U.S. patent application number 13/563905 was filed with the patent office on 2012-12-20 for composition for the treatment of atherosclerosis.
This patent application is currently assigned to Institut National De La Sante Et De La Recherche Medicale (Inserm). Invention is credited to Ziad MALLAT, Alain TEDGUI.
Application Number | 20120321650 13/563905 |
Document ID | / |
Family ID | 34941865 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120321650 |
Kind Code |
A1 |
TEDGUI; Alain ; et
al. |
December 20, 2012 |
COMPOSITION FOR THE TREATMENT OF ATHEROSCLEROSIS
Abstract
Epitopes derived from a protein present in an atherosclerotic
plaque, such as apolipoprotein B-100, are presented. The epitopes
can be used in a method of treating atherosclerosis by continuous
subcutaneous or transcutaneous administration of a therapeutically
effective amount of the epitope to a subject. Administering the
epitope to the subject can induce a specific regulatory immune
response, such as a Treg response. A composition or patch
containing the epitope and adapted for the prophylactic or
therapeutic treatment of a subject are also presented.
Inventors: |
TEDGUI; Alain; (Paris,
FR) ; MALLAT; Ziad; (Herbeville, FR) |
Assignee: |
Institut National De La Sante Et De
La Recherche Medicale (Inserm)
Paris Cedex
FR
|
Family ID: |
34941865 |
Appl. No.: |
13/563905 |
Filed: |
August 1, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11813071 |
Mar 3, 2008 |
|
|
|
PCT/IB06/00291 |
Jan 4, 2006 |
|
|
|
13563905 |
|
|
|
|
60667431 |
Apr 1, 2005 |
|
|
|
Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61P 37/00 20180101;
A61K 9/127 20130101; A61K 9/7023 20130101; A61K 9/0014 20130101;
A61K 38/10 20130101; A61K 38/17 20130101; Y02A 50/406 20180101;
A61P 9/10 20180101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 9/10 20060101 A61P009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 4, 2005 |
EP |
05290008.1 |
Claims
1. A method for treating atherosclerosis in a subject, comprising a
continuous subcutaneous or transcutaneous administration to the
subject of a therapeutically effective amount of at least one
epitope, wherein the at least one epitope is administered for a
period of time and at a daily dose that are sufficient to induce a
specific regulatory immune response, the at least one epitope being
administered for a period of time within a range of 7 to 30 days
and at a daily dose within a range of 0.05 to 5000 .mu.g per kg
body weight per day, the epitope is a synthetic peptide derived
from apolipoprotein B-100 (apoB-100), and the synthetic peptide is
selected from the group consisting of: TABLE-US-00005 (SEQ ID NO:
1) FLDTVYGNCSTHFTVKTRKG; (SEQ ID NO: 2) PQCSTHILQWLKRVHANPLL; (SEQ
ID NO: 3) VISIPRLQAEARSEILAHWS; (SEQ ID NO: 4)
KLVKEALKESQLPTVMDFRK; (SEQ ID NO: 5) LFVTQAEGAKQTEATMTFK; (SEQ ID
NO: 6) DGSLRHKFLDSNIKFSHVEK; (SEQ ID NO: 7) KGTYGLSCQRDPNTGRLNGE;
(SEQ ID NO: 8) RLNGESNLRFNSSYLQGTNQ; (SEQ ID NO: 9)
SLTSTSDLQSGIIKNTASLK; (SEQ ID NO: 10) TASLKYENYELTLKSDTNGK; (SEQ ID
NO: 11) DMTSFKQNALLRSEYQADYE; (SEQ ID NO: 12) MKVKIIRTIDQMQNSELQWP;
(SEQ ID NO: 13) IALDDAKINFNEKLSQLQTY; (SEQ ID NO: 14)
KTTKQSFDLSVKAQYKKNKH; (SEQ ID NO: 15) EEEMLENVSLVCPKDATRFK; (SEQ ID
NO: 16) GSTSHHLVSRKSISAALEHK; (SEQ ID NO: 17) IENIDFNKSGSSTASWIQNV;
(SEQ ID NO: 18) IREVTQRLNGEIQALELPQK; (SEQ ID NO: 19)
EVDVLTKYSQPEDSLIPFFE; (SEQ ID NO: 20) HTFLIYITELLKKLQSTTVM; (SEQ ID
NO: 21) LLDIANYLMEQIQDDCTGDE; (SEQ ID NO: 22) CTGDEDYTYKIKRVIGNMGQ;
(SEQ ID NO: 23) GNMGQTMEQLTPELKSSILK; (SEQ ID NO: 24)
SSILKCVQSTKPSLMIQKAA; (SEQ ID NO: 25) IQKAAIQALRKMEPKDKDQE; (SEQ ID
NO: 26) RLNGESNLRFNSSYLQGTNQ; (SEQ ID NO: 27) SLNSHGLELNADILGTDKIN;
(SEQ ID NO: 28) WIQNVDTKYQIRIQIQEKLQ; (SEQ ID NO: 29)
TYISDWWTLAAKNLTDFAEQ; (SEQ ID NO: 30) EATLQRIYSLWEHSTKNHLQ; (SEQ ID
NO: 31) ALLVPPETEEAKQVLFLDTV; (SEQ ID NO: 32) IEIGLEGKGFEPTLEALFGF;
(SEQ ID NO: 33) SGASMKLTTNGRFREHNAKF; (SEQ ID NO: 34)
NLIGDFEVAEKINAFRAKVH; (SEQ ID NO: 35) GHSVLTAKGMALFGEGKAEF; (SEQ ID
NO: 36) FKSSVITLNTNAELFNQSDI; (SEQ ID NO: 37) FPDLGQEVALNANTKNQKIR;
(SEQ ID NO: 38) ATRFKHLRKYTYNYQAQSSS;
and any of SEQ ID NO: 1-38 modified to mimic a modification of
apoB-100 protein that may occur during oxidation or non-oxidative
modification of LDL, selected from the group consisting of
oxidation by exposure to copper, oxidation after
aldehyde-modification or acetylation.
2. The method according to claim 1, wherein the subject has been
diagnosed as presenting one of the following coronary disorders: an
asymptomatic coronary artery coronary disease with silent ischemia
or without ischemia; a chronic ischemic disorder without myocardial
necrosis; an acute ischemic disorder with myocardial necrosis; and
an ischemic disorder with myocardial necrosis.
3. The method according to claim 1, wherein the at least one
epitope is administered for a period of time within the range of 10
days to 20 days.
4. The method according to claim 1, wherein the at least one
epitope is administered at a daily dose within the range of 0.5 to
1000 .mu.g per kg body weight per day.
5. The method according to claim 2, wherein the chronic ischemic
disorder without myocardial necrosis is stable or effort angina
pectoris.
6. The method according to claim 2, wherein the acute ischemic
disorder with myocardial necrosis is unstable angina pectoris.
7. The method according to claim 2, wherein the ischemic disorder
with myocardial necrosis is ST segment elevation myocardial
infarction or non-ST segment elevation myocardial infarction.
Description
[0001] The present invention relates to the prevention or treatment
of atherosclerosis, in particular to a composition comprising at
least one epitope derived from a protein present in the
atherosclerotic, which can be administrated to a subject suffering
from atherosclerosis and use thereof.
[0002] Atherosclerosis is the most common cause of death in western
societies and is predicted to become the leading cause of
cardiovascular disease in the world within two decades.
[0003] Atherosclerosis can be considered to be a form of chronic
inflammation resulting from interaction between modified
lipoproteins, monocyte-derived macrophages, T cells and the normal
cellular elements of the arterial wall. This inflammatory process
can ultimately lead to the development of complex lesions or
plaques that may protrude into the arterial lumen. Plaque
rupture/erosion and thrombosis results in the acute clinical
complications of myocardial infarction and stroke (ROSS, N. Eng. J.
Med., vol.340, p: 115-126, 1999; LIBBY, Nature, vol:420, p: 868-74,
2002; VIRMANI et al., Arterioscler. Thromb. Vasc. Biol., vol. 20,
p: 1262-1275, 2000).
[0004] The disease is initiated by accumulation of lipoproteins
particles in the extra-cellular matrix of the vessel. The principal
lipid components of lipoprotein particles are cholesterol,
triglycerides and phospholipids. Cholesterol is required for normal
cellular function and forms an important component of cell
membranes. Cholesterol exits in various forms in the circulation
and the major component is low-density lipoprotein cholesterol
(LDLc, approximately 60% of total serum cholesterol), with about
25% in the form of high-density lipoprotein cholesterol (HDLc) and
the remainder circulating in very low-density lipoprotein
cholesterol (VLDLc) and other lipoprotein particles. Plasma lipid
levels are determined by both genetic and environmental factors
such as the LDL receptor locus, apolipoprotein B, genetic
polymorphisms, diet, obesity and alcohol intake. Common
abnormalities of lipid levels include raised LDLc, a low HDLc and
high triglyceride level or a combination of these lipid
disturbances.
[0005] The common abnormalities of plasma lipid levels described
above contribute to the development of atherosclerotic vascular
diseases (AVD) which may affect the coronary arteries (causing
ischaemic heart disease), the cerebral circulation (causing
cerebrovascular disease), the aorta (producing aneurysms that are
prone to thrombosis and rupture) and peripheral blood vessels,
typically the legs (causing peripheral vascular disease and
intermittent claudication).
[0006] Ischaemic heart disease (IHD) includes angina (chest pain
caused by insufficient blood supply to cardiac muscle) and
myocardial infarction (death of cardiac muscle) and cerebrovascular
disease includes stroke and transient ischaemic attacks. One in
three men and one in four women will die from IHD and the death
rate for IHD was 58 per 100,000 in 1990.
[0007] HDLc levels are inversely associated with risk of AVD and
patterns of lipid abnormalities reflected by an increased ratio of
total cholesterol to HDLc, combined with raised fasting
triglyceride levels, are a better predictor of risk of IHD than
total cholesterol levels alone. A high ratio in combination with
increased fasting triglyceride levels is frequently associated with
the atherogenic lipoprotein phenotype (ALP), which also includes
the presence of increased concentrations of small dense LDL
particles. Other cardiovascular risk factors are known to
predispose to atherosclerosis, including hypertension, smoking,
diabetes, obesity, sedentarity.
