled
release porous polymeric microparticles for the delivery of
pharmaceutical compositions in PCT Application Publication WO
93/15722.
[0234] Suitable materials for this purpose include polylactides
(see, e.g., U.S. Pat. No. 3,773,919), polymers of
poly-(a-hydroxycarboxylic acids), such as
poly-D-(-)-3-hydroxybutyric acid (EP 133,988A), copolymers of
L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al.,
Biopolymers, 22: 547-556 (1983)), poly
(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12: 98-105
(1982)), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric
acid. Other biodegradable polymers include poly(lactones),
poly(acetals), poly(orthoesters), and poly(orthocarbonates).
Sustained-release compositions also may include liposomes, which
can be prepared by any of several methods known in the art (see,
e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92
(1985)). The carrier itself, or its degradation products, should be
nontoxic in the target tissue and should not further aggravate the
condition. This can be determined by routine screening in animal
models of the target disorder or, if such models are unavailable,
in normal animals.
[0235] Microencapsulation of recombinant proteins for sustained
release has been performed successfully with human growth hormone
(rhGH), interferon- (rhIFN-), interleukin-2, and MN rgp120. Johnson
et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther.,
27:1221-1223 (1993); Hora et al., Bio/Technology. 8:755-758 (1990);
Cleland, "Design and Production of Single Immunization Vaccines
Using Polylactide Polyglycolide Microsphere Systems," in Vaccine
Design: The Subunit and Adjuvant Approach, Powell and Newman, eds,
(Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO
96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010. The
sustained-release formulations of these proteins were developed
using poly-lactic-coglycolic acid (PLGA) polymer due to its
biocompatibility and wide range of biodegradable properties. The
degradation products of PLGA, lactic and glycolic acids can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be depending on its molecular weight and
composition. Lewis, "Controlled release of bioactive agents from
lactide/glycolide polymer," in: M. Chasin and R. Langer (Eds.),
Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New
York, 1990), pp. 1-41. Additional examples of sustained release
compositions include, for example, EP 58,481A, U.S. Pat. No.
3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et
al., Biopolymers 22, 547 [1983], R. Langer et al., Chem. Tech. 12,
98 [1982], Sinha et al., J. Control. Release 90, 261 [2003], Zhu et
al., Nat. Biotechnol. 18, 24 [2000], and Dai et al., Colloids Surf
B Biointerfaces 41, 117 [2005].
[0236] Bioadhesive polymers are also contemplated for use in or
with compositions of the present disclosure. Bioadhesives are
synthetic and naturally occurring materials able to adhere to
biological substrates for extended time periods. For example,
Carbopol and polycarbophil are both synthetic cross-linked
derivatives of poly(acrylic acid). Bioadhesive delivery systems
based on naturally occurring substances include for example
hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a
naturally occurring mucopolysaccharide consisting of residues of
D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acid is found
in the extracellular tissue matrix of vertebrates, including in
connective tissues, as well as in synovial fluid and in the
vitreous and aqueous humour of the eye. Esterified derivatives of
hyaluronic acid have been used to produce microspheres for use in
delivery that are biocompatible and biodegrable (see for example,
Cortivo et al., Biomaterials (1991) 12:727-730; European
Publication No. 517,565; International Publication No. WO 96/29998;
Illum et al., J. Controlled Rel. (1994) 29:133-141). Exemplary
hyaluronic acid containing compositions of the present disclosure
comprise a hyaluronic acid ester polymer in an amount of
approximately 0.1% to about 40% (w/w) of an IL-1.beta. binding
antibody or fragment to hyaluronic acid polymer.
[0237] Both biodegradable and non-biodegradable polymeric matrices
can be used to deliver compositions in accordance with the present
disclosure, and such polymeric matrices may comprise natural or
synthetic polymers. Biodegradable matrices are preferred. The
period of time over which release occurs is based on selection of
the polymer. Typically, release over a period ranging from between
a few hours and three to twelve months is most desirable. Exemplary
synthetic polymers which can be used to form the biodegradable
delivery system include: polymers of lactic acid and glycolic acid,
polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,
polyalkylene oxides, polyalkylene terepthalates, polyvinyl
alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides,
polyvinylpyrrolidone, polyglycolides, polysiloxanes,
polyanhydrides, polyurethanes and co-polymers thereof, poly(butic
acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl
celluloses, cellulose ethers, cellulose esters, nitro celluloses,
polymers of acrylic and methacrylic esters, methyl cellulose, ethyl
cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl
cellulose, hydroxybutyl methyl cellulose, cellulose acetate,
cellulose propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly(methyl methacrylate), poly(ethyl
methacrylate), poly(butylmethacrylate), poly(isobutyl
methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.
Exemplary natural polymers include alginate and other
polysaccharides including dextran and cellulose, collagen, chemical
derivatives thereof (substitutions, additions of chemical groups,
for example, alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art), albumin
and other hydrophilic proteins, zein and other prolamines and
hydrophobic proteins, copolymers and mixtures thereof. In general,
these materials degrade either by enzymatic hydrolysis or exposure
to water in vivo, by surface or bulk erosion. The polymer
optionally is in the form of a hydrogel (see for example WO
04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26,
581-587) that can absorb up to about 90% of its weight in water and
further, optionally is cross-linked with multi-valent ions or other
polymers.
[0238] Delivery systems also include non-polymer systems that are
lipids including sterols such as cholesterol, cholesterol esters
and fatty acids or neutral fats such as mono- di- and
tri-glycerides; hydrogel release systems; silastic systems; peptide
based systems; wax coatings; compressed tablets using conventional
binders and excipients; partially fused implants; and the like.
Specific examples include, but are not limited to: (a) erosional
systems in which the product is contained in a form within a matrix
such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189 and
5,736,152 and (b) diffusional systems in which a product permeates
at a controlled rate from a polymer such as described in U.S. Pat.
Nos. 3,854,480, 5,133,974 and 5,407,686. Liposomes containing the
product may be prepared by methods known methods, such as for
example (U.S. Pat. No. DE 3,218,121; Epstein et al., Proc. Natl.
Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl.
Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP
88,046; EP 143,949; EP 142,641; Japanese patent application
83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324).
[0239] A pharmaceutical composition comprising an IL-1.beta.
binding antibody or fragment can be formulated for inhalation, such
as for example, as a dry powder. Inhalation solutions also can be
formulated in a liquefied propellant for aerosol delivery. In yet
another formulation, solutions may be nebulized. Additional
pharmaceutical composition for pulmonary administration include,
those described, for example, in PCT Application Publication WO
94/20069, which discloses pulmonary delivery of chemically modified
proteins. For pulmonary delivery, the particle size should be
suitable for delivery to the distal lung. For example, the particle
size can be from 1 .mu.m to 5 .mu.m; however, larger particles may
be used, for example, if each particle is fairly porous.
[0240] Certain formulations containing IL-1.beta. binding
antibodies or antibody fragments can be administered orally.
Formulations administered in this fashion can be formulated with or
without those carriers customarily used in the compounding of solid
dosage forms such as tablets and capsules. For example, a capsule
can be designed to release the active portion of the formulation at
the point in the gastrointestinal tract when bioavailability is
maximized and pre-systemic degradation is minimized. Additional
agents can be included to facilitate absorption of a selective
binding agent. Diluents, flavorings, low melting point waxes,
vegetable oils, lubricants, suspending agents, tablet
disintegrating agents, and binders also can be employed.
[0241] Another preparation can involve an effective quantity of an
IL-1.beta. binding antibody or fragment in a mixture with non-toxic
excipients which are suitable for the manufacture of tablets. By
dissolving the tablets in sterile water, or another appropriate
vehicle, solutions can be prepared in unit dose form. Suitable
excipients include, but are not limited to, inert diluents, such as
calcium carbonate, sodium carbonate or bicarbonate, lactose, or
calcium phosphate; or binding agents, such as starch, gelatin, or
acacia; or lubricating agents such as magnesium stearate, stearic
acid, or talc.
[0242] Suitable and/or preferred pharmaceutical formulations can be
determined in view of the present disclosure and general knowledge
of formulation technology, depending upon the intended route of
administration, delivery format, and desired dosage. Regardless of
the manner of administration, an effective dose can be calculated
according to patient body weight, body surface area, or organ size.
Further refinement of the calculations for determining the
appropriate dosage for treatment involving each of the formulations
described herein are routinely made in the art and is within the
ambit of tasks routinely performed in the art. Appropriate dosages
can be ascertained through use of appropriate dose-response
data.
[0243] Additional formulations will be evident in light of the
present disclosure, including formulations involving IL-1.beta.
binding antibodies and fragments in combination with one or more
other therapeutic agents. For example, in some formulations, an
IL-1.beta. binding antibody, antibody fragment, nucleic acid, or
vector of the disclosure is formulated with a second inhibitor of
an IL-1 signaling pathway Representative second inhibitors include,
but are not limited to, antibodies, antibody fragments, peptides,
polypeptides, compounds, nucleic acids, vectors and pharmaceutical
compositions, such as, for example, those described in U.S. Pat.
No. 6,899,878, US 2003022869, US 20060094663, US 20050186615, US
20030166069, WO/04022718, WO/05084696, WO/05019259. For example, a
composition may comprise an IL-1.beta. binding antibody, antibody
fragment, nucleic acid, or vector of the disclosure in combination
with an IL-1.beta. binding antibody, fragment, or a nucleic acid or
vector encoding such an antibody or fragment.
Methods of Use
[0244] Anti-IL-1.beta. binding antibodies or binding fragments
thereof in a therapeutically effective amount may be used as
disclosed by the methods herein for the treatment and/or prevention
of cardiovascular disease, including, for example, acute
cardiovascular disease or chronic cardiovascular disease. Such
methods, as well as pharmaceutical compositions for use in such
methods, may be used for reducing, treating or preventing a
cardiovascular event, such as myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina, or a revascularization procedure
in a subject, including in a subject with a history of a risk
factor for cardiovascular disease. The methods and pharmaceutical
compositions may also be used to reduce mortality following a
cardiovascular event in a subject. The present disclosure also
contemplates the use of other IL-1 pathway inhibitors, as an
alternative or in addition to the anti-IL-1.beta. antibodies or
fragments.
[0245] The terms "prevention", "prevent", "preventing",
"suppression", "suppress", "suppressing", "inhibit" and
"inhibition" as used herein with respect to methods as described
refer to preventing, suppressing or reducing, either temporarily or
permanently, the onset of a clinical symptoms or manifestation of
an event, disease or condition, such as, for example, a
cardiovascular event or disease, (e.g., acute or chronic
cardiovascular disease). Such preventing, suppressing or reducing
need not be absolute to be useful.
[0246] The terms "reduce", "reducing" and "reduction" as used
herein with respect to the methods as described refer to delaying
the time to an event or disease, decreasing the likelihood or risk
of an event or disease, decreasing the incidence of an event or
disease (e.g., in a treatment group), preventing the occurrence of
an event or disease (e.g., prevention of a cardiovascular event or
disease), decreasing the magnitude or severity of an event or
disease (except in the case where the event is death), and/or
decreasing the time to recovery from an event or disease (except in
the case where the event is death), such as, for example, treating
or treatment of a cardiovascular event or disease, (e.g., acute or
chronic cardiovascular disease).