[0008] Plasma levels of several mediators of inflammation or
endothelial dysfunction have been found to predict future
cardiovascular events. These biomarkers include, but are not
restricted to, hsCRP, IL-6, CD40L, IL-10, IL-18, MMP9, PlGF,
circulating microparticles, secretory PLA2, circulating endothelial
cells, circulating endothelial progenitor cells.
[0009] Atherosclerotic plaques begin as fatty streaks underlying
the endothelium of large arteries. Recruitment of macrophages and
their subsequent uptake of LDL-derived cholesterol are the major
cellular events contributing to fatty streak formation. Many lines
of evidence suggest that oxidative or non-oxidative modifications
in the lipid and apolipoprotein B (apo B) components of LDL drive
the initial formation of fatty streaks (NAVAB et al., Arterioscler.
Thromb. Vasc. Biol., vol.16, p: 831-842, 1996). The specific
properties of oxidized LDL (oxLDL), usually studied following
oxidation of native LDL in vitro, depend on the extent of
modification. This can range from "minimal" modification (mmLDL)
wherein the LDL particle can still be recognized by LDL receptors,
to extensive oxidation wherein the apoB component is fragmented and
lysine residues are covalently modified with reactive breakdown
products of oxidized lipids. Such particles are not bound by the
LDL receptor but rather by so-called scavenger receptors expressed
on macrophages and smooth muscle cells. A large number of
proinflammatory and proatherogenic properties have been ascribed to
mmLDL and oxLDL and their components. For instance,
lysophosphatidylcholine or oxidized phospholipids increase
monocyte's adhesion, monocyte and T cell chemotaxis and can induce
proinflammatory gene expression. Although the recruitment of
monocytes to the arterial wall and their subsequent differentiation
into macrophages may initially serve a function by removing
cytotoxic and proinflammatory oxLDL particles or apoptotic cells,
progressive accumulation of macrophages and their uptake of oxLDL
ultimately leads to development of atherosclerotic lesions.
[0010] As used herein, the term "T cells" includes lymphocytes
which express phenotypic markers and rearrangements of the
TCR.beta. locus with or without rearrangements of the TCR.alpha..
Phenotypic markers include expression of CD4 and/or CD8.
[0011] The transition from the relatively simple fatty streak to
the more complex plaque is characterized by the migration of smooth
muscle cells from the medial layer of the artery wall to the
internal elastic lamina and to intimal or subendothelial space, or
by recruitment of smooth muscle cell progenitors. Intimal smooth
muscle cells may proliferate and take up modified lipoproteins,
thus contributing to foam cell formation, and synthesize
extracellular matrix proteins that lead to the development of the
fibrous cap (ROSS, 1999, aforementioned; PAULSSON et al.,
Arterioscler. Thromb. Vasc. Biol., vol.20, p: 10-17, 2000). Thus,
the advanced atherosclerotic plaque is schematically divided into
two portions: the fibrous cap making up the surface layer and a
lipid core making up the deep layer. This extra-cellular matrix
(ECM) is composed of vastly different macromolecules including
collagen, elastin, glycoproteins and proteoglycans (KATSUDA and
KAJI, J. Atheroscler. Thromb., vol.10(5), p: 267-274, 2003). Large
amounts of ECM are deposited in the fibrous cap, with the strength
of the plaque maintained, whereas in the lipid core in addition to
lipid deposition, ECM degradation is enhanced, leading to increased
tissue fragility. This plaque fragility gives rise to plaque
vulnerability in turn becoming a cause of plaque rupture.
[0012] This phase of plaque development is influenced by
interactions between monocyte/macrophages and T cells that result
in a broad range of cellular and humoral responses and the
acquisition of many features of a chronic inflammatory state.
Significant cross talk appears to occur among the cellular elements
of developing lesions. Lesional T cells appear to be activated and
express both Th1 and Th2 cytokines (HANSSON et al., Circ. Res.,
vol.91(1), p: 281-91, 2002). Similarly, macrophages, endothelial
cells and smooth muscle cells appear to be activated based on their
expression of MHC class II molecules and numerous inflammatory
products such as TNF, IL-6 and MCP 1.
[0013] Most of the T cells in atherosclerotic lesions are CD3+CD4+
T-cell receptor (TCR) .alpha..beta.+ cells (JONASSON et al.,
Arteriosclerosis, vol.6, p: 131-138 , 1986; STEMME et al.,
Arterioscler. Thromb., vol.12, p: 206-211, 1992). This implies that
they recognize protein antigens presented to them by macrophages
after uptake and processing through the endosomal pathway. Most of
them are of the T-helper (Th1) subtype) which secretes IFN-.gamma.,
IL-2, TNF.alpha. and -.beta., and which causes macrophage
activation, vascular activation and inflammation (FROSTEGARD et
al., Atherosclerosis, vol.145, p: 33-43, 1999). For instance,
IFN-.gamma. induces expression of inflammatory cytokines and of
secretory phopsholipase A2 which can lead to the production of
inflammatory lipid mediators such as eicosanoids,
lysophosphatidylcholine and platelet activating factor (PAF). At
least three important stimuli for Thi differentiation are present
in the atherosclerotic plaque. The cytokine IL-12 and IL-18 which
are produced by many lesion cells is an important stimulus for Th1
differentiation, and both have been shown to promote plaque
progression and instability (UYEMURA et al., J. Clin. Invest.,
vol.97, p: 2130-8, 1996; MALLAT et al., Circ. Res., vol.89, p:
e41-e45, 2001). Osteopontin, also called early T-lymphocyte
activation protein-1 (Eta-1), is needed for Th1 responses and
promotes IL-12 expression and granuloma formation (ASHKAR et al.,
Science, vol.287, p: 860-4, 2000). It is expressed by macrophages,
endothelial cells and smooth muscle cells in plaques (O'BRIEN et
al., Arterioscler. Thromb., vol.14, p: 1648-54, 1994) and may be
important for local immunity as well as for mineralization. Th2
cytokines such as IL-4, IL-5 and IL-10 are less abundant than
cytokines of the Th1 type in end-stage human lesions (FROSTERGARD
et al., 1999, aforementioned). Deficiency in IL-4, the prototypic
Th2-related cytokine, has been associated with a decrease in
atherosclerotic lesion formation, thus suggesting a pro-atherogenic
role of Th2, and exaggerated Th2 responses promoted atherosclerotic
plaque progression (KING et al., Arterioscler. Thromb. Vasc. Biol.,
vol.22, p: 456-461, 2002) as well as aneurysm formation (SHIMIZU et
al., J. Clin. Invest., vol.114, p: 300-308, 2004). Mice showing
up-regulation of both Th1 and Th2 responses display enhanced plaque
inflammation (GOJOVA et al., Blood, vol.102, p: 4052-4058, 2003;
ROBERTSON et al., J. Clin. Invest., vol.112:1342-1350, 2003).
Therefore, even though atherosclerosis occurs mostly in a
Th1-related pathogenic context, no direct and solid evidence is
available suggesting that promotion of Th2 responses would
invariably lead to limitation of disease progression. Moreover,
frequent association in humans between atherosclerosis, a
Th1-predominant disease, and aortic aneurysm, a Th2-predominant
process, suggests a deregulation in both Th1- and Th2-mediated
responses (MALLAT and TEDGUI, Expert. Opin. Biol. Ther., vol.4, p:
1387-1393, 2004).
[0014] As used herein, "Th1 cells" refer to a subset of CD4+ T that
produces IL-2, IFN.gamma. and lymphotoxin (LT also called
TNF.beta.). Thi differentiation from naive T cell is favoured by
the presence of exogenous IFN.gamma., IL-12 and IL-18. The
expression of the IL-12R .beta.2 subunit and IL-18r may be
considered as a marker for Th1 cells. Thi play important roles in
cellular immune functions such as delayed-type hypersensitivity or
in the defence against intracellular organisms such as
parasites.
[0015] As used herein, "Th2 cells" refer to another subset of cells
that produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13, but not IL-12
and IFN.gamma.. The essential cytokine for the development of Th2
cells is IL-4. Th2 cells generally enhance antibody production from
B cells.
[0016] An existing approach for the treatment of atherosclerosis
result from the identification of circulating auto-antibodies
against oxidized LDL in humans (PALINSKI et al., Proc. Natl. Acad.
Sci. USA., vol.86(4), p: 1372-6, 1989) and from the observation
that an immunization with oxidized LDL had a effect reducing
atherosclerosis with about 50%. In this approach the athero-effect
is mediated by antibodies generated against peptidic sequences
present in oxLDL. It has been supposed that these antibodies could
facilitate the removal of oxLDL by macrophage. Thus, the
application WO 02/080954 describes the identification of epitopes
in apolipoprotein B that give rise to "antibody formation" in man
and the use of efficient quantities of such epitopes in order to
induce an humoral immune response corresponding to the production
of antibodies.
[0017] Another approach is based on evidence that the Th1 and Th2
pathways appear to play a key role. Thus immunomodulatory treatment
that promotes regulatory immunity can represent an attractive tool
for treating and/or preventing atherosclerosis. This might be
accomplished by promoting regulatory T cell generation such as Tr1
cells, CD4+CD25+cells or Th3 cells. In that context, IL-10 and
TGF.beta. appears to be two of the most interesting cytokines,
which are capable of downregulating the inflammatory process
trigerred by Th1 cells. Indeed IL-10 is a pleitropic cytokine that
is expressed in human atherosclerotic plaques and is mainly
produced by macrophages, T helper (Th) type 2 and T regulatory
type-1 lymphocytes (Tr1 cells). It has been shown that ex vivo
repeated stimulation of naive T cells with ovalbumin (OVA) and
IL-10 results in the generation of T cells clones with
immunosuppressives properties, which can prevent in vivo Th1
response after cells injection (GROUX et al., Nature,
vol.389(6652), p: 737-42, 1997). On the basis of these
observations, a recent study has shown that intraperitoneal
administration of OVA-Tr1 cells (expanded in vitro) with their
cognate antigen to female apo-E null mice results in a significant
reduction of atherosclerotic plaque size (MALLAT et al.,
Circulation, vol.108(10), p: 1232-7, 2003). Nevertheless, all of
these methods actually implicate the administration of the T
regulatory cells in the atherosclerotic subjects. Thus, these
methods are far to be simple and reliable because they first
involve the isolation of T cell from the subject, the stimulation
of said T cells with antigen and IL-10 in order to induce
regulatory T cells, the expansion of said regulatory T cells and
finally the administration of said cells to the subject.
[0018] So, there is a recognized and permanent need in the art for
new reliable method for triggering a regulatory immune response in
order to enhance IL-10 and/or TGF.beta. to dampen the inflammatory
response in atherosclerotic lesions.
[0019] The purpose of the present invention is to fulfil this need
by providing a composition for generating a specific regulatory
immune response, preferably regulatory T cell response, within the
atherosclerotic lesion sites to prevent the unwanted Th1/Th2
pro-atherogenic immunity.
[0020] Unexpectedly, the inventors have demonstrated that prolonged
subcutaneous administration to apoE-null mice of apo B-100
peptides, with a concentration which does not induce an humoral
response corresponding to the production of antibodies, by means of
ALZET mini pumps, results in the induction of a Treg response which
is responsible of plaque antigen specific tolerance.
[0021] WO 02/080954 describes a treatment of atherosclerosis by the
injection of one or more epitopes derived from apoB-100 in a dose
efficient for the induction of specific antibodies' expression.
Thus, WO 02/080954 does not describe nor suggest a treatment of
atherosclerosis with a "continuous" administration without
induction of an humoral response corresponding to the production of
specific antibodies. By this way, the present invention limits the
risk of inflammatory complications associated with the induction of
antibodies' expression.
[0022] In one aspect the present invention relates to a method of
prophylactic or therapeutic treatment of a subject suffering from
atherosclerosis comprising the step of administrating a composition
comprising at least one epitope derived from a protein present in
the atherosclerotic plaque to said subject with a continuous
subcutaneous or transcutaneous administration.
[0023] As used herein, the term "subject" denotes a Mammal, such as
a rodent, a feline, a canine and a primate. The subject is an
animal such as cow, pig, horse, chicken, cat, dog and most
preferably a human.
[0024] Inflammatory and thrombotic processes are major determinants
of atherosclerotic plaque complications leading to acute coronary
syndrome (ACS) and sudden death. In addition to pathological
studies showing an important association between plaque
inflammation and plaque rupture in humans, and experimental data
showing a critical role of the immuno-inflammatory response both in
plaque development and composition, the last decade has witnessed
an increasing interest in the study of the role of the systemic
inflammatory markers and their relation to the severe complications
of atherosclerosis. Several circulating inflammatory markers,
including CRP, IL-6, IL-1 receptor antagonist (IL-1ra), vascular
cell adhesion molecule (VCAM)-1, and more recently myeloperoxidase,
have been shown to be elevated in patients with ACS and to be
associated with adverse clinical outcomes at follow-up.
[0025] Since the landmark study of LIUZZO et al. (N. Engl. J. Med.,
vol.331(7), p: 417-24, 1994), numerous studies have addressed the
prognostic value of CRP in patients with ACS. Higher CRP levels
were associated with increased risk at follow-up in several
randomized trials of patients with unstable angina or NSTEMI,
including TIMI IIa (MORROW et al., J. Am. Coll. Cardiol., vol
31(7), p: 1460-5, 1998), CAPTURE (HEESCHEN et al., J. Am. Coll.
Cardiol., vol 35(6), p: 1535-42, 2000), FRISC (LINDAHL et al., N.
Engl. J. Med., vol.343(16), p: 1139-47, 2000) and GUSTO-IV (JAMES
et al., Circulation, vol.108(3), p; 275-81, 2003). CRP levels in
ACS patients assigned to early invasive revascularization
procedures also predict adverse outcomes (MUELLER, Circulation,
vol.105(12), p: 1412-1415, 2002). On the basis of previous
experimental data by the inventors and others showing a potent
pro-atherogenic role for endogenous IL-18 (MALLAT et al.,
Circulation, vol.104(4), p: 1598-603, 2001; MALLAT et al.,
Circulation Res., vol.89(7), E41-5, 2001; WHITMAN et al.,
Circulation Res., vol.90(2), E34-8, 2002), plasma levels have been
measured in patients with a history of coronary artery disease and
in healthy middle-aged men and was found to be an independent
predictor of coronary events (BLANKENBERG et al., Circulation,
vol.107(12), p: 1579-85, 2003; BLANKENBERG et al., Circulation,
vol.108(20), p: 2453-9, 2003).
[0026] The method according to the present invention can be
supplied to a subject, which has been diagnosed as presenting one
of the following coronary disorders: [0027] asymptomatic coronary
artery coronary diseases with silent ischemia or without ischemia;
[0028] chronic ischemic disorders without myocardial necrosis, such
as stable or effort angina pectoris; [0029] acute ischemic
disorders myocardial necrosis, such as unstable angina pectoris;
[0030] ischemic disorders with myocardial necrosis, such as ST
segment elevation myocardial infarction or non-ST segment elevation
myocardial infarction.
[0031] Tissue ischemia occurs when the needs in oxygen exceed the
delivery of oxygen to tissues. Myocardial ischemia can be diagnosed
clinically (e.g. chest pain), biologically (e.g. increase in
myeloperoxidase activity), metabolically, using scintigraphy, or by
use of an electrocardiogram (typical modifications of the ST
segment, upper or lower ST segment deviation, typical changes in T
waves such as T wave inversion or steep asymmetric or high
amplitude positives T waves). Silent ischemia is typically
diagnosed using scintigraphy or a 24 h electrocardiogram
recording.
[0032] Chronic stable angina results from fixed stenoses in
epicardial coronary arteries, which do not limit blood flow at
rest, may become flow-limiting during periods of increased
myocardial oxygen demand. Stenoses producing a 50% reduction in
diameter, or a 70% reduction in cross-sectional area, are
sufficient to impair the hyperaemic response that occurs with
increasing cardiac work. Selective coronary arteriography is the
gold standard for detecting such lesions, although it may
underestimate the severity of CAD in more diffusely diseased
arteries. Angina pectoris is classically described as a
retrosternal pressure that may radiate to the jaw, back, or left
arm or shoulder. It may be associated with nausea, diaphoresis and
a sense of impending doom. Stable angina is typically brought on by
physical exertion, emotional stress, food and exposure to cold.
Angina was originally studied in men, and the presentation of
angina in women and the elderly tends to be less straightforward.
Provocative stress testing with physical or pharmacological stress
attempts to induce myocardial ischaemia in a controlled setting.
The ischaemic zone may be detected as an electrically abnormal area
on electrocardiagraphy (ECG), an area of impaired radionuclide
intake with single photon emission computed tomography (SPECT), or
as a wall motion abnormality on echocardiography. Patients with
typical angina have a high pretest probability of CAD, but a
negative test does not exclude the diagnosis.
[0033] Unstable angina is defined as new-onset angina, angina at
rest, angina of increasing frequency and severity, or angina in the
early post-MI setting. The pathological correlate of unstable
angina is rupture of an atherosclerotic plaque with formation of a
flow-limiting, but nonocclusive, intracoronary platelet-rich
thrombus. Vulnerable plaques are thought to have a thick lipid core
with a thin fibrous cap and a preponderance of inflammatory cells.
The ability to identify which plaques are unstable is limited.
Intravascular ultrasonography and intracoronary catheters that can
detect temperature differences in plaques are two methods under
investigation. Unstable angina and non Q-wave myocardial infarction
(NQWMI) are diagnosed by history, examination, ECG and laboratory
studies. As noted above, rest pain is the hallmark of unstable
angina. The ECG may show ST segment depression in the area of
ischaemia, caused by abnormalities of polarization in the ischaemic
tissue. The lack of pathologic Q waves on ECG signifies that the
infarction is nontransmural. Prolonged ischaemia results in
myocardial necrosis and release of the cardiac specific molecule
troponin and creatine kinase (CK-MB) into the bloodstream. These
markers of necrosis define the presence of MI and are usually
evident only in retrospect. Therefore, unstable angina and NQWMI
are usually grouped together for purposes of initial
management.
[0034] Acute myocardial infarction is also known as Q-wave MI,
transmural MI, or ST-elevation. It may occur suddenly or be
preceded by unstable angina. The pathological correlate of acute MI
is rupture of an atherosclerotic plaque with occlusive fibrin and
platelet-rich intracoronary thrombus. Complete occlusion of the
vessel results in transmural myocardial injury. The clinical
presentation of patients with acute MI is protean, ranging from
mild chest pain to cardiogenic shock or sudden cardiac death.
Physical examination may reveal only abnormal heart sounds or may
demonstrate hypotension and pulmonary oedema. The ECG reveals
ST-segment elevation in the area of ischaemia representing
myocardial injury, and Q waves in areas of infarction. CK-MB and
troponin levels are increased. Clinical features that predict a
poor outcome include advanced patient age, tachycardia, low blood
pressure, and the presence of pulmonary oedema and impaired tissue
perfusion.
[0035] Preferably, the continuous administration of said
composition is carried out for a period of time and at a daily dose
of said at least one epitope, which are sufficient to induce a
specific regulatory immune response, preferably a Treg
response.
[0036] As used herein, a specific regulatory immune response is a
cellular immune response corresponding to an induction of tolerance
for the administrated epitope, which is also present in the
atherosclerotic plaque, which can also dampen the immune response
against any other epitope present in the micro-environment of the
specific epitope by the so called bystander regulatory immune
response and inhibit the local inflammatory response, through the
release of IL-10 and of TGF-.beta..