[0247] The phrase "mortality following a cardiovascular event" as
used herein refers to mortality (i.e., death) that occurs after a
cardiovascular event (e.g., after initiation of the cardiovascular
event), and which may be, but need not be, directly or indirectly
caused by or influenced by the cardiovascular event. Following also
refers to the proximity in time (e.g., measurable) between the
cardiovascular event and mortality, regardless of whether or not a
direct or indirect causal link can be determined. The proximity in
time between the cardiovascular event and mortality may vary and
include an amount of time that is approaching being
simultaneous.
[0248] The terms "treatment", "treat" and "treating" as used with
respect to methods as described herein refers eliminating,
reducing, suppressing or ameliorating, either temporarily or
permanently, a clinical symptom, manifestation or progression of an
event, disease or condition, such as, for example, a cardiovascular
event or disease, (e.g., acute or chronic cardiovascular disease).
Such treating need not be absolute to be useful.
[0249] The term "in need of treatment" as used herein refers to a
judgment made by a caregiver that a patient requires or will
benefit from treatment. This judgment is made based on a variety of
factors that are in the realm of a caregiver's expertise, but that
includes the knowledge that the patient is ill, or will be ill, as
the result of a condition that is treatable by a method or compound
of the disclosure.
[0250] The term "in need of prevention" as used herein refers to a
judgment made by a caregiver that a patient requires or will
benefit from prevention. This judgment is made based on a variety
of factors that are in the realm of a caregiver's expertise, but
that includes the knowledge that the patient will be ill or may
become ill, as the result of a condition that is preventable by a
method or compound of the disclosure.
[0251] The term "therapeutically effective amount" as used herein
refers to an amount of a compound (e.g., antibody), either alone or
as a part of a pharmaceutical composition, that is capable of
having any detectable, positive effect on any symptom, aspect, or
characteristics of a disease state or condition when administered
to a subject (e.g., as one or more doses), including, for example,
reducing a cardiovascular event or disease, or reducing mortality
following a cardiovascular event or disease, (e.g., acute or
chronic cardiovascular disease). Such effect need not be absolute
to be beneficial.
[0252] The present disclosure provides methods of treating a
subject with cardiovascular disease, including, for example, acute
cardiovascular disease or chronic cardiovascular disease,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof.
[0253] The present disclosure provides methods of reducing a
cardiovascular event in a subject, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof, wherein the subject
is a subject with a history of a previous cardiovascular event or a
history of at least one risk factor for cardiovascular disease, and
wherein the cardiovascular event is myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina, or a revascularization
procedure.
[0254] The present disclosure also provides methods of reducing a
cardiovascular event (e.g., delaying time to event, reducing
likelihood or risk of event, preventing an event, reducing severity
of event, reducing time to recovery) in a subject with a history of
at least one risk factor for cardiovascular disease, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof,
and wherein the cardiovascular event is myocardial infarction,
stroke, cardiovascular death, congestive heart failure, cardiac
arrest, acute coronary syndrome, angina, or a revascularization
procedure.
[0255] The present disclosure also provides methods of reducing a
cardiovascular event (e.g., delaying time to event, reducing
likelihood or risk of event, preventing an event, reducing severity
of event, reducing time to recovery) in a subject with a history of
a previous cardiovascular event, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1n binding
antibody or binding fragment thereof, and wherein said
cardiovascular event is myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina or a revascularization procedure.
In some embodiments, the previous cardiovascular event is a first
cardiovascular event. In some embodiments, the previous or first
cardiovascular event is selected from the group consisting of
myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome, angina and a revascularization procedure.
[0256] In some embodiments, the previous or first cardiovascular
event is myocardial infarction or acute coronary syndrome. In some
embodiments, the myocardial infarction is myocardial infarction
with ST elevation (e.g., ST-segment elevation myocardial
infarction, STEMI). In some embodiments, the myocardial infarction
is myocardial infarction without ST elevation (e.g., non-ST-segment
elevation myocardial infarction, NSTEMI). In some embodiments the
presence or absence of ST elevation is determined by
electrocardiogram (e.g., ECG, EKG).
[0257] In some embodiments, the method of reducing a cardiovascular
event is a method of reducing a second or subsequent cardiovascular
event. In some embodiments, the cardiovascular event (e.g., second
or subsequent cardiovascular event) is selected from the group
consisting of myocardial infarction, stroke, cardiovascular death,
congestive heart failure, cardiac arrest, acute coronary syndrome,
angina and a revascularization procedure. In some embodiments, the
first cardiovascular event and second cardiovascular event are the
same types of cardiovascular events. In some embodiments, the first
cardiovascular event and second cardiovascular event are different
types of cardiovascular events.
[0258] In some embodiments, the revascularization procedure is a
coronary, carotid or peripheral arterial revascularization
procedure. In some embodiments, the coronary, carotid or peripheral
arterial revascularization procedure is a percutaneous coronary
intervention (PCI), a stent implant, coronary artery bypass graft
(CABG), carotid endarterectomy, peripheral vascular disease bypass
surgery, or peripheral angioplasty surgery.
[0259] In some preferred embodiments, the therapeutically effective
amount of an anti-IL-1.beta. binding antibody or binding fragment
thereof is first administered within 1 week of the cardiovascular
event, within 96 hours of the cardiovascular event, within 72 hours
of the cardiovascular event, within 48 hours of the cardiovascular
event, within 24 hours of the cardiovascular event, or within 12
hours of the cardiovascular event. In some embodiments, the subject
also has a history of at least one risk factor for cardiovascular
disease. In some embodiments, the risk factor is manifest coronary
heart disease, coronary artery disease, thrombosis, transient
ischaemic attack, left ventricular hypertrophy, arteriosclerosis,
restenosis, tobacco smoking or peripheral vascular disease. In some
embodiments the peripheral vascular disease is clinically apparent
(e.g., peripheral artery disease of Fontaine Class II or greater).
In some embodiments, the risk factor is elevated triglycerides,
systemic inflammation, high blood phosphorus levels, high
parathyroid hormone levels, microalbuminuria, or high homocysteine
levels. In some embodiments, the risk factor is obesity,
hyperglycemia, chronic renal failure, high blood glucose, chronic
kidney disease, or metabolic syndrome. In some embodiments, the
risk factor is end stage renal disease. In some embodiments, the
risk factor is hypertension, dyslipidemia, hyperlipidemia, elevated
total cholesterol, elevated LDL cholesterol, or low HDL cholesterol
or atherosclerosis. In some embodiments, the hypertension is
manifested as a blood pressure of greater than or equal to 180/110
mm Hg. In some other embodiments, the hypertension is
mild-to-moderate, with systolic blood pressure (SBP) of 140 to 180
mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mm Hg. In
some embodiments, the subject has elevated levels of C-reactive
protein (CRP). In some embodiments, the subject is older than 55
years. In some embodiments, the subject is older than 65 years. In
some embodiments, the subject is non-hypertensive. In some
embodiments, the subject has poorly controlled hypertension. In
some embodiments, the subject has a "Type A" personality. In some
embodiments, the subject has a sedentary lifestyle. In some
embodiments, the subject has diabetes mellitus. In some
embodiments, the diabetes mellitus is Type 2 diabetes. In some
embodiments, the subject has a history of two or more said risk
factors. In some embodiments, the subject has a history of three or
more said risk factors.
[0260] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP
levels.
[0261] The present disclosure also provides methods of reducing
mortality following a cardiovascular event in a subject, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof. In
some embodiments, the cardiovascular event is myocardial
infarction, stroke, cardiac arrest, congestive heart failure,
cardiovascular death, acute coronary syndrome (e.g., diagnosed),
angina or a revascularization procedure. In some embodiments, the
cardiovascular event is myocardial infarction or acute coronary
syndrome. In some embodiments, the myocardial infarction is
myocardial infarction with ST elevation (e.g., ST-segment elevation
myocardial infarction, STEMI). In some embodiments, the myocardial
infarction is myocardial infarction without ST elevation (e.g.,
non-ST-segment elevation myocardial infarction, NSTEMI). In some
embodiments the presence or absence of ST elevation is determined
by electrocardiogram (e.g., ECG, EKG).
[0262] In some embodiments, mortality is death from cardiovascular
causes. In other embodiments, mortality is death from any cause. In
some embodiments, the cardiovascular event is myocardial
infarction, stroke, congestive heart failure, acute coronary
syndrome, angina or a revascularization procedure. In some
embodiments, the revascularization procedure is a coronary, carotid
or peripheral arterial revascularization procedure. In some
embodiments, the coronary, carotid or peripheral arterial
revascularization procedure is a percutaneous coronary intervention
(PCI), a stent implant, coronary artery bypass graft (CABG),
carotid endarterectomy, peripheral vascular disease bypass surgery,
or peripheral angioplasty surgery.
[0263] In some preferred embodiments the therapeutically effective
amount of an anti-IL-1.beta. binding antibody or binding fragment
thereof is first administered within 1 week of the cardiovascular
event, within 96 hours of the cardiovascular event, within 72 hours
of the cardiovascular event, within 48 hours of the cardiovascular
event, within 24 hours of the cardiovascular event, or within 12
hours of the cardiovascular event. In some embodiments, the subject
does not have Type 2 diabetes. In some embodiments, the subject has
survived a previous cardiovascular event of myocardial infarction
or stroke. In some embodiments, the occurrence of said
cardiovascular event is a reoccurrence of a cardiovascular event of
myocardial infarction or stroke.
[0264] In some embodiments, the subject has a history of one or
more risk factors for cardiovascular disease. In some embodiments,
the risk factor is manifest coronary heart disease, coronary artery
disease, thrombosis, transient ischaemic attack, left ventricular
hypertrophy, arteriosclerosis, restenosis, tobacco smoking or
peripheral vascular disease. In some embodiments the peripheral
vascular disease is clinically apparent (e.g., peripheral artery
disease of Fontaine Class II or greater). In some embodiments, the
risk factor is elevated triglycerides, systemic inflammation, high
blood phosphorus levels, high parathyroid hormone levels,
microalbuminuria, or high homocysteine levels. In some embodiments,
the risk factor is obesity, hyperglycemia, chronic renal failure,
high blood glucose, chronic kidney disease, or metabolic syndrome.
In some embodiments, the risk factor is hypertension, dyslipidemia,
hyperlipidemia, elevated total cholesterol, elevated LDL
cholesterol, or low HDL cholesterol or atherosclerosis. In some
embodiments, the hypertension is manifested as a blood pressure of
greater than or equal to 180/110 mm Hg. In some other embodiments,
the hypertension is mild-to-moderate, with systolic blood pressure
(SBP) of 140 to 180 mm Hg and/or diastolic blood pressure (DBP) of
90 to 110 mm Hg.
[0265] In some embodiments, the subject is non-hypertensive. In
some embodiments, the subject has poorly controlled hypertension.
In some embodiments, the subject has a "Type A" personality. In
some embodiments, the subject has a sedentary lifestyle. In some
embodiments, the subject has a history of two or more said risk
factors. In some embodiments, the subject has a history of three or
more said risk factors.
[0266] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP
levels.
[0267] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of at least one
risk factor for cardiovascular disease, comprising administering to
said subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof, and
wherein said risk factor is not Type 2 diabetes, obesity,
hyperglycemia, dyslipidemia, hyperlipidemia, chronic renal failure,
high blood glucose, chronic kidney disease, hypertension,
atherosclerosis or metabolic syndrome.
[0268] In some embodiments, the cardiovascular event is myocardial
infarction, stroke, cardiac arrest, congestive heart failure,
cardiovascular death, acute coronary syndrome (e.g., diagnosed),
angina or a revascularization procedure. In some embodiments, the
revascularization procedure is a coronary, carotid or peripheral
arterial revascularization procedure. In some embodiments, the
coronary, carotid or peripheral arterial revascularization
procedure is a percutaneous coronary intervention (PCI), a stent
implant, coronary artery bypass graft (CABG), carotid
endarterectomy, peripheral vascular disease bypass surgery, or
peripheral angioplasty surgery.