[0037] As used herein, a "Treg response" corresponds to a response
specific of "Treg cells" or "Tr cells" which refer to a distinct
subset of T cells. Tr cells exhibit a cytokine pattern distinct
from Th1 and Th2 cells. In particular, Tr cells secrete high amount
of IL-10 and subsequent of TGF-.beta.. Tr cells play a major role
in induction of tolerance, largely by their ability to suppress
responses mediated by other populations of T cells, especially Th1
cells. Tr cells include Tr1 cells and Th3 cells, which are
characterized by the secretion of high amount of TGF-.beta.. Tr1
cells secrete high levels of IL-10 and/or TGF-.beta., with or
without IL-5 or IL-13, but little or no IL-2. There exists another
subset of Tr cell that is antigen specific and can induce
tolerance: CD4+CD25+ T cells. CD4+CD25+ T cells comprise 5-10% of
the peripheral T cell pool and exhibit immunosuppressive abilities
both in vitro and in vivo. Specifically, CD4+CD25+ T cells express
the Foxp3 gene. This regulatory activity may depend on TGFP or
cell-cell contact. Other Th1- or Th2-like cells may exert
regulatory activity. As used herein, "Treg response" may be
preceded or associated by the induction of tolerogenic antigen
presenting cell response.
[0038] Advantageously, said continuous administration of said
composition is carried out for a period of time within the range of
7 days to 30 days, preferably of 10 days to 20, most preferably
about 14 days. This period might be repeated one or several
times.
[0039] Advantageously, said continuous administration of said
composition corresponds to a daily dose within the range of 0.05 to
5000 .mu.g per kg body weight per day, preferably of 0.5 to 1000
.mu.g and more preferably of 5 to 500 .mu.g. This daily dose is
sufficient to induce a regulatory immune response but insufficient
to induce a humoral immune response corresponding to the production
of antibodies, specifically of protective antibodies generated
against said epitope, said protective antibodies can for example
facilitate the removal of oxidatively damaged LDL particles by
macrophages receptor when the used epitope is a peptide derived
from apolipoprotein B.
[0040] Proteins present in the atherosclerotic plaque are well
known from one of skills in the art. Such proteins can include
apolipoprotein B-100 (apoB-100, human, Accession number P04114),
collagen type I (human, Accession number CAA67261; AAB59577), type
III (human, Accession number P02458), type IV (human, Accession
number P02462) and type V (human, Accession number CAI17260),
elastin (human, Accession number P15502), laminin (human, Accession
number P024043; Q16363), entactin/nidogen (human, Accession number
P14543), fibronectin (human, Accession number NP.sub.--997647;
NP.sub.--997643; NP.sub.--997641; NP.sub.--997640; NP.sub.--997639;
NP.sub.--997635), thrombospondin (human, Accession number
NP.sub.--003237), vitronectin (human, Accession number P04004),
tenascin (human, Accession number P24821), osteopontin (human,
Accession number NP.sub.--000573), proteoglycans [glycorin, decorin
(human, Accession number AAV38603), versican, hyaluronan], medin
(human, Accession number Q08431), lactadherin (human, Accession
number Q08431), .beta.-amyloid (human, Accession number P05067),
HSP 60 (human, Accession number AAA36022.1) or HSP 70 (human,
Accession number BAA24847.1). Preferably, the protein is selected
among apoB-100, HSP 60 and HSP 70, most preferably the protein is
apoB-100.
[0041] Epitopes from these proteins can be simply identified by one
of skills in the art. These epitopes correspond to proteins present
in the atherosclerotic plaque or to synthetic peptides from more
than 10 amino acids length, more preferably from more than 15 amino
acids, derived from such a protein and which can be presented in a
MHC class II context. Such epitopes corresponding to proteins
present in' the atherosclerotic plaque can be obtained by grinding
an atherosclerotic plaque as described in XU et al. (Arterioscler
Thromb. Vol.12(7), p: 789-799, 1992).
[0042] Preferably, theses epitopes correspond to synthetic
peptides. Typically, the length of these peptides is comprised
between 15 and 25 amino acids. Such epitopes can include the
apoB-100 epitopes described in WO 02/080954, the HSP 60 and HSP 70
epitopes described in WYSOCKI et al. (Cardiovasc. Pathol. vol.11,
p: 238-243, 2002) and in CHAN et al. (Eur. J. Vasc. Endovasc. Surg.
Vol.18, p: 381-385, 1999).
[0043] For the epitopes derived from apoB-100, the peptides can be
used in their native state, or after incorporation in phopholipid
liposomes, after a modification of the amino acids that mimics the
different modifications of apoB-100 protein that may occur during
oxidation or non-oxidative modification of LDL. Preferably, this
modification is selected among oxidation by exposure to copper,
oxidation after aldehyde-modification, like malone dealdehyde
(MDA), hydroxynonenal or other aldehydes, or acetylation, most
preferably this modification corresponds to oxidation after malone
dealdehyde (MDA)-modification.
[0044] Preferably, these epitopes correspond to the peptides
below:
TABLE-US-00001 (SEQ ID NO: 1) FLDTVYGNCSTHFTVKTRKG; (SEQ ID NO: 2)
PQCSTHILQWLKRVHANPLL; (SEQ ID NO: 3) VISIPRLQAEARSEILAHWS; (SEQ ID
NO: 4) KLVKEALKESQLPTVMDFRK; (SEQ ID NO: 5) LFVTQAEGAKQTEATMTFK;
(SEQ ID NO: 6) DGSLRHKFLDSNIKFSHVEK; (SEQ ID NO: 7)
KGTYGLSCQRDPNTGRLNGE; (SEQ ID NO: 8) RLNGESNLRFNSSYLQGTNQ; (SEQ ID
NO: 9) SLTSTSDLQSGIIKNTASLK; (SEQ ID NO: 10) TASLKYENYELTLKSDTNGK;
(SEQ ID NO: 11) DMTSFKQNALLRSEYQADYE; (SEQ ID NO: 12)
MKVKIIRTIDQMQNSELQWP; (SEQ ID NO: 13) IALDDAKINFNEKLSQLQTY; (SEQ ID
NO: 14) KTTKQSFDLSVKAQYKKNKH; (SEQ ID NO: 15) EEEMLENVSLVCPKDATRFK;
(SEQ ID NO: 16) GSTSHHLVSRKSISAALEHK; (SEQ ID NO: 17)
IENIDFNKSGSSTASWIQNV; (SEQ ID NO: 18) IREVTQRLNGEIQALELPQK; (SEQ ID
NO: 19) EVDVLTKYSQPEDSLIPFFE; (SEQ ID NO: 20) HTFLIYITELLKKLQSTTVM;
(SEQ ID NO: 21) LLDIANYLMEQIQDDCTGDE; (SEQ ID NO: 22)
CTGDEDYTYKIKRVIGNMGQ; (SEQ ID NO: 23) GNMGQTMEQLTPELKSSILK; (SEQ ID
NO: 24) SSILKCVQSTKPSLMIQKAA; (SEQ ID NO: 25) IQKAAIQALRKMEPKDKDQE;
(SEQ ID NO: 26) RLNGESNLRFNSSYLQGTNQ; (SEQ ID NO: 27)
SLNSHGLELNADILGTDKIN; (SEQ ID NO: 28) WIQNVDTKYQIRIQIQEKLQ; (SEQ ID
NO: 29) TYISDWWTLAAKNLTDFAEQ; (SEQ ID NO: 30) EATLQRIYSLWEHSTKNHLQ;
(SEQ ID NO: 31) ALLVPPETEEAKQVLFLDTV; (SEQ ID NO: 32)
IEIGLEGKGFEPTLEALFGF; (SEQ ID NO: 33) SGASMKLTTNGRFREHNAKF; (SEQ ID
NO: 34) NLIGDFEVAEKINAFRAKVH; (SEQ ID NO: 35) GHSVLTAKGMALFGEGKAEF;
(SEQ ID NO: 36) FKSSVITLNTNAELFNQSDI; (SEQ ID NO: 37)
FPDLGQEVALNANTKNQKIR; (SEQ ID NO: 38) ATRFKHLRKYTYNYQAQSSS.
[0045] More preferably, the peptides are selected among
IALDDAKINFNEKLSQLQTY (SEQ ID NO: 13), and KTTKQSFDLSVKAQYKKNKH (SEQ
ID NO: 14).
[0046] Advantageously, said at least on epitope is not
administrated with any adjuvant or any effective amount of such an
adjuvant likely to induce antibodies' expression.
[0047] In another aspect the present invention relates to a kit
adapted for the prophylactic or therapeutic treatment of a subject
suffering from atherosclerosis comprising: [0048] (i) a composition
comprising an effective amount of at least one epitope derived from
a protein present in the atherosclerotic plaque; and [0049] (ii)
means for administrating subcutaneously or transcutaneously said
composition in a continuous manner, whereby said continuous
administration to a subject induces a specific regulatory immune
response, preferably a Treg response.
[0050] Means for administrating subcutaneously or transcutaneously
a composition in a continuous manner are well known of one of skill
in the art. Such means include needle with pump.
[0051] Said means enable an administration of said composition for
a period of time within the range of 7 days to 30 days, preferably
of 10 days to 20, most preferably about 14 days. This period might
be repeated one or several times.
[0052] Moreover, said means enable a continuous administration of
said composition corresponding to a daily dose within the range of
0.05 to 5000 .mu.g per kg body weight per day, preferably of 0.5 to
1000 .mu.g and more preferably of 5 to 500 .mu.g.
[0053] Advantageously, the composition of said kit does not contain
any adjuvant or any effective amount of such an adjuvant likely to
induce antibodies' expression. Such adjuvants are well known from
one of skills in the art (e.g. DNA with non methylated CG
nucleotides).
[0054] The composition of the kit according to the present
invention can be supplied to a subject, which has been diagnosed as
presenting one of the following coronary disorders: [0055]
asymptomatic coronary artery coronary diseases with silent ischemia
or without ischemia; [0056] chronic ischemic disorders without
myocardial necrosis, such as stable or effort angina pectoris;
[0057] acute ischemic disorders myocardial necrosis, such as
unstable angina pectoris; [0058] ischemic disorders with myocardial
necrosis, such as ST segment elevation myocardial infarction or
non-ST segment elevation myocardial infarction.
[0059] Advantageously, said composition comprises at least one
epitope derived from a protein present in the atherosclerotic
plaque in an amount within the range of 0.05 .mu.g to 250 mg per
millilitre, preferably of 0.5 .mu.g to 50 mg per millilitre and
more preferably of 5 .mu.g to 25 mg.
[0060] Proteins present in the atherosclerotic plaque are well
known from one of skills in the art. Such proteins can include
apolipoprotein B-100 (apoB-100, human, Accession number P04114),
collagen type I (human, Accession number CAA67261; AAB59577), type
III (human, Accession number P02458), type IV (human, Accession
number P02462) and type V (human, Accession number CAI17260),
elastin (human, Accession number P15502), laminin (human, Accession
number P024043; Q16363), entactin/nidogen (human, Accession number
P14543), fibronectin (human, Accession number NP.sub.--997647;
NP.sub.--997643; NP.sub.--997641; NP.sub.--997640; NP.sub.--997639;
NP.sub.--997635), thrombospondin (human, Accession number
NP.sub.--003237), vitronectin (human, Accession number P04004),
tenascin (human, Accession number P24821), osteopontin (human,
Accession number NP.sub.--000573), proteoglycans [glycorin, decorin
(human, Accession number AAV38603), versican, hyaluronan], medin
(human, Accession number Q08431), lactadherin (human, Accession
number Q08431), .beta.-amyloid (human, Accession number P05067),
HSP 60 (human, Accession number AAA36022.1) or HSP 70 (human,
Accession number BAA24847.1). Preferably, the protein is selected
among apoB-100, HSP 60 and HSP 70, most preferably the protein is
apoB-100.
[0061] Epitopes from these proteins can be simply identified by one
of skills in the art. These epitopes correspond to proteins present
in the atherosclerotic plaque or to synthetic peptides from more
than 10 amino acids length, more preferably from more than 15 amino
acids, derived from such a protein and which can be presented in a
MHC class II context. Such epitopes corresponding to proteins
present in the atherosclerotic plaque can be obtained by grinding
an atherosclerotic plaque as described in XU et al. (1992,
abovementioned).
[0062] Preferably, these epitopes correspond to synthetic peptides.
Typically, the length of these peptides is comprised between 15 and
25 amino acids. Such epitopes can include the apoB-100 epitopes
described in WO 02/080954, the HSP 60 and HSP 70 epitopes described
in WYSOCKI et al. (2002, abovementioned) and in CHAN et al. (1999,
abovementioned).
[0063] For the epitopes derived from apoB-100, the peptides can be
used in their native state, or after incorporation in phopholipid
liposomes, after a modification of the amino acids that mimics the
different modifications of apoB-100 protein that may occur during
oxidation or non-oxidative modification of LDL. Preferably, this
modification is selected among oxidation by exposure to copper,
oxidation after aldehyde-modification, like malone dealdehyde
(MDA), hydroxynonenal or other aldehydes, or acetylation, most
preferably this modification corresponds to oxidation after malone
dealdehyde (MDA)-modification.
[0064] Preferably, these epitopes correspond to the peptides
below:
TABLE-US-00002 (SEQ ID NO: 1) FLDTVYGNCSTHFTVKTRKG; (SEQ ID NO: 2)
PQCSTHILQWLKRVHANPLL; (SEQ ID NO: 3) VISIPRLQAEARSEILAHWS; (SEQ ID
NO: 4) KLVKEALKESQLPTVMDFRK; (SEQ ID NO: 5) LFVTQAEGAKQTEATMTFK;
(SEQ ID NO: 6) DGSLRHKFLDSNIKFSHVEK; (SEQ ID NO: 7)
KGTYGLSCQRDPNTGRLNGE; (SEQ ID NO: 8) RLNGESNLRFNSSYLQGTNQ; (SEQ ID
NO: 9) SLTSTSDLQSGIIKNTASLK; (SEQ ID NO: 10) TASLKYENYELTLKSDTNGK;
(SEQ ID NO: 11) DMTSFKQNALLRSEYQADYE; (SEQ ID NO: 12)
MKVKIIRTIDQMQNSELQWP; (SEQ ID NO: 13) IALDDAKINFNEKLSQLQTY; (SEQ ID
NO: 14) KTTKQSFDLSVKAQYKKNKH; (SEQ ID NO: 15) EEEMLENVSLVCPKDATRFK;
(SEQ ID NO: 16) GSTSHHLVSRKSISAALEHK; (SEQ ID NO: 17)
IENIDFNKSGSSTASWIQNV; (SEQ ID NO: 18) IREVTQRLNGEIQALELPQK; (SEQ ID
NO: 19) EVDVLTKYSQPEDSLIPFFE; (SEQ ID NO: 20) HTFLIYITELLKKLQSTTVM;
(SEQ ID NO: 21) LLDIANYLMEQIQDDCTGDE; (SEQ ID NO: 22)
CTGDEDYTYKIKRVIGNMGQ; (SEQ ID NO: 23) GNMGQTMEQLTPELKSSILK; (SEQ ID
NO: 24) SSILKCVQSTKPSLMIQKAA; (SEQ ID NO: 25) IQKAAIQALRKMEPKDKDQE;
(SEQ ID NO: 26) RLNGESNLRFNSSYLQGTNQ; (SEQ ID NO: 27)
SLNSHGLELNADILGTDKIN; (SEQ ID NO: 28) WIQNVDTKYQIRIQIQEKLQ; (SEQ ID
NO: 29) TYISDWWTLAAKNLTDFAEQ; (SEQ ID NO: 30) EATLQRIYSLWEHSTKNHLQ;
(SEQ ID NO: 31) ALLVPPETEEAKQVLFLDTV; (SEQ ID NO: 32)
IEIGLEGKGFEPTLEALFGF; (SEQ ID NO: 33) SGASMKLTTNGRFREHNAKF; (SEQ ID
NO: 34) NLIGDFEVAEKINAFRAKVH; (SEQ ID NO: 35) GHSVLTAKGMALFGEGKAEF;
(SEQ ID NO: 36) FKSSVITLNTNAELFNQSDI; (SEQ ID NO: 37)
FPDLGQEVALNANTKNQKIR; (SEQ ID NO: 38) ATRFKHLRKYTYNYQAQSSS.
[0065] More preferably, the peptides are selected among
IALDDAKINFNEKLSQLQTY (SEQ ID NO: 13), and KTTKQSFDLSVKAQYKKNKH (SEQ
ID NO: 14).
[0066] The composition may comprise a vehicle. For example, the
composition may comprise emulsions, microemulsions, oil-in-water
emulsions, anhydrous lipids and oil-in-water emulsions, other types
of emulsions. The composition may also comprise one or more
additives (e.g., diluents, excipients, stabilizers, preservatives).
See, generally, Ullmann's Encyclopedia of Industrial Chemistry,
6.sup.th Ed. (various editors, 1989-1998, Marcel Dekker); and
Pharmaceutical Dosage Forms and Drug Delivery Systems (ANSEL et
al., 1994, WILLIAMS & WILKINS).
[0067] Epitope may be solubilized in a buffer or water or
incorporated in emulsions and microemulsions. Suitable buffers
include, but are not limited to, phosphate buffered saline
Ca.sup.++/Mg.sup.++ free (PBS), phosphate buffered saline (PBS),
normal saline (150 mM NaCl in water), and Tris buffer.
[0068] There are numerous causes of peptide instability or
degradation, including hydrolysis and denaturation. Hydrophobic
interaction may cause clumping of molecules together (i.e.
aggregation). This result may entail diminution of the induction of
a Treg response. Stabilizers may be added to lessen or prevent such
problems.
[0069] Stabilizers include cyclodextrine and derivatives thereof
(see U.S. Pat. No. 5,730,969). Suitable preservatives such as
sucrose, mannitol, sorbitol, trehalose, dextran and glycerin can
also be added to stabilize the final formulation. A stabilizer
selected from non-ionic surfactants, D-glucose, D-galactose,
D-xylose, D-galacturonic acid, trehalose, dextrans, hydroxyethyl
starches, and mixtures thereof may be added to the formulation.
Addition of alkali metal salt or magnesium chloride may stabilize a
peptide. The peptide may also be stabilized by contacting it with a
saccharide selected from the group consisting of dextran,
chondroitin sulphuric acid, starch, glycogen, dextrin, and alginic
acid salt. Other sugars that can be added include monosaccharides,
disaccharides, sugar alcohols, and mixtures thereof (E.g., glucose,
mannose, galactose, fructose, sucrose, maltose, lactose, mannitol,
xylitol). Polyols may stabilize a peptide, and are water-miscible
or water-soluble. Suitable polyols may be polyhydroxy alcohols,
monosaccharides and disaccharides including mannitol, glycrol,
ethylene glycol, propylene glycol, trimethyl glycol, vinyl
pyrrolidone, glucose, fructose, arabinose, mannose, maltose,
sucrose, and polymers thereof. Various excipients may also
stabilize peptides, including serum albumin, amino acids, heparin,
fatty acids and phospholipids, surfactants, metals, polyols,
reducing agents, metal chelating agents, polyvinyl pyrrolidone,
hydrolysed gelatin, and ammonium sulfate.
[0070] In another aspect the invention relates to a patch adapted
for the prophylactic or therapeutic treatment by continuous
transcutaneous administration of a subject, suffering from
atherosclerosis, said patch comprising: [0071] (a) a dressing, and
[0072] (b) an effective amount of at least one epitope derived from
a protein present in the atherosclerotic plaque,
[0073] whereby application of said patch to intact skin induces a
specific regulatory immune response, preferably a Treg
response.
[0074] The production of such a patch is described in US patent
application 2004/0028727 A1 in the name of Gregory M. GLENN.