[0269] In some embodiments, the risk factor is manifest coronary
heart disease, coronary artery disease, thrombosis, transient
ischaemic attack, left ventricular hypertrophy, arteriosclerosis,
restenosis, tobacco smoking or peripheral vascular disease. In some
embodiments, the risk factor is elevated triglycerides, systemic
inflammation, high blood phosphorus levels, high parathyroid
hormone levels, microalbuminuria, or high homocysteine levels.
[0270] In some embodiments, the subject has elevated levels of
C-reactive protein (CRP). In some embodiments, the subject is older
than 55 years. In some embodiments, the subject is older than 65
years. In some embodiments, the subject has a history of two or
more said risk factors. In some embodiments, the subject has a
history of three or more said risk factors.
[0271] In some embodiments, administering said therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof is sufficient to achieve a decrease in CRP
levels.
[0272] The present disclosure also provides methods of treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof and at least one other
pharmaceutical composition comprising an active agent other than an
IL-1.beta. antibody or fragment.
[0273] In some embodiments, the active agent of said at least one
other pharmaceutical composition is a cholesterol lowering agent, a
statin, an HMG-CoA reductase inhibitor, a calcium channel blocker,
a beta blocker, an antihypertensive, a diuretic, aspirin, niacin,
an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II
receptor blocker, a vasodilator, an anticoagulant, a inhibitor of
platelet aggregation, a thrombolytic or digitalis.
[0274] The present disclosure also provides methods of treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof and a
revascularization procedure. In some embodiments, the
revascularization procedure is a coronary, carotid or peripheral
arterial revascularization procedure.
[0275] The present disclosure also provides methods of reducing
restenosis in a subject following a revascularization procedure,
comprising administering to said subject a therapeutically
effective amount of an anti-IL-1.beta. binding antibody or binding
fragment thereof. In some embodiments, the revascularization
procedure is a coronary, carotid or peripheral arterial
revascularization procedure.
[0276] The present disclosure also provides methods of treating
acute hypertension in a subject comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof and one or more
antihypertensive agents. In some embodiments, the subject has a
blood pressure of greater than or equal to 180/110 mm Hg. In some
other embodiments, the subject has mild-to-moderate hypertension,
with systolic blood pressure (SBP) of 140 to 180 mm Hg and/or
diastolic blood pressure (DBP) of 90 to 110 mm Hg. In some
embodiments, the antihypertensive agent is administered
intravenously. In some embodiments, the antihypertensive agent is
selected from the group consisting of alpha/beta-adrenergic
blocking agents, angiotensin-converting enzyme inhibitors,
angiotensin II receptor antagonists, antiadrenergic agents,
beta-adrenergic blocking agents, calcium-channel blocking agents,
diuretics, and vasodilators. In some embodiments, the
antihypertensive agent is carvedilol, labetalol, benazepril,
captopril, enalapril, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, trandolapril, candesartan,
eprosartan, irbesartan, losartan, telmisartan, valsartan,
clonidine, doxazosin, guanabenz, guanadrel, guanethidine,
guanfacine, mecamylamine, methyldopa, prazosin, reserpine,
terazosin, acebutolol, atenolol, betaxolol, bisoprolol, carteolol,
metoprolol, nadolol, penbutolol, pindolol, propranolol, timolol,
amlodipine, diltiazem, felodipine, isradipine, nicardipine,
nifedipine, nisoldipine, verapamil, amiloride, benzthiazide,
chlorothiazide, chlorthalidone, furosemide, hydrochlorothiazide,
indapamide, metolazone, polythiazide, spironolactone, torsemide,
trichlormethiazide, hydralazine, nitroglycerin, sodium
nitroprusside, clevidipine or minoxidil. In some embodiments, the
antihypertensive agent is labetalol, metoprolol, hydralazine,
nitroglycerin, nicardipine, sodium nitroprusside or
clevidipine.
[0277] The present disclosure also provides methods of reducing,
preventing or treating a cardiovascular event or disease in a
subject comprising administering to the subject an anti-IL-1.beta.
binding antibody or binding fragment thereof in combination with
(e.g., before, during or after) a medical or surgical intervention.
Such antibodies may be administered in therapeutically effective
amounts. Such interventions may be therapeutically effective. In
some embodiments, a medical intervention is an active agent, such
as a drug or a biologic, including, for example, any one or more of
the active agents described herein. In some embodiments, a medical
intervention is an out-patient medical treatment or procedure. In
some embodiments, a medical intervention is an in-patient
hospitalization. In some embodiments, a surgical intervention is a
revascularization procedure, including, for example, any one or
more of the revascularization procedures described herein. In some
embodiments, a surgical intervention involves a heart valve repair
or replacement, coronary bypass surgery, heart transplant or heart
pump. In some embodiments, a surgical intervention involves a
biventricular cardiac pacemaker, internal cardiac defibrillator
(ICD) or myectomy. In some embodiments, a medical intervention is
smoking cessation medication or smoking cessation counseling.
[0278] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of at least one
risk factor for cardiovascular disease, comprising (a) identifying,
diagnosing or selecting the subject with the history of at least
one risk factor for cardiovascular disease and (b) administering to
the subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof, and
wherein the cardiovascular event is myocardial infarction, stroke,
cardiovascular death, congestive heart failure, cardiac arrest,
acute coronary syndrome, angina, or a revascularization
procedure.
[0279] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of a previous
cardiovascular event, comprising (a) identifying, diagnosing or
selecting the subject with the history of the previous
cardiovascular event and (b) administering to the subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof, and wherein the
cardiovascular event is myocardial infarction, stroke, acute
coronary syndrome, angina or a revascularization procedure.
[0280] The present disclosure also provides methods of reducing
mortality following a cardiovascular event in a subject, comprising
(a) identifying, diagnosing or selecting the subject having the
cardiovascular event and (b) administering to the subject a
therapeutically effective amount of an anti-IL-1.beta. binding
antibody or binding fragment thereof.
[0281] The present disclosure also provides methods of reducing a
cardiovascular event in a subject with a history of at least one
risk factor for cardiovascular disease, comprising (a) identifying,
diagnosing or selecting the subject with the history of at least
one risk factor for cardiovascular disease and (b) administering to
the subject a therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof, and
wherein the risk factor is not Type 2 diabetes, obesity,
hyperglycemia, dyslipidemia, hyperlipidemia, chronic renal failure,
high blood glucose, chronic kidney disease, hypertension,
atherosclerosis or metabolic syndrome.
[0282] The present disclosure also provides methods of treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising (a) identifying, diagnosing
or selecting the subject with the cardiovascular event and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
at least one other pharmaceutical composition comprising an active
agent other than an IL-1.beta. antibody or fragment.
[0283] The present disclosure also provides methods for treating a
cardiovascular event in a subject, wherein the cardiovascular event
is myocardial infarction, stroke, congestive heart failure, acute
coronary syndrome or angina, comprising (a) identifying, diagnosing
or selecting the subject with the cardiovascular event and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
a revascularization procedure.
[0284] The present disclosure also provides methods of reducing
restenosis in a subject following a revascularization procedure,
comprising (a) identifying, diagnosing or selecting the subject
with the revascularization procedure and (b) administering to the
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof.
[0285] The present disclosure also provides methods of treating
acute hypertension in a subject comprising (a) identifying,
diagnosing or selecting the subject with acute hypertension and (b)
administering to the subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof and
one or more antihypertensive agents. In some embodiments, the
hypertension is manifested as a blood pressure of greater than or
equal to 180/110 mm Hg. In some other embodiments, the hypertension
is mild-to-moderate, with systolic blood pressure (SBP) of 140 to
180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mm
Hg.
[0286] The present disclosure also provides methods of reducing in
a subject with a history of recent myocardial infarction (MI),
recent stroke, or established peripheral arterial disease, the rate
of a combined endpoint of new ischemic stroke (fatal or not), new
MI (fatal or not), and other vascular death, comprising
administering to said subject a therapeutically effective amount of
an anti-IL-1.beta. binding antibody or binding fragment thereof. In
some embodiments, the subject has acute coronary syndrome without
ST segment elevation (e.g., unstable angina or non-Q-wave
myocardial infarction). In some embodiments, the anti-IL-1.beta.
binding antibody or binding fragment thereof is administered
initially within 12, 24, 48 or 72 hours of onset of the most recent
episode of chest pain or symptoms consistent with ischemia. In some
embodiments, the subject has either ECG changes compatible with new
ischemia (e.g., without ST segment elevation) or elevated cardiac
enzymes or troponin I or T to at least twice the upper limit of
normal.
[0287] The present disclosure also provides methods of reducing
atherothrombotic events in a subject with a history of recent
myocardial infarction (MI), recent stroke, or established
peripheral arterial disease, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof. In some embodiments,
the subject has acute coronary syndrome without ST segment
elevation (e.g., unstable angina or non-Q-wave myocardial
infarction). In some embodiments, the anti-IL-1.beta. binding
antibody or binding fragment thereof is administered initially
within 72 hrs, or preferably 48 hours, or more preferably 24, 12, 6
or 3 hours of onset of the most recent episode of chest pain or
symptoms consistent with ischemia. In some embodiments, the subject
has either ECG changes compatible with new ischemia (e.g., without
ST segment elevation) or elevated cardiac enzymes or troponin I or
T to at least twice the upper limit of normal.
[0288] The present disclosure also provides methods of reducing in
subjects with ST-segment elevation acute myocardial infarction, the
rate of death from any cause and the rate of a combined endpoint of
death, re-infarction or stroke, comprising administering to said
subject a therapeutically effective amount of an anti-IL-1.beta.
binding antibody or binding fragment thereof. In some embodiments,
the anti-IL-1.beta. binding antibody or binding fragment thereof is
administered initially within 72 hrs, or preferably 48 hours, or
more preferably 24, 12, 6 or 3 hours of the subject presenting with
symptoms of myocardial infarction.
[0289] In some embodiments of any of the methods described above,
the subject is a patient with cardiovascular disease, including,
for example, acute cardiovascular disease or chronic cardiovascular
disease.
[0290] In some embodiments of any of the methods described above,
administering said therapeutically effective amount of an
anti-IL-1.beta. binding antibody or binding fragment thereof is
sufficient to achieve a decrease in CRP levels.
[0291] The present disclosure also provides uses of an
anti-IL-1.beta. binding antibody or binding fragment thereof which
has a lower IC.sub.50 than an IL-1.beta. receptor antagonist in a
human whole blood IL-1.beta. inhibition assay that measures
IL-1.beta. induced production of IL-8, in the manufacture of a
composition for use in the reduction, prevention or treatment of a
cardiac event or a cardiovascular disease.
[0292] The disclosure also provides that a reduction of a
cardiovascular event (e.g., delaying time to event, reducing
likelihood or risk of event, preventing an event, reducing severity
of event, reducing time to recovery) may be evaluated in subjects
over a period of 2 or more years, 3 or more years, 4 or more years,
or 5 or more years following the cardiovascular event and/or
initial administration of the IL-1.beta. binding antibody or
binding fragment thereof.