[0075] The patch according to the present invention can be applied
to a subject, which has been diagnosed as presenting one of the
following coronary disorders: [0076] asymptomatic coronary artery
coronary diseases with silent ischemia or without ischemia; [0077]
chronic ischemic disorders without myocardial necrosis, such as
stable or effort angina pectoris; [0078] acute ischemic disorders
myocardial necrosis, such as unstable angina pectoris; [0079]
ischemic disorders with myocardial necrosis, such as ST segment
elevation myocardial infarction or non-ST segment elevation
myocardial infarction.
[0080] The dressing may be occlusive or non-occlusive.
[0081] Advantageously, said effective amount of at least one
epitope is adapted for obtaining an administration of a daily dose
of said at least one epitope within the range of 0.05 to 5000 .mu.g
per kg body weight per day, preferably of 0.5 to 1000 .mu.g and
more preferably of 5 to 500 .mu.g. This daily dose is sufficient to
induce a regulatory immune response but insufficient to induce an
humoral immune response corresponding to the production of
antibodies, specifically of protective antibodies generated against
said epitope, which could for example facilitate the removal of
oxidatively damaged LDL particles by macrophages receptor when the
used epitope is a peptide derived from apolipoprotein B.
[0082] For effective treatment, multiples patches may be applied at
frequent intervals or constantly over a period of time within the
range of 7 days to 30 days, preferably of 10 days to 20, most
preferably about 14 days. (see U.S. Pat. No. 5,049,387 and Example
1 for a detailed description of a patch); or may be applied
simultaneously.
[0083] Proteins present in the atherosclerotic plaque are well
known from one of skills in the art. Such proteins can include
apolipoprotein B-100 (apoB-100, human, Accession number P04114),
collagen type I (human, Accession number CAA67261; AAB59577), type
III (human, Accession number P02458), type IV (human, Accession
number P02462) and type V (human, Accession number CAI17260),
elastin (human, Accession number P15502), laminin (human, Accession
number P024043; Q16363), entactin/nidogen (human, Accession number
P14543), fibronectin (human, Accession number NP.sub.--997647;
NP.sub.--997643; NP.sub.--997641; NP.sub.--997640; NP.sub.--997639;
NP.sub.--997635), thrombospondin (human, Accession number
NP.sub.--003237), vitronectin (human, Accession number P04004),
tenascin (human, Accession number P24821), osteopontin (human,
Accession number NP.sub.--000573), proteoglycans [glycorin, decorin
(human, Accession number AAV38603), versican, hyaluronan], medin
(human, Accession number Q08431), lactadherin (human, Accession
number Q08431), .beta.-amyloid (human, Accession number P05067),
HSP 60 (human, Accession number AAA36022.1) or HSP 70 (human,
Accession number BAA24847.1). Preferably, the protein is selected
among apoB-100, HSP 60 and HSP 70, most preferably the protein is
apoB-100.
[0084] Epitopes from these proteins can be simply identified by one
of skills in the art. These epitopes correspond to proteins present
in the atherosclerotic plaque or to synthetic peptides from more
than 10 amino acids length, more preferably from more than 15 amino
acids, derived from such a protein and which can be presented in a
MHC class II context. Such epitopes corresponding to proteins
present in the atherosclerotic plaque can be obtained by grinding
an atherosclerotic plaque as described in XU et al. (1992,
abovementioned).
[0085] Preferably, these epitopes correspond to synthetic peptides.
Typically, the length of these peptides is comprised between 15 and
25 amino acids. Such epitopes can include the apoB-100 epitopes
described in WO 02/080954, the HSP 60 and HSP 70 epitopes described
in WYSOCKI et al. (2002, abovementioned) and in CHAN et al. (1999,
abovementioned).
[0086] For the epitopes derived from apoB-100, the peptides can be
used in their native state, or after incorporation in phopholipid
liposomes, after a modification of the amino acids that mimics the
different modifications of apoB-100 protein that may occur during
oxidation or non-oxidative modification of LDL. Preferably, this
modification is selected among oxidation by exposure to copper,
oxidation after aldehyde-modification, like malone dealdehyde
(MDA), hydroxynonenal or other aldehydes, or acetylation, most
preferably this modification corresponds to oxidation after malone
dealdehyde (MDA)-modification.
[0087] Preferably, these epitopes correspond to the peptides
below:
TABLE-US-00003 (SEQ ID NO: 1) FLDTVYGNCSTHFTVKTRKG; (SEQ ID NO: 2)
PQCSTHILQWLKRVHANPLL; (SEQ ID NO: 3) VISIPRLQAEARSEILAHWS; (SEQ ID
NO: 4) KLVKEALKESQLPTVMDFRK; (SEQ ID NO: 5) LFVTQAEGAKQTEATMTFK;
(SEQ ID NO: 6) DGSLRHKFLDSNIKFSHVEK; (SEQ ID NO: 7)
KGTYGLSCQRDPNTGRLNGE; (SEQ ID NO: 8) RLNGESNLRFNSSYLQGTNQ; (SEQ ID
NO: 9) SLTSTSDLQSGIIKNTASLK; (SEQ ID NO: 10) TASLKYENYELTLKSDTNGK;
(SEQ ID NO: 11) DMTSFKQNALLRSEYQADYE; (SEQ ID NO: 12)
MKVKIIRTIDQMQNSELQWP; (SEQ ID NO: 13) IALDDAKINFNEKLSQLQTY; (SEQ ID
NO: 14) KTTKQSFDLSVKAQYKKNKH; (SEQ ID NO: 15) EEEMLENVSLVCPKDATRFK;
(SEQ ID NO: 16) GSTSHHLVSRKSISAALEHK; (SEQ ID NO: 17)
IENIDFNKSGSSTASWIQNV; (SEQ ID NO: 18) IREVTQRLNGEIQALELPQK; (SEQ ID
NO: 19) EVDVLTKYSQPEDSLIPFFE; (SEQ ID NO: 20) HTFLIYITELLKKLQSTTVM;
(SEQ ID NO: 21) LLDIANYLMEQIQDDCTGDE; (SEQ ID NO: 22)
CTGDEDYTYKIKRVIGNMGQ; (SEQ ID NO: 23) GNMGQTMEQLTPELKSSILK; (SEQ ID
NO: 24) SSILKCVQSTKPSLMIQKAA; (SEQ ID NO: 25) IQKAAIQALRKMEPKDKDQE;
(SEQ ID NO: 26) RLNGESNLRFNSSYLQGTNQ; (SEQ ID NO: 27)
SLNSHGLELNADILGTDKIN; (SEQ ID NO: 28) WIQNVDTKYQIRIQIQEKLQ; (SEQ ID
NO: 29) TYISDWWTLAAKNLTDFAEQ; (SEQ ID NO: 30) EATLQRIYSLWEHSTKNHLQ;
(SEQ ID NO: 31) ALLVPPETEEAKQVLFLDTV; (SEQ ID NO: 32)
IEIGLEGKGFEPTLEALFGF; (SEQ ID NO: 33) SGASMKLTTNGRFREHNAKF; (SEQ ID
NO: 34) NLIGDFEVAEKINAFRAKVH; (SEQ ID NO: 35) GHSVLTAKGMALFGEGKAEF;
(SEQ ID NO: 36) FKSSVITLNTNAELFNQSDI; (SEQ ID NO: 37)
FPDLGQEVALNANTKNQKIR; (SEQ ID NO: 38) ATRFKHLRKYTYNYQAQSSS.
[0088] More preferably, the peptides are selected among
IALDDAKINFNEKLSQLQTY (SEQ ID NO: 13), and KTTKQSFDLSVKAQYKKNKH (SEQ
ID NO: 14).
[0089] The patch may include a controlled, released reservoir or, a
matrix or rate controlling membrane, which allows stepped release
of epitope. Such a patch is described in EP 0318385. Preferably
said epitope is maintained in a dry form prior to administration.
Subsequent release of liquid from a reservoir or entry of liquid
into the reservoir containing the dry ingredient of the formulation
will at least partially dissolve that ingredient.
[0090] The formulation may comprise a vehicle. For example, the
formulation may comprise AQUAFOR (an emulsion of petrolatum,
mineral oil, mineral wax, wool wax, panthenol, bisabol, and
glycerin as shown in WO 98/20734), emulsions, microemulsions, gels,
oil-in-water emulsions, anhydrous lipids and oil-in-water
emulsions, other types of emulsions, fats waxes, oil, silicones,
gels and humectants. The formulation may also comprise one or more
additives (e.g., diluents, binders, excipients, stabilizers,
dessicants, preservatives, coloring). See, generally, Ullmann's
Encyclopedia of Industrial Chemistry, (abovementioned) and
Pharmaceutical Dosage Forms and Drug Delivery Systems
(abovementioned).
[0091] Advantageously, said formulation does not contain any
adjuvant or any effective amount of such an adjuvant likely to
induce antibodies' expression.
[0092] Epitope may be solubilized in a buffer or water or organic
solvents such as alcohol or DMSO, or incorporated in gels,
emulsions, microemulsions, and creams. Suitable buffers include,
but are not limited to, phosphate buffered saline
Ca.sup.++/Mg.sup.++ free (PBS), phosphate buffered saline (PBS),
normal saline (150 mM NaCl in water), and Tris buffer. Epitope not
soluble in neutral buffer can be solubilized in 10 mM acetic acid
and then diluted to the desired volume with a neutral buffer such
as PBS. In the case of epitope soluble only at acid pH, acetate-PBS
at acid pH may be used as a diluent after solubilization in dilute
acetic acid. Glycerol may be a suitable non-aqueous buffer for use
in the invention.
[0093] There are numerous causes of peptide instability or
degradation, including hydrolysis and denaturation. Hydrophobic
interaction may cause clumping of molecules together (i.e.
aggregation). This result may entail diminution of the induction of
a Treg response. Stabilizers may be added to lessen or prevent such
problems.
[0094] This formulation, or any intermediate in its production, may
be pretreated with agents (i.e., cryoprotectants and dry
stabilizers) and then subjected to cooling rates and final
temperatures that minimize ice crystal formation. By proper
selection of cryo agents and use of pre-selected drying parameters,
almost any formulation might be cryoprepared for a suitable desired
end use.
[0095] Stabilizers include cyclodextrine and derivatives thereof
(see U.S. Pat. No. 5,730,969). Suitable preservatives such as
sucrose, mannitol, sorbitol, trehalose, dextran and glycerin can
also be added to stabilize the final formulation. A stabilizer
selected from non-ionic surfactants, D-glucose, D-galactose,
D-xylose, D-galacturonic acid, trehalose, dextrans, hydroxyethyl
starches, and mixtures thereof may be added to the formulation.
Addition of alkali metal salt or magnesium chloride may stabilize a
peptide. The peptide may also be stabilized by contacting it with a
saccharide selected from the group consisting of dextran,
chondroitin sulphuric acid, starch, glycogen, dextrin, and alginic
acid salt. Other sugars that can be added include monosaccharides,
disaccharides, sugar alcohols, and mixtures thereof (E.g., glucose,
mannose, galactose, fructose, sucrose, maltose, lactose, mannitol,
xylitol). Polyols may stabilize a peptide, and are water-miscible
or water-soluble. Suitable polyols may be polyhydroxy alcohols,
monosaccharides and disaccharides including mannitol, glycrol,
ethylene glycol, propylene glycol, trimethyl glycol, vinyl
pyrrolidone, glucose, fructose, arabinose, mannose, maltose,
sucrose, and polymers thereof. Various excipients may also
stabilize peptides, including serum albumin, amino acids, heparin,
fatty acids and phospholipids, surfactants, metals, polyols,
reducing agents, metal chelating agents, polyvinyl pyrrolidone,
hydrolysed gelatin, and ammonium sulfate.
[0096] In another aspect the present invention relates to the Use
of at least one epitope derived from a protein present in the
atherosclerotic plaque for the manufacture of a medicament for use
in the prevention or treatment by continuous subcutaneous or
transcutaneous administration, which induce a specific regulatory
immune response, preferably a Treg response, to a subject suffering
from atherosclerosis.
[0097] Said medicament can be administrated to a subject, which has
been diagnosed as presenting one of the following coronary
disorders: [0098] asymptomatic coronary artery coronary diseases
with silent ischemia or without ischemia; [0099] chronic ischemic
disorders without myocardial necrosis, such as stable or effort
angina pectoris; [0100] acute ischemic disorders myocardial
necrosis, such as unstable angina pectoris; [0101] ischemic
disorders with myocardial necrosis, such as ST segment elevation
myocardial infarction or non-ST segment elevation myocardial
infarction.
[0102] Preferably, said medicament is administrated for a period of
time and at a daily dose of said at least one epitope, which are
sufficient to induce a regulatory immune response, preferably a
Treg response.
[0103] Proteins present in the atherosclerotic plaque are well
known from one of skills in the art. Such proteins can include
apolipoprotein B-100 (apoB-100, human, Accession number P04114),
collagen type I (human, Accession number CAA67261; AAB59577), type
III (human, Accession number P02458), type IV (human, Accession
number P02462) and type V (human, Accession number CA117260),
elastin (human, Accession number P15502), laminin (human, Accession
number P024043; Q16363), entactin/nidogen (human, Accession number
P14543), fibronectin (human, Accession number NP.sub.--997647;
NP.sub.--997643; NP.sub.--997641; NP.sub.--997640; NP.sub.--997639;
NP.sub.--997635), thrombospondin (human, Accession number
NP.sub.--003237), vitronectin (human, Accession number P04004),
tenascin (human, Accession number P24821), osteopontin (human,
Accession number NP.sub.--000573), proteoglycans [glycorin, decorin
(human, Accession number AAV38603), versican, hyaluronan], medin
(human, Accession number Q08431), lactadherin (human, Accession
number Q08431), .beta.-amyloid (human, Accession number P05067),
HSP 60 (human, Accession number AAA36022.1) or HSP 70 (human,
Accession number BAA24847.1). Preferably, the protein is selected
among apoB-100, HSP 60 and HSP 70, most preferably the protein is
apoB-100.
[0104] Epitopes from these proteins can be simply identified by one
of skills in the art. These epitopes correspond to proteins present
in the atherosclerotic plaque or to synthetic peptides from more
than 10 amino acids length, more preferably from more than 15 amino
acids, derived from such a protein and which can be presented in a
MHC class II context. Such epitopes corresponding to proteins
present in the atherosclerotic plaque can be obtained by grinding
an atherosclerotic plaque as described in XU et al.(2002,
abovementioned).
[0105] Preferably, these epitopes correspond to synthetic peptides.
Typically, the length of these peptides is comprised between 15 and
25 amino acids. Such epitopes can include the apoB-100 epitopes
described in WO 02/080954, the HSP 60 and HSP 70 epitopes described
in WYSOCKI et al. (2002, abovementioned) and in CHAN et al. (1999,
abovementioned).
[0106] For the epitopes derived from apoB-100, the peptides can be
used in their native state, or after incorporation in phopholipid
liposomes, after a modification of the amino acids that mimics the
different modifications of apoB-100 protein that may occur during
oxidation or non-oxidative modification of LDL. Preferably, this
modification is selected among oxidation by exposure to copper,
oxidation after aldehyde-modification, like malone dealdehyde
(MDA), hydroxynonenal or other aldehydes, or acetylation, most
preferably this modification corresponds to oxidation after malone
dealdehyde (MDA)-modification.
[0107] Preferably, these epitopes correspond to the peptides
below:
TABLE-US-00004 (SEQ ID NO: 1) FLDTVYGNCSTHFTVKTRKG; (SEQ ID NO: 2)
PQCSTHILQWLKRVHANPLL; (SEQ ID NO: 3) VISIPRLQAEARSEILAHWS; (SEQ ID
NO: 4) KLVKEALKESQLPTVMDFRK; (SEQ ID NO: 5) LFVTQAEGAKQTEATMTFK;
(SEQ ID NO: 6) DGSLRHKFLDSNIKFSHVEK; (SEQ ID NO: 7)
KGTYGLSCQRDPNTGRLNGE; (SEQ ID NO: 8) RLNGESNLRFNSSYLQGTNQ; (SEQ ID
NO: 9) SLTSTSDLQSGIIKNTASLK; (SEQ ID NO: 10) TASLKYENYELTLKSDTNGK;
(SEQ ID NO: 11) DMTSFKQNALLRSEYQADYE; (SEQ ID NO: 12)
MKVKIIRTIDQMQNSELQWP; (SEQ ID NO: 13) IALDDAKINFNEKLSQLQTY; (SEQ ID
NO: 14) KTTKQSFDLSVKAQYKKNKH; (SEQ ID NO: 15) EEEMLENVSLVCPKDATRFK;
(SEQ ID NO: 16) GSTSHHLVSRKSISAALEHK; (SEQ ID NO: 17)
IENIDFNKSGSSTASWIQNV; (SEQ ID NO: 18) IREVTQRLNGEIQALELPQK; (SEQ ID
NO: 19) EVDVLTKYSQPEDSLIPFFE; (SEQ ID NO: 20) HTFLIYITELLKKLQSTTVM;
(SEQ ID NO: 21) LLDIANYLMEQIQDDCTGDE; (SEQ ID NO: 22)
CTGDEDYTYKIKRVIGNMGQ; (SEQ ID NO: 23) GNMGQTMEQLTPELKSSILK; (SEQ ID
NO: 24) SSILKCVQSTKPSLMIQKAA; (SEQ ID NO: 25) IQKAAIQALRKMEPKDKDQE;
(SEQ ID NO: 26) RLNGESNLRFNSSYLQGTNQ; (SEQ ID NO: 27)
SLNSHGLELNADILGTDKIN; (SEQ ID NO: 28) WIQNVDTKYQIRIQIQEKLQ; (SEQ ID
NO: 29) TYISDWWTLAAKNLTDFAEQ; (SEQ ID NO: 30) EATLQRIYSLWEHSTKNHLQ;
(SEQ ID NO: 31) ALLVPPETEEAKQVLFLDTV; (SEQ ID NO: 32)
IEIGLEGKGFEPTLEALFGF; (SEQ ID NO: 33) SGASMKLTTNGRFREHNAKF; (SEQ ID
NO: 34) NLIGDFEVAEKINAFRAKVH; (SEQ ID NO: 35) GHSVLTAKGMALFGEGKAEF;
(SEQ ID NO: 36) FKSSVITLNTNAELFNQSDI; (SEQ ID NO: 37)
FPDLGQEVALNANTKNQKIR; (SEQ ID NO: 38) ATRFKHLRKYTYNYQAQSSS.
[0108] More preferably, the peptides are selected among
IALDDAKINFNEKLSQLQTY (SEQ ID NO: 13), and KTTKQSFDLSVKAQYKKNKH (SEQ
ID NO: 14).
[0109] Advantageously, said medicament does not contain any
adjuvant or any effective amount of such an adjuvant likely to
induce antibodies' expression.
[0110] Advantageously, said medicament is administrated for a
period of time within the range of 7 days to 30 days, preferably of
10 days to 20, most preferably about 14 days. This period might be
repeated one or several times.
[0111] Advantageously, said medicament is administrated at a daily
dose within the range of 0.05 to 5000 .mu.g per kg body weight per
day, preferably of 0.5 to 1000 .mu.g and more preferably of 5 to
500 .mu.g. This daily dose is sufficient to induce a regulatory
immune response but insufficient to induce an humoral immune
response corresponding to the production of antibodies,
specifically of protective antibodies generated against said
epitope, which could for example facilitate the removal of
oxidatively damaged LDL particles by macrophages receptor when the
used epitope is a peptide derived from apolipoprotein B.
[0112] The invention is further illustrated below by the following
Examples, which are not intended to limit its scope.
EXAMPLE 1
[0113] 1. Peptides.
[0114] P210: Human apoB-100 derived peptide (SEQ ID NO: 14,
KTTKQSFDLSVKAQYKKNKH, amino acids 3136 to 3155). The homology
between human (Accession number: P04114) and mouse (Accession
number: XP.sub.--137955) sequences is 90% for this peptide.