[0293] In addition, the disclosure further provides that a
therapeutically effective amount of anti-IL-1.beta. binding
antibody or binding fragment thereof may also be sufficient to
achieve a decrease in C-reactive protein (CRP) levels. The
reduction in CRP levels is readily measured using standard assays
(e.g., high-sensitivity CRP, ultra-sensitive CRP). As provided by
the methods disclosed herein, the decrease in C-reactive protein
levels may, for example, be a decrease of .gtoreq.0.2, .gtoreq.0.4,
.gtoreq.0.6, .gtoreq.0.8, .gtoreq.1.0, .gtoreq.1.4, .gtoreq.1.8,
.gtoreq.2.2, .gtoreq.2.6, .gtoreq.3.0 mg/L from pre-treatment
levels. Alternatively, the decrease in C-reactive protein levels
may, for example, be a decrease of >20%, >30%, >40%,
>50%, >60%, >70%, >80%, >90%, >95% from
pre-treatment levels.
[0294] Alternatively, or in addition, subjects treated as disclosed
herein may experience a measurable improvement in lipid profile
(e.g., decrease in serum lipids, change in ratio of HDL and LDL).
Such measurements of serum lipids and/or lipid profile may include,
for example a decrease in cholesterol, a decrease in low-density
lipoprotein cholesterol (LDL), a decrease in very-low-density
lipoprotein cholesterol (VLDL), a decrease in triglycerides, a
decrease in free fatty acids, a decrease in apolipoprotein B (Apo
B), an increase in high-density lipoprotein cholesterol (HDL),
maintaining the level of high-density lipoprotein cholesterol (HDL)
compared to pre-treatment level, and/or an increase in
apolipoprotein A (Apo A). Measurements may be using standard
techniques known in the art. For example, a decrease in the level
of cholesterol (e.g., total cholesterol) may be a decrease of at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or
more from the pre-treatment level. A decrease in the level of
low-density lipoprotein cholesterol may be a decrease of at least
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more
from the pre-treatment level. A decrease in the triglyceride level
in the blood of the subject may be a decrease of at least 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more
from the pre-treatment level. A decrease in the level of free fatty
acids may be a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, or more from the pre-treatment level.
An increase in the level of high-density lipoprotein cholesterol
may be an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%,
12%, 14%, 16%, or more from the pre-treatment level.
[0295] The aforementioned diagnoses and measurements may be made
using standard medical practices known in the art and/or any of a
variety of standard assays known in the art, such as for example
assays published in Chernecky C C, Berger B J, eds. (2004).
Laboratory Tests and Diagnostic Procedures, 4th ed. Philadelphia:
Saunders; Fischbach F T, Dunning M B III, eds. (2004). Manual of
Laboratory and Diagnostic Tests, 7th ed. Philadelphia: Lippincott
Williams and Wilkins; Genest J, et al. (2003). Recommendations for
the management of dyslipidemia and the prevention of cardiovascular
disease: Summary of the 2003 update. Canadian Medical Association
Journal, 169(9): 921-924. Also available online:
http://www.cmaj.ca/cgi/content/full/169/9/921/DC1; Handbook of
Diagnostic Tests (2003). 3rd ed. Philadelphia: Lippincott Williams
and Wilkins; and Pagana K D, Pagana T J (2002). Mosby's Manual of
Diagnostic and Laboratory Tests, 2nd ed. St. Louis: Mosby.
Dosing
[0296] Anti-IL-1.beta. binding antibodies or binding fragments
thereof for use in any and/or all of the aforementioned methods may
be administered in one or more doses (e.g., initial dose and one or
more subsequent doses). In some embodiments, the anti-IL-1.beta.
binding antibody or binding fragment thereof is administered in one
or more doses of 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or
less, or 2 mg/kg or less of antibody or fragment. In some
embodiments, the anti-IL-1.beta. binding antibody or binding
fragment thereof is administered in one or more doses of 1 mg/kg or
less, one or more doses of 0.5 mg/kg or less, one or more doses of
0.3 mg/kg or less, one or more doses of 0.1 mg/kg or less, or one
or more doses of 0.03 mg/kg or less of antibody or fragment. In
some of the aforementioned embodiments, the one or more doses are
at least 0.01 mg/kg of anti-IL-1.beta. binding antibody or binding
fragment thereof. In some embodiments, the anti-IL-1.beta. binding
antibody or binding fragment thereof is administered in one or more
doses of about 0.01 mg/kg to 1 mg/kg, about 0.03 mg/kg to 1 mg/kg,
about 0.01 mg/kg to 0.3 mg/kg, or about 0.1 mg/kg to 0.3 mg/kg. In
some embodiments, the anti-IL-1.beta. binding antibody or binding
fragment thereof is administered in one or more doses of about
0.001 mg/kg to 0.3 mg/kg, about 0.001 mg/kg to 0.1 mg/kg, about
0.001 mg/kg to 0.03 mg/kg or about 0.001 mg/kg to 0.01 mg/kg.
[0297] In other embodiments, the initial dose and one or more
subsequent doses of anti-IL-1.beta. binding antibody or binding
fragment thereof are each from about 0.01 mg/kg to about 10 mg/kg
of antibody, from about 0.05 to about 5 mg/kg of antibody, from
about 0.05 mg/kg to about 3 mg/kg of antibody, from about 0.1 mg/kg
to about 3 mg/kg of antibody, from about 0.1 mg/kg to about 1 mg/kg
of antibody, from about 0.1 mg/kg to about 0.5 mg/kg of antibody,
from about 0.3 mg/kg to about 5 mg/kg of antibody, from about 0.3
mg/kg to about 3 mg/kg of antibody, from about 0.3 mg/kg to about 1
mg/kg of antibody, from about 0.5 mg/kg to about 5 mg/kg of
antibody, from about 0.5 mg/kg to about 3 mg/kg of antibody, from
about 0.5 mg/kg to about 1 mg/kg of antibody, from about 1 mg/kg to
about 5 mg/kg of antibody, or from about 1 mg/kg to about 3 mg/kg
of antibody. In certain embodiments, two or more, three or more,
four or more, five or more, six or more, seven or more, eight or
more, nine or more, ten or more or eleven or more subsequent doses
of the antibody are administered. The aforementioned dosage amounts
refer to mg (antibody or fragment)/kg (weight of the individual to
be treated).
[0298] Anti-IL-1.beta. binding antibodies or binding fragment
thereof for use in any and/or all of the aforementioned methods may
be administered as a fixed dose, independent of a dose per subject
weight ratio. In some embodiments, the anti-IL-1.beta. binding
antibody or binding fragment thereof is administered in one or more
fixed doses of 1000 mg or less, 500 mg or less, or 250 mg or less
of antibody or fragment. In some embodiments, the anti-IL-13
binding antibody or binding fragment thereof is administered in one
or more fixed doses of 100 mg or less, 25 mg or less, or 10 mg or
less of antibody or fragment. In some embodiments, the
anti-IL-1.beta. binding antibody or binding fragment thereof is
administered in one or more doses of at least 0.5 mg of antibody or
fragment. In some embodiments, the anti-IL-1.beta. binding antibody
or binding fragment thereof is administered in one or more doses of
at least 1 mg of antibody or fragment. In some embodiments, the
anti-IL-1.beta. binding antibody or binding fragment thereof is
administered in one or more doses of at least 10 mg of antibody or
fragment. In some embodiments, the anti-IL-1.beta. binding antibody
or binding fragment thereof is administered in one or more doses of
1 mg to 100 mg of antibody or fragment.
[0299] In certain embodiments, the fixed dose of anti-IL-1.beta.
binding antibody or binding fragment thereof is from about 1 mg to
about 10 mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg,
about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 25
mg to about 50 mg, about 25 mg to about 100 mg, about 50 mg to
about 100 mg, about 50 mg to about 150 mg, about 100 mg to about
150 mg, about 100 mg to about 200 mg, about 150 mg to about 200 mg,
about 150 mg to about 250 mg, about 200 mg to about 250 mg, about
200 mg to about 300 mg, about 250 mg to about 300 mg, about 250 mg
to about 500 mg, about 300 mg to about 400 mg, about 400 mg to
about 500 mg, about 400 mg to about 600 mg, about 500 mg to about
750 mg, about 600 mg to about 750 mg, about 700 mg to about 800 mg,
or about 750 mg to about 1000 mg. In other embodiments, the fixed
dose anti-IL-1.beta. binding antibody or binding fragment thereof
is from about 1 mg to about 10 mg, about 1 mg to about 25 mg, about
10 mg to about 25 mg, about 10 mg to about 100 mg, about 25 mg to
about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 150
mg, about 150 mg to about 200 mg, or about 200 mg to about 250
mg.
[0300] In some embodiments of any and/or all of the aforementioned
methods, the fixed dose of anti-IL-1.beta. binding antibody or
binding fragment thereof is administered using a pre-filled syringe
or delivery device.
[0301] In some embodiments of any and/or all of the aforementioned
methods, the anti-IL-1.beta. binding antibody or binding fragment
thereof is administered by subcutaneous, intravenous or
intramuscular injection.
[0302] In some embodiments of any and/or all of the aforementioned
methods, administration of an initial dose of anti-IL-1.beta.
binding antibody or binding fragment thereof is followed by the
administration of one or more subsequent doses. In some
embodiments, the initial dose and one or more subsequent doses are
administered at an interval of about once every week to about once
every 12 months. In some embodiments, the initial dose and one or
more subsequent doses are administered at an interval of about once
every two weeks to about once every 6 months. In some embodiments,
the initial dose and one or more subsequent doses are administered
at an interval of about once every month to about once every 6
months. In some embodiments, the initial dose and one or more
subsequent doses are administered at an interval of about once
every month to about once every 3 months. In some embodiments, the
initial dose and one or more subsequent doses are administered at
an interval of about once every 3 months to about once every 6
months.
[0303] The disclosure also provides dosing regimens for use in any
and/or all of the aforementioned methods, wherein the dosing
regimens comprise more than one dosing interval for administration
of an IL-1.beta. binding antibody or binding fragment thereof. In
some embodiments, the dosage regimen comprises at least two (e.g.,
two, three, four, five, six) different dosing intervals for
administration of the IL-1.beta. antibody or fragment thereof. In
some embodiments, the dosage regimen comprises two different dosing
intervals for administration of the IL-1.beta. antibody or fragment
thereof. In some embodiments, the dosing regimen comprises two
different dosing intervals for administration of the IL-1.beta.
binding antibody or binding fragment thereof, wherein a first
dosing interval comprises administration of one or more doses of
the IL-1 f3 antibody or fragment thereof and a second dosing
interval comprises administration of one or more doses of the
IL-1.beta. antibody or fragment thereof, and wherein the first
dosing interval is shorter in time than the second dosing interval.
For example, the first dosing interval may be days or weeks, and
the second dosing interval may be months. In some embodiments, the
first dosing interval is about 5 days to about 28 days, about 7
days to about 21 days, about 12 days to about 16 days, or about 14
days. In some embodiments, the second dosing interval is about 1
month to about 3 months, about 1 month to about 2 months, or about
1 month. In some embodiments, the first dosing interval is about 7
days and the second dosing interval is about 1 month.
[0304] In some embodiments, administration of an initial dose of
anti-IL-1.beta. binding antibody or binding fragment thereof is
followed by administration of one or more subsequent doses, and
wherein the dosing intervals between administration of the initial
dose and a second dose, and the second dose and a third dose are
about 7 days to about 21 days, and wherein the dosing intervals
between administration of subsequent doses is about 1 month to
about 3 months. In some embodiments, the dosing intervals between
administration of the initial dose and a second dose, and the
second dose and a third dose are about 12 to 16 days, and the
dosing intervals between administration of subsequent doses is
about 1 month to about 2 months. In some embodiments, the dosing
intervals between administration of the initial dose and a second
dose, and the second dose and a third dose are about 14 days, and
the dosing intervals between administration of subsequent doses is
about 1 month. In some embodiments of any and/or all of the
aforementioned methods, the anti-IL-1.beta. binding antibody or
binding fragment thereof is administered to a subject such that the
interval between doses is a time sufficient to maintain a plasma
concentration of said antibody or antibody fragment in the subject
at a level of at least about 0.1 ug/mL. In some embodiments, the
anti-IL-1.beta. binding antibody or binding fragment thereof is
administered to a subject such that the interval between doses is a
time sufficient to maintain a plasma concentration of said antibody
or antibody fragment in the subject at a level of at least about
0.3 ug/mL. In some embodiments, the anti-IL-1.beta. binding
antibody or binding fragment thereof is administered to a subject
such that the interval between doses is a time sufficient to
maintain a plasma concentration of said antibody or antibody
fragment in the subject at a level of at least about 1 ug/mL. In
some embodiments, these plasma concentration values refer to values
obtained for an individual that is treated with the antibody of
fragment in accordance with the disclosure herein.
[0305] In some embodiments of any and/or all of the aforementioned
methods, administration of an initial dose of the anti-IL-1.beta.
binding antibody or binding fragment thereof is followed by the
administration of one or more subsequent doses, and wherein said
one or more subsequent doses are in an amount that is approximately
the same or less than the initial dose.
[0306] In some embodiments of any and/or all of the aforementioned
methods, administration of an initial dose of the anti-IL-1.beta.
binding antibody or binding fragment thereof is followed by the
administration of one or more subsequent doses, and wherein at
least one of the subsequent doses is in an amount that is more than
the initial dose.
[0307] In some embodiments of any and/or all of the aforementioned
methods, the anti-IL-1.beta. binding antibody or binding fragment
thereof has a lower IC.sub.50 than an IL-1.beta. receptor
antagonist in a human whole blood IL-1.beta. inhibition assay that
measures IL-1.beta. induced production of IL-8. In some
embodiments, the IL-1.beta. receptor antagonist is anakinra.
[0308] In some embodiments of any and/or all of the aforementioned
methods, an anti-IL-1.beta. binding antibody or binding fragment is
administered, wherein administration of an initial dose of the
antibody or antibody fragment is followed by the administration of
one or more subsequent doses, and wherein the plasma concentration
of said antibody or antibody fragment in the human is permitted to
decrease below a level of about 0.1 ug/mL for a period of time
greater than about 1 week and less than about 6 months between
administrations during a course of treatment with said initial dose
and one or more subsequent doses. In some embodiments, the plasma
concentration of said antibody or antibody fragment is permitted to
decrease below a level of about 0.07 ug/mL, about 0.05 ug/mL, about
0.03 ug/mL or about 0.01 ug/mL for a period of time greater than
about 1 week and less than about 5 months, about 4 months, about 3
months, about 2 months, about 1 month, about 3 weeks, or about 2
weeks between administrations. In some embodiments, the plasma
concentration values refer to values obtained for an individual
that is treated with the antibody of fragment in accordance with
the disclosure herein.
Combinations
[0309] The disclosure also provides that pharmaceutical
compositions comprising one or more other active agents may be
administered in conjunction with (e.g., separately from) the
IL-1.beta. binding antibodies or fragments, and such
administrations may be performed at the same point or different
points in time, such as for example the same or different days.
Administration of the other active agents may be according to
standard medical practices known in the art (e.g., current standard
of care), or the administration may be modified (e.g., longer
intervals, smaller dosages, delayed initiation) when used in
conjunction with administration of IL-1.beta. binding antibodies or
fragments, such as disclosed herein. The active agents set forth
below are exemplary and not intended to be limiting. combinations
can also include more than one additional agent, e.g., two or three
additional agents.
[0310] Anti-IL-1.beta. antibodies or fragments thereof administered
to a subject in as disclosed herein may be administered in
combination with treatment with at least one additional active
agent, such as for example any of the active agents provided
herein. In one embodiment, treatment with the at least one active
agent is maintained. In another embodiment, treatment with the at
least one active agent is reduced or discontinued (e.g., when the
subject is stable), while treatment with the anti-IL-1.beta.
antibody or fragment is maintained at a constant dosing regimen. In
another embodiment, treatment with the at least one active agent is
reduced or discontinued (e.g., when the subject is stable), and
treatment with the anti-IL-1.beta. antibody or fragment is reduced
(e.g., lower dose, less frequent dosing, shorter treatment
regimen). In another embodiment, treatment with the at least one
active agent is is reduced or discontinued (e.g., when the subject
is stable), and treatment with the anti-IL-1.beta. antibody or
fragment is increased (e.g., higher dose, more frequent dosing,
longer treatment regimen). In yet another embodiment, treatment
with the at least one active agent is maintained and treatment with
the anti-IL-1.beta. antibody or fragment is reduced or discontinued
(e.g., lower dose, less frequent dosing, shorter treatment
regimen). In yet another embodiment, treatment with the at least
one active agent and treatment with the anti-IL-1.beta. antibody or
fragment are reduced or discontinued (e.g., lower dose, less
frequent dosing, shorter treatment regimen).
[0311] In some embodiments, any of the methods described above may
further comprise administering at least one other pharmaceutical
composition comprising an active agent other than an
anti-IL-1.beta. binding antibody or binding fragment thereof. In
some embodiments, the active agent of said at least one other
pharmaceutical composition is a cholesterol lowering agent. In some
embodiments, the active agent of said at least one other
pharmaceutical composition is a statin or an HMG-CoA reductase
inhibitor (e.g., lovastatin, pravastatin, simvastatin, fluvastatin,
atorvastatin, cerivastatin, mevastatin, pitavastatin, rosuvastatin
or mixtures thereof or mixtures with Ezetimibe, niacin, Amlodipine
Besylate). In some embodiments, the active agent of said at least
one other pharmaceutical composition is a calcium channel blocker
(e.g., amlodipine, diltiazem, nifedipine, nicardipine, verapamil)
or a beta blocker (e.g., esmolol, metoprolol, nadolol, penbutolol).
In some embodiments, the active agent of said at least one other
pharmaceutical composition is an antihypertensive (e.g., labetalol,
metoprolol, hydralazine, nitroglycerin, nicardipine, sodium
nitroprusside, clevidipine), a diuretic (e.g., a thiazide diuretic,
chlorthalidone, furosemide, hydrochlorothiazide, indapamide,
metolazone, amiloride hydrochloride, spironolactone, triamterene)
or aspirin. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an angiotensin-converting
enzyme (ACE) inhibitor (e.g. ramipril, ramiprilat, captopril,
lisinopril) or an angiotensin II receptor blocker (e.g., losartan,
olmesartan, valsartan). In some embodiments, the active agent of
said at least one other pharmaceutical composition is a
vasodilator. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an anticoagulant (e.g.,
acenocoumarol, phenprocoumon, warfarin heparin, low molecular
weight heparin) or inhibitor of platelet aggregation (e.g.,
clopidogrel, ticlopidine, cilostazol, dipyridamole, eptifibatide,
aspirin, abciximab, eptifibatide, tirofiban). In some embodiments,
the active agent of said at least one other pharmaceutical
composition is a thrombolytic (e.g., streptokinase, urokinase,
alteplase, reteplase, tenecteplase). In some embodiments, the
active agent of said at least one other pharmaceutical composition
is digitalis. In some embodiments, the active agent of said at
least one other pharmaceutical composition is digoxin or
nesiritide. In some embodiments, the active agent of said at least
one other pharmaceutical composition is oxygen. In some
embodiments, the active agent of said at least one other
pharmaceutical composition is a thrombin inhibitor (e.g., hirudin,
bivalirudin). In some embodiments, the active agent of said at
least one other pharmaceutical composition is a nitrate (e.g.,
glyceryl trinitrate (GTN)/nitroglycerin, isosorbide dinitrate,
isosorbide mononitrate). In some embodiments, the active agent of
said at least one other pharmaceutical composition is an analgesic
(e.g., morphine sulfate). In some embodiments, the active agent of
said at least one other pharmaceutical composition is a renin
inhibitor. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an endothelin A receptor
inhibitor. In some embodiments, the active agent of said at least
one other pharmaceutical composition is an aldosterone
inhibitor.
[0312] In another embodiment, the use of the IL-1.beta. antibodies
or binding fragments is contemplated in the manufacture of a
medicament for treating or preventing a disease or condition as
disclosed herein (e.g., for the reduction, prevention or treatment
of cardiovascular events and/or cardiovascular diseases). In any of
the uses, the medicament can be coordinated with treatment using a
second active agent.
[0313] In yet another aspect of the present disclosure, an article
of manufacture is provided, comprising a container, a composition
within the container comprising an anti-IL-1.beta. antibody or
fragment thereof, and a package insert containing instructions to
administer the antibody or fragment to a subject (e.g., human) as
disclosed herein (e.g., for the reduction, prevention or treatment
of cardiovascular events and/or cardiovascular diseases). In one
embodiment, the container further comprises a pharmaceutically
suitable carrier, excipient or diluent. In a related embodiment,
the composition within the container further comprises a second
active agent.
[0314] Kits are also contemplated by the disclosure. In one
embodiment, a kit comprises a therapeutically or prophylactically
effective amount of an anti-IL-1.beta. antibody or fragment
thereof, packaged in a container, such as a vial or bottle, and
further comprising a label attached to or packaged with the
container, the label describing the contents of the container and
providing indications and/or instructions regarding use of the
contents of the container as disclosed herein (e.g., for the
reduction, prevention or treatment of cardiovascular events and/or
cardiovascular diseases). In one embodiment, the container further
comprises a pharmaceutically suitable carrier, excipient or
diluent. In a related embodiment, the container further contains a
second active agent.
[0315] In one embodiment, the article of manufacture, kit or
medicament is for the treatment or prevention of a disease or
condition in a subject (e.g., human) as disclosed herein (e.g., for
the reduction, prevention or treatment of cardiovascular events
and/or cardiovascular diseases). In another embodiment, the
instructions of a package insert of an article of manufacture or
label of a kit comprise instructions for administration of the
antibody or fragment according to any of the aforementioned dose
amounts and/or dosing regiments. In yet another embodiment, the
container of kit or article of manufacture is a pre-filled
syringe.
EXAMPLES
[0316] The following examples are intended merely to further
illustrate the practice of the present disclosure, but should not
be construed as in any way limiting its scope. The disclosures of
all patent and scientific literatures cited within are hereby
expressly incorporated in their entirety by reference.
Example 1
Administration of an IL-43 Antibody to Human Subjects
[0317] IL-1.beta. binding antibodies or binding fragments thereof
may be administered to a subject for the aforementioned uses.
Specifically, in one example, an IL-1.beta. antibody designated AB7
(described above) was administered to human subjects to evaluate
safety, pharmacokinetics, and in vivo biological activity. A
double-blind, placebo controlled clinical study was performed in
human subjects with Type 2 diabetes. Groups of subjects were given
the antibody by either the intravenous (IV) or subcutaneous (SC)
route, and either as a single doses or as multiple doses over a
period of time.
[0318] The treatment groups and numbers of subjects for the study
are shown in the following table for a single dose by the IV route
of administration.
TABLE-US-00005 IV Route Antibody Placebo Group # Subjects Dose #
Subjects 1 5 0.01 mg/kg 1 2 5 0.03 mg/kg 1 3 5 0.1 mg/kg 1 4 5 0.3
mg/kg 1 5 5 1.0 mg/kg 1 6 5 3.0 me/kg 1
[0319] Similarly, treatment groups and numbers of subjects are
shown in the following table for single and multiple (3 times,
biweekly) doses by the SC route of administration.
TABLE-US-00006 SC Route Antibody Placebo Group # Subjects Dose #
Subjects Single Dose 1 5 0.03 mg/kg 1 2 5 0.1 mg/kg 1 3 5 0.3 mg/kg
1 Multi Dose 4 5 0.03 mg/kg 1 5 5 0.3 mg/kg 1
On study Day 1, antibody or placebo was administered via constant
rate IV infusion or SC injection (e.g., anterior abdomen, arm,
thigh). Safety assessments, including the recording of adverse
events, physical examinations, vital signs, and clinical laboratory
tests (e.g., blood chemistry, hematology, urinalysis) were
conducted using standard medical practices known in the art. Blood
samples were collected pre-dose administration and at multiple time
periods post-administration to assess various parameters, including
C-reactive protein.
[0320] Alternatively or in addition, study groups also may be
included to evaluate, for example, the administration of additional
numbers of subsequent doses at the same or longer intervals (e.g.,
monthly interval), alternative dose amounts, and/or increased group
sizes.
Example 2
Pharmacokinetics of an IL-1.beta. Antibody in Human Subjects
[0321] Samples are obtained for pharmacokinetic analysis at days 0,
1, 2, 3, 4, 7, 9.+-.1, 11.+-.1, 14.+-.1, 21.+-.2, 28.+-.2, 42.+-.3,
and 56.+-.3. Interim analysis of pharmacokinetic data following IV
administration of a single dose of antibody at the 0.01, 0.03, 0.1,
0.3, or 1.0 mg/kg dose levels showed serum concentration-time
profiles with a terminal half-life of 22 days, clearance of 2.54
mL/day/kg and volume of distribution of the central compartment of
41.3 mL/kg, very similar to serum volume (FIG. 1).
[0322] Similarly, samples were analyzed for the single dose SC
administration groups. As shown in FIG. 2, administration of the
antibody at 0.03, 0.1 and 0.3 mg/kg dose levels yielded profiles
with a terminal half-life of 22.7 days, clearance of 2.4 mL/day/kg
and volume distribution of the central compartment of 40.7
mL/kg.
Example 3
Effect of an IL-1.beta. Antibody on CRP in Human Subjects
[0323] C-reactive protein also was measured in serum at the same
time points as the PK samples. A single dose of antibody reduced
ultrasensitive C-reactive protein (usCRP) levels in each of the
antibody treatment dose groups compared to placebo. As shown in
FIG. 3, at 28 days after a single IV dose of antibody, the median
percent reductions in usCRP were 33, 46, 47, 36, and 26 for the
0.01, 0.03, 0.1, 0.3, and 1.0 mg/kg dose groups, respectively,
compared to 4 percent for placebo.
Example 4
Evaluation of an IL-1.beta. Antibody in a Cardiovascular Event
Model (Acute Myocardial Infarction)
[0324] To determine the cardioprotective effect (e.g., inhibiting
adverse cardiac remodeling) of an IL-1.beta. antibody or binding
fragment thereof, a rodent model of acute myocardial infarction
(MI) may be used (see for example, Wang et al., 2006, Tex. Heart
Inst. J. 33:290-293; Salloum et al., 2009, Cardiovasc. Drugs Ther.
23:129-135). Improvements in measurements of heart function, such
as for example in the MI model, are related to the chance of a
subsequent cardiovascular event (e.g., congestive heart failure).
Outbred mice (e.g., Institute of Cancer Research mice) and/or rats
(e.g., Wistar rats) are used in the rodent MI model. Prior to
surgery, the animals are evaluated by transthoracic
echocardiography (TTE), for example, using a Vevo770 imaging system
(VisualSonics, Toronto, Canada) or Acuson C256, to obtain
measurements for the following parameters: [0325] Left ventricular
end-diastolic diameter (LVEDD) [0326] Left ventricular end-systolic
diameter (LVESD) [0327] Anterior wall diastolic thickness (AWDT)
[0328] Posterior wall diastolic thickness (PWDT) [0329] Anterior
wall systolic thickness (AWST) [0330] Posterior wall systolic
thickness (PWST) Left ventricular fractional-shortening (FS) is
calculated as:
[0330] (LVEDD-LVESD)/LVEDD.times.100
[0331] Adult animals under anesthesia are subjected to coronary
artery ligation. After thoracotomy to expose the heart, MI is
induced by ligation of the proximal left descending coronary artery
using a silk ligature placed around the vessel. Control animals
(sham operation) are subjected to the same surgical procedure, but
without the coronary ligation (see following table). Animals that
die during or immediately after the postoperative period are not
included in the analyses.
TABLE-US-00007 TTE Surgery Antibody Txt Repeat TTE Group 1 Yes Yes,
MI induction High dose (t = 0) 24 hr, 7 d, 14 d Group 2 Yes Yes, MI
induction Low dose (t = 0) 24 hr, 7 d, 14 d Group 3 Yes Yes, MI
induction High dose (24 hr) 24 hr, 7 d, 14 d Group 4 Yes Yes, MI
induction Low dose (24 hr) 24 hr, 7 d, 14 d Group 3 Yes Yes, MI
induction Placebo 24 hr, 7 d, 14 d Group 4 Yes Yes, Sham N/A 24 hr,
7 d, 14 d operation
[0332] Animals then receive either the treatment antibody or
placebo (e.g., control antibody) administered intraperitoneally or
intravenously at one or more pre-determined times during and/or
following ischemia. For example, in one group the antibody is
administered during ischemia (t=0) and in another group, the
antibody is administered 24 hours after ischemia.
[0333] The animals are observed and numbers of deaths during the
study period are recorded. The remaining animals again are
evaluated by TTE at pre-determined post-treatment days (e.g., 24
hr, Day 7, Day 14). Systolic BP also may be measured in conscious
awake awake, for example, using a noninvasive computerized
tail-cuff system (BP-2000, Visitech Systems), which has been found
to correlate closely with direct intraarterial measurement of BP.
Animals are sacrificed, blood collected for serum, and the infarct
area (size) determined. After removal, the heart is subjected to
staining with Evans blue dye or 0.5% nitroblue tetrazolium (NBT),
rinsed with saline and photographed to determine infarct size. The
tissue is then fixed in 4% paraformaldehyde, embedded in paraffin
and sectioned for staining with hematoxylin and eosin for
histologic evaluation of tissue damage. Alternatively or
additionally, tissue is fixed and sectioned to quantitate the level
of cardiomyocyte cell death (e.g., TUNEL to determine
apoptosis).
[0334] Alternatively, studies to evaluate the effect of an
IL-1.beta. antibody or fragment on heart function and/or adverse
cardiac remodeling (e.g., chance of a subsequent cardiovascular
event, such as for example, congestive heart failure) may be
performed in adult male out-bred ICR mice (e.g., Harlan
Laboratories (Indianapolis, Ind.)). CD-1 mice underwent
experimental myocardial infarction as previously described (Abbate
et al., 2008, Circulation 117:2670-2683). Mice were anesthetized
with pentobarbital (70 mg/kg, IP), intubated orotracheally, and
ventilated on a positive-pressure ventilator. Left thoracotomy was
performed at the fourth intercostal space and the heart was exposed
by stripping the pericardium. The left descending coronary artery
was then identified with a surgical microscope (Leica F40) and
ligated with a 7.0 silk ligature. A group of 4 mice underwent sham
operation as previously described (Abbate, ibid). After surgery,
mice were randomly assigned to treatment with the anti-IL-1.beta.
antibody XMA052 MG1K, administered intraperitoneally (0.05 mg/kg,
0.5 mg/kg, 5 mg/kg doses) or a control IgG (n=6 per group)
immediately after surgery and then again 7 days later. The effect
of pretreatment with an additional dose of the antibody (0.5 mg/kg)
48 hours prior to surgery also was tested.
[0335] All mice underwent transthoracic echocardiography before
surgery and at 7, 14 and 28 days after coronary ligation. Doppler
echocardiography was performed with the Vevo770 imaging system
(VisualSonics Inc, Toronto, Ontario, Canada) and a 30-MHz probe.
The heart was first imaged in the 2-dimensional mode in the
parasternal and apical views and measurements were performed
according to the to the American Society of Echocardiography
recommendations (Gardin et al., 2002, J Am Soc Echocardiography
15:275-290). The left ventricular (LV) end-diastolic diameter
(LVEDD), LV end-systolic diameters (LVESD), anterior wall diastolic
thickness (AWDT), anterior wall systolic thickness (AWST),
posterior wall diastolic thickness (PWDT), and posterior wall
systolic thickness (PWST) were measured at M-mode. LV fractional
shortening (LVFS) was calculated as follows:
FS=(LVEDD-LVESD)/LVEDD.times.100. The number of akinetic segments
(which correlates with infarct size) was determined using a
17-segment map. An apical view was used to measure the ejection
time (ET), the time interval between the end of the transmitral A
wave and the following E wave (AE). The myocardial performance
index (MPI, or Tei index) was then computed (MPI=[AE-ET]/ET). The
tricuspidal annular plane systolic excursion was also measured as a
marker of right ventricular function. The investigator performing
and reading the echocardiogram was blinded to the treatment
allocation. The SPSS 11.0 (Chicago, Ill.) was used for the
statistical analysis, using ANOVA for multiple comparisons with
post-hoc T-test to explore between group differences. For
comparisons of interval changes between multiple groups, random
effects ANOVA for repeated-measures was used to determine the main
effect of time, group, and time-by-group interaction. Statistical
differences were considered significant if the P value was
<0.05.
[0336] Baseline echocardiographic values were similar in all
groups. As expected, significant increases in LV diameters (LVEDD
and LVESD) and a significant decrease in LVFS were observed as
early as 7 days after surgery compared to baseline in all groups
(except sham-operated mice). Mice receiving the XMA052 MG1K
antibody had smaller increase in LVEDD, LVESD and smaller decrease
in LVFS compared to controls (FIG. 4).
[0337] The number of akinetic segments, a surrogate for infarct
size, was 3.9.+-.0.4 in the saline-treated mice, and it was not
affected by treatment (FIG. 5). Accordingly, the anterior wall
(infarct) thickness was 0.52.+-.0.05 mm in the saline-treated and
unaffected by treatment (FIG. 5). The MPI or Tei index, a marker of
combined systolic and diastolic dysfunction and a surrogate marker
for heart failure related mortality, was significantly increased
after AMI (reflecting poor function) and preserved in the mice
treated with the XMA052 MG1K antibody (FIG. 5). Similarly, the
TAPSE, a marker of right ventricular function and a surrogate
marker for AMI related mortality, was significantly decreased after
AMI (reflecting poor function) and partially preserved in the mice
treated with the XMA052 MG1K antibody (FIG. 5). Thus, blockade of
IL-1.beta. using the antibody ameliorates cardiac enlargement and
dysfunction following AMI in the mouse, independent of infarct
size. Pretreatment with an additional dose of the XMA052 MG1K
antibody 48 hours prior to surgery offered no advantage over
treatment after surgery in this animal model (data not shown).
Example 5
Evaluation of an IL-1.beta. Antibody in a Cardiovascular Event
Model (Stroke)
[0338] Rodent (e.g., mice, rats) models of stoke may be used to
evaluate the effect of an IL-1.beta. antibody or binding fragment
thereof. For example, in one model, adult male Fischer rats are
used (see for example, Morales et al., 2008, Circulation
118:1450-1459). In another model, C57BL/6 mice are used (see for
example, Royl et al., 2009, Brain Res. 1265:148-157). Experiments
are performed in a randomized fashion by investigators blinded to
treatment groups. Permanent focal cerebral ischemia is induced by
occlusion of the middle cerebral artery (MCAO), such as by
cauterization or monofilament occlusion. Rats/mice in which the MCA
was exposed but not occluded serve as sham-operated controls.
[0339] Control animal groups and MCAO groups then receive the
treatment antibody or placebo (e.g., control antibody) administered
intraperitoneally or intravenously at one or more pre-determined
times following the procedure. For example, in one group the
antibody is administered immediately following the procedure and in
another group, the antibody is administered 24 hours later.
TABLE-US-00008 MCAO Antibody Txt In-life Tests MRI Histology Group
1 Sham N/A Yes Yes Yes Group 2 Yes Placebo Yes Yes Yes Group 3 Yes
Low dose (t = 0) Yes Yes Yes Group 4 Yes High dose (t = 0) Yes Yes
Yes Group 3 Yes Low dose (24 hr) Yes Yes Yes Group 4 Yes High dose
(24 hr) Yes Yes Yes
[0340] Animals are evaluated for survival and body weight changes,
as well as functional recovery (e.g., sensorimotor, behavioral
testing, such as pole test, wire hanging test and/or neurological
deficit score) and measurement of brain lesion size using MRI
during the in-life stage (e.g., T2-weighted MRI), followed by
histological examination (e.g., HE staining and GFAP staining of
coronal brain cryostat sections) post-sacrifice (e.g., at 4 weeks).
Additionally, a computer-assisted hemisphere volumetry may be
performed, based on T2-weighted MRI and HE-stained coronal brain
cryostat sections. Additional test groups may be evaluated to
determine the effect on acute reperfusion after MCAO by measuring
hemispheric cerebral blood flow with MRI (e.g., FAIR MRI).
Example 6
Evaluation of an IL-1.beta. Antibody in a Model of Peripheral
Vascular Disease
[0341] To determine the effect of an IL-1.beta. antibody or binding
fragment thereof on peripheral vascular disease, an animal model of
limb ischemia may be used (see for example, Park et al.,
Endocrinology 149:483-491, 2008). For example, limb ischemia is
induced in C57BL/6 male mice by the ligation of one femoral artery
in anesthetized animals. Mice in which the artery is exposed but
not ligated serve as sham-operated controls.
[0342] Control animal groups and artery ligation groups then
receive the treatment antibody or placebo (e.g., control antibody)
administered intraperitoneally or intravenously at one or more
pre-determined times following the procedure. For example, in one
group the antibody is administered immediately following the
procedure and in another group, the antibody is administered 24
hours later.
TABLE-US-00009 Ligation Antibody Txt LDPI Histology Group 1 Sham
N/A Yes Yes Group 2 Yes Placebo Yes Yes Group 3 Yes Low dose (t =
0) Yes Yes Group 4 Yes High dose (t = 0) Yes Yes Group 3 Yes Low
dose (24 hr) Yes Yes Group 4 Yes High dose (24 hr) Yes Yes
[0343] The blood flow in both hind legs is assessed with a laser
Doppler perfusion image (LDPI) analyzer (Moor Instruments, Devon,
UK), and the blood flow recovery is assessed by the ischemic limb
to normal limb ratio of blood flow. Serial blood flow measurements
by LDPI are observed at regular intervals (e.g., daily for two
weeks). Mice are euthanized and the ischemic hind limb isolated for
histological analysis.
[0344] After fixation with 4% paraformaldehyde, ischemic lower legs
are embedded in OCT compound and frozen for cryostat sectioning.
Tissue sections are stained with rat anti-mouse platelet EC
adhesion molecule-1 (PECAM-1) (PharMingen), mouse anti-.alpha.
smooth muscle actin (SMA) (Sigma), and rat anti-mouse CD45
(PharMingen), rabbit anti-cGKI (Calbiochem). To assess capillary
density and inflammation, four random fields on two different
sections (.apprxeq.=3 mm apart) from each mouse are photographed
and by computer-assisted analysis, capillary density is calculated
as the mean number of capillaries stained with PECAM-1 (endothelial
marker) or a SMA (vascular smooth muscle marker). The mean number
of infiltrating CD45-positive leukocytes is counted as the
assessment of inflammation.
Example 7
Evaluation of an IL-1.beta. Antibody in a Model of
Atherosclerosis
[0345] The effect of an IL-1.beta. antibody (XOMA 052) on
macrophage-induced cytokine production from endothelial cells and
smooth muscle cells was evaluated in a co-culture system. In this
model, THP-1 cells were pre-activated to a macrophage-like
phenotype with 200 nM PMA for 12 hours, washed once and added to
pre-plated human umbilical vein endothelial cells (HUVEC) or human
coronary artery smooth muscle cells (CASMC) at a ratio of (10:1;
10.sup.6 THP-1 and 10.sup.5 HUVEC or CASMC) in the presence or
absence of XOMA 052, as indicated. Alternatively, cells were
incubated with rhIL-1.beta. (R&D Systems) in the presence or
absence of XOMA 052, as indicated. After 48 hours, supernatants
were removed and assessed for cytokine or enzyme content by ELISA
(R&D Systems). All assays were performed in triplicate. The
data demonstrate that XOMA 052 inhibits the release of
IL-1.beta.-induced pro-inflammatory molecules, such as IL-6, IL-8,
MCP-1 and PAI-1 from endothelial cells (p<0.05, FIG. 6, left
panel). In addition, the data show that XOMA 052 inhibits the
release of IL-6 and IL-8 from smooth muscle cells, as well as
IL-1.beta.-driven MMP-3 and MMP-9 (p<0.05, FIG. 7, left panel).
Importantly, it was also observed that XOMA 052 potently reduces
the induction of these factors in the context of macrophage/EC or
macrophage/SMC co-culture systems (p<0.05, FIGS. 6 & 7,
right panel).
[0346] The ApoE knockout mouse is a well validated model of
atherosclerosis that follows a similar pattern of progression to
that of human. Male ApoE.sup.-/- mice on a C57BL/6 background were
fed an atherogenic diet for 16 weeks beginning at 6 weeks and
treated with an IL-1.beta. antibody, XMA052 MG1K (i.p., twice
weekly as indicated), control mouse IgG (i.p., twice weekly, 1.0
mg/kg; Jackson ImmunoResearch), or quinapril (subQ, 10 mg/kg,
daily) for the duration of the study. En face analysis was carried
out using Sudan IV staining as described previously (Calkin et al.,
2007, Atherosclerosis 195:17-22) and as follows. Aortas were
divided into arch, thoracic and abdominal aorta then cut
longitudinally. After pinning en face onto wax, aortas were
photographed and analyzed. Total and segmental plaque area was
quantified as percentage area visualized red as stained by Sudan
IV. Aortas were subsequently embedded in paraffin and sections cut
for cross-sectional analysis.XMA052 MG1K inhibited the formation of
atherosclerotic lesions in ApoE knockout mice by 22-37% across the
three doses tested (p<0.05, FIG. 8, 9).
[0347] Alternatively, plaque progression and in vivo coronary
artery function is assessed using noninvasive high-resolution
ultrasound techniques (see for example, Gronros et al., Am J
Physiol Heart Circ Physiol. 295:H2046-53, 2008). Eight-week-old
male ApoE mice are fed a high-fat diet with or without antibody
treatment for approximately 16 weeks. During the course of
treatment, total cholesterol levels are measured, as well as the
degree of retardation of lesion progression in the brachiocephalic
artery, as visualized in vivo using an ultrasound biomicroscope.
Histological analysis is also used to determine the reduction of
brachiocephalic atherosclerosis. Coronary artery function also may
be measured by volumetric flow, such as for example by simultaneous
recording of Doppler velocity signals and left coronary artery
morphology before and during adenosine infusion.
TABLE-US-00010 Antibody Txt Cholesterol Ultrasound Histology Group
1 Placebo Yes Yes Yes Group 2 Low dose Yes Yes Yes Group 3 Med dose
Yes Yes Yes Group 4 High dose Yes Yes Yes
[0348] To further characterize the impact of IL-1.beta. antibody on
the formation of atherosclerotic lesions in the ApoE-knockout
model, the aortic sinus and/or brachiocephalic artery is sectioned
and assessed for lesion cross-sectional area and content (Zhou et
al., 2008, Eur. J. Pharmacol. 590:297-302; Calkin et al., 2007,
Atherosclerosis 195:17-22; Kirii et al., 2003, Arterioscler.
Thromb. Vasc. Biol. 23:656-660). Serial 3-.mu.m paraffin sections
are dewaxed and rehydrated. Endogenous peroxidase activity is
inhibited by incubation with 3% hydrogen peroxide. After blocking
sections with 20% (v/v) goat serum in phosphate-buffered saline,
sections are incubated overnight at 4.degree. C. with antibodies
against .alpha.-smooth muscle actin, inflammatory markers, such as
IL-6, IL-8, MCP-1, ICAM-1 and VCAM-1, degradative enzymes, such as
MMP-3, MMP-9 and cathepsin S or thrombotic factors, such as tissue
factor or PAI-1. Sections are then incubated with the appropriate
secondary antibodies. Positive areas are counted and expressed as a
percentage of the whole plaque area. A negative control, in which
the primary antibody is replaced with either mouse or rat IgG at
the same dilution, is included. Sections are also evaluated for
lipid content by staining with the lipophilic dye Oil Red 0 and
macrophage infiltration is quantified by immunohistochemistry by
staining with antibodies against CD68 (Kirii et al., 2003,
Arterioscler. Thromb. Vasc. Biol. 23:656-660). Blinded analysis of
positive immunostained sections is performed with an image-analysis
program (Image Pro Plus, Media Cybernetics).
[0349] Alternatively, markers of inflammation and matrix
degradation are interrogated by quantitative gene expression
analysis (Calkin et al., 2007, Atherosclerosis 195:17-22). RNA is
extracted from whole aorta by homogenization using Trizol and DNAse
treated. Quantitative real time RT-PCR is carried out using the
Taqman system on an ABI Prism 7700 Sequence Detector. Gene
expression of the aforementioned genes are normalized to 18S mRNA
and reported as ratios compared to the level of expression in
untreated control mice. For statistical purposes, non-parametric
data are handled as their log derivative. Differences in expression
are compared using Student's t-tests (two groups) or one-way ANOVA
(three or more groups).
[0350] The influence of IL-1.beta. antibody on the aforementioned
markers of inflammation, degradation and thrombosis are also
assessed in the serum of antibody-treated ApoE knockout mice by
ELISA or using the Mesoscale Discovery (MSD) platform. Serum
obtained by cardiac puncture at the time of sacrifice is analyzed
for serum lipids as described (Warnick, 1986, Methods Enzymol.
129:101-23). All lipid assays are performed in triplicate
determinations. An external control sample with known analyte
concentration is run for each assay to assure accuracy. Free plasma
glycerol concentrations is also determined and used to correct the
triglyceride values.
[0351] To quantitatively evaluate stability of atherosclerotic
lesions, sections of 5 .mu.m thickness are selected and quantified.
Sections are serially cut every 50 .mu.m from the cardiac base
cross-section until the ascending aorta appears. Approximately six
serial 5-.mu.m sections per mouse are used for morphometric and
immunohistochemical analysis. Collagen and foam cells in plaques
are stained with a modified Movat pentachrome stain. Stained
sections are inspected for buried fibrous caps within the plaque,
which are also counted. Morphometry is performed with a
computerized image-analysis program (Image Pro Plus, Media
Cybernetics). Plaque composition, including extracellular lipids,
foam cells and collagen is determined as a percentage of plaque
area. The plaque area is measured directly and subtracted from the
area enclosed by the internal elastic lamina to derive the patent
lumen area corrected by dividing internal elastic lamina
surrounding area. The effect of IL-1.beta. antibody on plaque
stability is evaluated by calculating the vulnerability index
((foam cells+extracellular lipids)/(collagens+smooth muscle cells))
and the average number of buried fibrous caps.
[0352] Systolic and diastolic blood pressure are measured using a
tail-cuff system and mean blood pressure calculated (Chamberlain et
al., 2009, PLoS ONE 4(4): e5073). To ensure stress levels of mice
are kept to a minimum, a single handler is used throughout the
experiment and mice are subjected to one week of training (blood
pressure and pulse readings are taken, but the data discarded)
prior to starting analysis. Measurements are taken at the same
time, daily to avoid normal daily variance in blood pressure. In
addition, the blood pressure is taken on the same part of the tail
every day. During analysis, 10 measurements are taken each day, and
mean blood pressure and standard deviation calculated for each
`data day` and week (total of 50 readings per mouse per week, 10
per day). On each day, individual data points are rejected if the
blood pressure is below 40 or above 210 mmHg, or if it is outside
of 2 standard deviations from the mean. All data for a day is
rejected if there were less than 4 valid readings. Data for a week
is rejected if it does not have at least 3 valid days of
measurements. One week of baseline readings on chow diet are taken
for each mouse, prior to feeding of Western or WHC diets. Data are
analyzed by global non-linear regression. This statistical test
analyzes an entire family of data sets simultaneously sharing one
or more parameters between data sets. For each shared parameter,
global non-linear regression finds one best-fit value that applies
to all the data sets. In this case, blood pressure is determined
under control (chow fed) and treated (diet-fed) conditions, for
different mouse genotypes, and global non-linear regression
determines whether the difference between each blood pressure curve
is convincing. The test does not compare individual time points,
but instead treats the data globally to produce a single p value
per comparison.
[0353] These studies are further extended to evaluate the effect of
the IL-1.beta. antibody or fragment thereof on plaque rupture in
carotid artery lesions in the ApoE deficient murine atherosclerosis
model (see for example, Nakamura et al., Atherosclerosis, 2009,
Feb. 21 [Epub ahead of print]). ApoE-deficient 8-week-old mice
(C57BL/6) are anesthetized and subjected to ligation of the left
common carotid artery just proximal to its bifurcation. Four weeks
after ligation, a polyethylene cuff is applied just proximal to the
ligated site. Control groups are included in which the artery is
exposed but not ligated, as well as ligated but not subjected to
the polyethylene cuff.
[0354] Animals then receive the treatment antibody or placebo
(e.g., control antibody) administered intraperitoneally or
intravenously at one or more pre-determined times following the
procedure. For example, in one group the antibody is administered
24 or 48 hours preceding cuff placement. In another group, the
antibody is administered at the time of cuff placement.
TABLE-US-00011 Ligation Cuff Antibody Txt Histology Group 1 Sham
N/A N/A Yes Group 2 Yes No N/A Yes Group 3 Yes No Placebo Day 0
Group 4 Yes Yes Placebo Day 4 Group 5 Yes No Low dose (-24 hr) Day
0 Group 6 Yes No High dose (-24 hr) Day 0 Group 7 Yes Yes Low dose
(-24 hr) Day 4 Group 8 Yes Yes High dose (-24 hr) Day 4 Group 9 Yes
Yes Low dose (Day 0) Day 4 Group 10 Yes Yes High dose (Day 0) Day
4
[0355] Just before cuff placement (Day 0) and 4 days after cuff
placement, mice are perfused through the left cardiac ventricle
with isotonic saline and 4% paraformaldehyde in 0.01 M phosphate
buffer (pH 7.4) under physiological pressure. Carotid arteries are
collected and processed for histological analysis.
Cross-cryosections (6 .mu.m) are prepared from the intracuff region
of each carotid artery and stained with hematoxylin and eosin
(H&E), and picrosirius red for collagen. The corresponding
sections on separate slides are used for immunohistochemical
staining with antibodies against neutrophils.
[0356] The proportions of intraplaque hemorrhage and disruption in
the neointima accompanying the intramural thrombus are compared
between the antibody and control groups. Histological
classification of the plaque disruption at the intracuff region of
the carotid artery is done by dividing the lesions into three
groups, based on the analyses of 30 sections at 60-.mu.m intervals
in each sample tissue. When there are no cracks and no mural or
occlusive thrombus at the intracuff region, classification is "no
disruption". When intraplaque hemorrhage, or mural or occlusive
thrombus with cracks or erosion in the plaques are detected,
classified is "hemorrhage" or "disruption", respectively.
[0357] Neutrophil infiltration in the neointima and collagen
content is also determined. Collagen content is evaluated by the
picrosirius red-stained positive area which appears bright when
viewed with polarized light. Neutrophil infiltration in the intima
is assessed by the neutrophils positive area which was stained by
anti-neutrophil antibody (1:50; Serotec, MCA771GA).
Example 8
Cardiovascular Event Reduction in Subjects with a History of at
Least One Risk Factor for Cardiovascular Disease
[0358] To determine the effect of an IL-1.beta. antibody or binding
fragment thereof on reducing a cardiovascular event (e.g., time to
first event) in subjects with a history of at least one risk factor
for cardiovascular disease, a clinical study is performed. In one
study, an IL-1.beta. antibody is evaluated in an at risk
population, measuring reduction of (e.g., preventing) a primary
outcome that includes a composite of death from cardiovascular
causes, myocardial infarction, or stroke, as well as each outcome
separately. Measurements of reduction of (e.g., preventing) a
secondary outcome may include death from any cause, the need for a
revascularization procedure, heart failure, angina (e.g.,
hospitalization for angina, unstable angina), congestive heart
failure, and acute coronary syndrome.
[0359] For a double-blind study, subjects are randomly enrolled
into one of two IL-1.beta. antibody treatment dose groups (e.g.,
0.3 mg/kg, 0.1 mg/kg), or a matching placebo group. Antibody and
placebo treatments are administered in conjunction with standard of
care. Men and women of at least 55 years in age are included in the
study if they have a history of coronary artery disease (e.g.,
manifest coronary artery disease), peripheral vascular disease,
Type 2 diabetes, elevated total cholesterol, hypertension, low HDL
cholesterol levels, tobacco smoking, atherosclerosis and/or
microalbuminuria. Subjects are excluded if they are known to have
experienced a recent (e.g., within 6 months of enrollment)
cardiovascular event. Group sizes include sufficient numbers of
subjects to detect a reduction in the relative risk of a
cardiovascular event during the period of the study. All subjects
provide written informed consent.
[0360] Subjects are administered the IL-1.beta. antibody or placebo
at monthly intervals and outcomes monitored throughout the study
period (e.g., 3 year study period). Outcomes are determined by
standard clinical diagnoses accepted by the medical field. Results
indicative of an effect from the IL-1.beta. antibody include a
reduction in the relative risk of a cardiovascular event outcome
(e.g., 20 percent reduction in relative risk).
Example 9
Cardiovascular Event Reduction in Subjects with a History a
Previous Cardiovascular Event
[0361] To determine the effect of an IL-1.beta. antibody or binding
fragment thereof on reducing a cardiovascular event (e.g., time to
second event) in subjects with a history of a previous
cardiovascular event, a clinical study is performed. In one study,
an IL-1.beta. antibody is evaluated in subjects in the period after
the occurrence of a first documented cardiovascular event of
myocardial infarction or acute coronary syndrome. The study
measures reduction of (e.g., preventing) a primary cardiovascular
event outcome that includes a composite of death from
cardiovascular causes, myocardial infarction, or stroke, as well as
each outcome separately. Measurements for reduction of (e.g.,
preventing) a secondary outcome may include death from any cause,
the need for a revascularization procedure, heart failure, angina
(e.g., hospitalization for angina, unstable angina), congestive
heart failure, and acute coronary syndrome.
[0362] For a double-blind study, subjects are randomly enrolled
into one of two dose groups (e.g., 0.3 mg/kg, 0.1 mg/kg) for an
IL-1.beta. antibody, or a matching placebo group. Men and women are
enrolled in the study following a recent occurrence of a first
cardiovascular event (e.g., within 96 hours), as described above.
Group sizes include sufficient numbers of subjects to detect a
reduction in the relative risk of a subsequent cardiovascular event
during the period of the study. All subjects provide written
informed consent.
[0363] Subjects are administered the IL-1.beta. antibody or placebo
at monthly intervals and outcomes monitored throughout the study
period (e.g., 3 year study period). Outcomes are determined by
standard clinical diagnoses accepted by the medical field. Results
indicative of an effect from the IL-1.beta. antibody include a
reduction in the relative risk of a second cardiovascular event
outcome (e.g., 20 percent reduction in relative risk).
[0364] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0365] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Wherever an open-ended term is used to
describe a feature or element of the invention, it is specifically
contemplated that a closed-ended term can be used in place of the
open-ended term without departing from the spirit and scope of the
invention. Recitation of ranges of values herein are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the invention and does not pose a limitation on the
scope of the invention unless otherwise claimed. No language in the
specification should be construed as indicating any non-claimed
element as essential to the practice of the invention.
[0366] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those working in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
51107PRTArtificialSynthesized AB7 light chain 1Asp Ile Gln Met Thr
Gln Ser Thr Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Ser Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile 35 40 45Tyr Tyr
Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Gln65 70 75
80Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln Gly Lys Met Leu Pro Trp
85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
1052120PRTArtificialSynthesized AB7 heavy chain 2Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met
Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp
Leu Ala His Ile Trp Trp Asp Gly Asp Glu Ser Tyr Asn Pro Ser 50 55
60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65
70 75 80Ser Leu Lys Ile Thr Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Phe 85 90 95Cys Ala Arg Asn Arg Tyr Asp Pro Pro Trp Phe Val Asp Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120323PRTArtificialSynthesized Epitope corresponding to residues
83-105 of the mature IL-1 protein 3Glu Ser Val Asp Pro Lys Asn Tyr
Pro Lys Lys Lys Met Glu Lys Arg1 5 10 15Phe Val Phe Asn Lys Ile Glu
204107PRTArtificialSynthesized AB5 light chain 4Asp Ile Gln Met Thr
Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr
Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30Leu Ser Trp
Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr Tyr
Thr Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75
80Glu Asp Ile Ala Thr Tyr Phe Cys Leu Gln Gly Lys Met Leu Pro Trp
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
1055120PRTArtificialSynthesized AB5 heavy chain 5Gln Val Thr Leu
Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln1 5 10 15Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met
Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp
Leu Ala His Ile Trp Trp Asp Gly Asp Glu Ser Tyr Asn Pro Ser 50 55
60Leu Lys Thr Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val65
70 75 80Phe Leu Lys Ile Thr Ser Val Asp Thr Val Asp Thr Ala Thr Tyr
Phe 85 90 95Cys Ala Arg Asn Arg Tyr Asp Pro Pro Trp Phe Val Asp Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115 120
* * * * *
References