[0115] MDA P210: Malondialdehyde (MDA)-modified human apoB-100
derived peptide. A fraction of native P210 was modified by 0.5
mol/L MDA for 3 hours at 37.degree. C. The MDA-modified peptide was
dialysed against PBS containing 1 mmol/L EDTA with several changes
for 18 hours at 4.degree. C. The MDA modification of P210 was
assessed using the thiobarbituric acid reactive substances
assay.
[0116] P240: Human apoB-100 derived peptide (SEQ ID NO: 37,
FPDLGQEVALNANTKNQKIR, amino acids 3586 to 3605). The homology
between human (Accession number: P04114) and mouse (Accession
number: XP.sub.--137955) sequences is 86% amino acids 3591 to 3604
(SEQ ID NO: 39, QEVALNANTKNQKI).
[0117] Control: phosphate buffer saline (PBS).
[0118] 2. Peptide Delivery Pump Treatment.
[0119] Male ApoE -/- mice (B&M, RY, DENMARK, 11 week old) were
implanted subcutaneously with mini-osmotic pumps (ALZET1002, DURECT
CORPORATION) diffusing PBS or 10 .mu.g of P210, MDA-P210 or P240
per day and for 14 day, at a rate of 0.25 .mu.l/h.
[0120] Then, the mice are kept for another 6 weeks and killed at 19
weeks of age.
[0121] 3. Purification and Culture of Spleen and Lymph Node
Cells
[0122] T cells were purified from spleen or from draining lymph
nodes by negative selection with anti-CD11b (M1/70), anti B220,
anti-CD8 and anti-NK cells (DX5) followed by depletion with a
mixture of magnetic beads coated with anti-rat Ig (DYNAL). CD11c+
dendritic cells were purified by positive selection with anti-CD11c
using directly conjugated anti-CD11c beads (clone N418; MILTENYI
BIOTEC).
[0123] For cytokine measurements, purified T cells were cultured in
96-well plates in the presence of anti-CD3 (5 .mu.g/ml) and
anti-CD28 (1 .mu.g/ml) antibodies. Supernatants were collected at
24 hours (for IL-4 measurements) and at hours (for IL-5, IL-10 and
IFN-.gamma. measurements) and assayed for cytokine levels by
ELISA.
[0124] For the cell proliferation assay, purified CD4+ cells (50
000) were mixed with CD11c+ cells (10 000) and anti-CD3 antibodies
(3 .mu.g/ml) for 72 hours. [3H]-Thymidine (1 .mu.Ci; PERKIN ELMER)
was added for the last 18 hours of cell culture.
[0125] Co-culture experiments were performed to evaluate regulatory
T cell function. Isolated dendritic cells were also assessed for
their cytokine production
[0126] 4. Analysis of Atherosclerotic Plaque Size and
Composition.
[0127] Total Plasma and HDL cholesterol were measured with a
commercially available cholesterol kit (SIGMA) according to the
manufacturer's instructions. Morphometric and immuno-histochemical
studies were performed in the aortic sinus and in the thoracic
aorta (spanning from the brachiocephalic artery to the renal
arteries). Collagen fibers were stained with Sirius red.
Immuno-histochemical analysis were then performed. The following
primary antibodies were used: MOMA-2 (BIOSOURCE INTERNATIONAL) as a
specific marker for macrophages; anti-mouse CD3-(SANTA CRUZ);
anti-smooth muscle actin, alkaline phosphatase conjugate, clone 1A4
(SIGMA); and anti-IL-10 antibody (SANTA CRUZ). Morphometric
analysis was performed with an automated image processor (HISTOLAB,
MICROVISION).
[0128] 5. Determination of Antibody Titers Against Peptide.
[0129] Native or MDA-modified peptides 210 and 240 were used for
coating (10 .mu.g/ml of each in PBS pH 7.4) microtiter plates (Nunc
MaxiSorp, Nunc, Roskilde, Denmark) in an overnight incubation at
4.degree. C. Coated plates were washed with PBS with 0.05% Tween-20
and thereafter blocked with SuperBiock in Tris-buffered saline
(TBS, Pierce) for 5 minutes at room temperature followed by an
incubation of mouse serum diluted 1:50 in TBS-0.05% Tween-20 for 2
hours at room temperature and overnight at 4.degree. C. After
rinsing, depositions were detected by using biotinylated goat
anti-mouse IgM or IgG antibodies (JACKSON IMMUNORESEARCH, West
Grove, Pa.) that were incubated for 2 hours at room temperature.
The plates were washed and bound biotinylated antibodies were
detected by alkaline phosphatase-conjugated streptavidin (SIGMA).
The color reaction was developed using phosphatase substrate kit
(Pierce). The absorbency at 405 nm was measured after 1 hour of
incubation at room temperature. Mean values were calculated after
subtraction of background absorbance.
[0130] 6. Determination of Functional Regulatory T Cell
Activity
[0131] CD4+CD25- cells in RPMI 1640 supplemented with Glutamax, 10%
FCS, 0.02 mM 2.beta.-mercaptoethanol and antibiotics were
co-cultured with CD11c+ dendritic cells and CD4+CD25+ regulatory T
cells at a ratio of CD25-/CD25+ of 1:1, 1:2, 1:4, 1:8, in
flat-bottomed 96-well microplates (0.5.times.10.sup.5 cells/well
;total volume 200 .mu.l/well). Cells were stimulated with purified
soluble CD3-specific antibody (1 .mu.g/ml, Pharmingen). Cells were
cultured at 37.degree. C. for 72 h and pulsed with 1 .mu.Ci of
[.sup.3H] thymidine (Amersham) for the last 18 h of culture.
Thymidine incorporation was assessed using a TopCount NXT
scintillation counter (Perkin Elmer).
[0132] 7. Results
[0133] Total plasma cholesterol levels were not different in the
different groups: 4.82.+-.0.60 g/l in control mice receiving PBS,
5.84.+-.0.61 g/l in mice receiving MDA-P210, 4.66.+-.0.45 g/l in
mice receiving P210, and 4.95.+-.0.59 g/l in mice receiving
P240.
[0134] Lesion size in the control group receiving PBS was 80
664.+-.14 541 .mu.m.sup.2. It was markedly reduced in mice
receiving MDA-P210 (26 479.+-.4 303 .mu.m.sup.2, p<0.0007) and
P210 (32 301.+-.11 307 .mu.m.sup.2, p<0.003), and less, albeit
significantly, decreased in mice receiving P240 (41 688.+-.10 301
.mu.m.sup.2, p<0.02).
[0135] Proliferation of CD4+ cells from control mice in the
presence of CD11c+ cells from mice receiving MDA-P210 was markedly
decreased compared with that in the presence of CD11c+ cells from
control mice receiving PBS (9500 cpm versus 20 000 cpm), indicating
that dendritic cells from mice receiving MDA-P210 acquired
tolerogenic potential.
[0136] IgG antibody levels against native or MDA-modified peptides
were not significantly different in the different groups. IgG
against P210 were 0.81.+-.0.22, 0.51.+-.0.07, 0.47.+-.0.09 and
0.94.+-.0.72 absorbance units in P210, MDA-P210, P240 and PBS
groups, respectively. IgG against MDA-P210 were 1.45.+-.0.49,
0.83.+-.0.28, 0.62.+-.0,11 and 0.82.+-.0.42 absorbance units in
P210, MDA-P210, P240 and PBS groups, respectively. IgG against P240
were undectable. IgG against MDA-P240 were 0.080.+-.0.038,
0.041.+-.0.007, 0.037.+-.0.007 and 0.049.+-.0.019 absorbance units
in P210, MDA-P210, P240 and PBS groups, respectively.
[0137] IgM antibody levels against native or MDA-modified peptides
were not significantly different in the different groups. IgM
against P210 were 1.17.+-.0.22, 0.55.+-.0.25, 0.79.+-.0.45 and
1.05.+-.0.30 absorbance units in P210, MDA-P210, P240 and PBS
groups, respectively. IgM against MDA-P210 were 1.38.+-.0.04,
1.30.+-.0.13, 0.79.+-.0.45 and 1.39.+-.0.08 absorbance units in
P210, MDA-P210, P240 and PBS groups, respectively. IgM against P240
were undectable. IgM against MDA-P240 were 1.80.+-.0.41,
1.24.+-.0.40, 1.34.+-.0.52 and 1.14.+-.0.62 absorbance units in
P210, MDA-P210, P240 and PBS groups, respectively.
EXAMPLE 2
[0138] 1. Peptides.
[0139] P210: Human apoB-100 derived peptide (SEQ ID NO: 14,
KTTKQSFDLSVKAQYKKNKH, amino acids 3136 to 3155). The homology
between human (Accession number: P04114) and mouse (Accession
number: XP.sub.--137955) sequences is 90% for this peptide.
[0140] MDA P210: Malondialdehyde (MDA)-modified human apoB-100
derived peptide. A fraction of native P210 was modified by 0.5
mol/L MDA for 3 hours at 37.degree. C. The MDA-modified peptide was
dialysed against PBS containing 1 mmol/L EDTA with several changes
for 18 hours at 4.degree. C. The MDA modification of P210 was
assessed using the thiobarbituric acid reactive substances
assay.
[0141] P240: Human apoB-100 peptide (SEQ ID NO: 37,
FPDLGQEVALNANTKNQKIR, amino acids 3586 to 3605). The homology
between human (Accession number: P04114) and mouse (Accession
number: XP.sub.--137955) sequences is 86% between amino acids 3591
and 3604 (SEQ ID NO: 39, QEVALNANTKNQKI).
[0142] Control: chicken ovalbumin (OVA) peptide (SEQ ID NO: 40,
ISQAVHAAHAEINEAGR, amino acids 323 to 339).
[0143] 2. Peptide Delivery Pump Treatment.
[0144] Male apoE-/- mice (B&M, RY, DENMARK, 11 week old) were
implanted subcutaneously with mini-osmotic pumps (ALZET1002, DURECT
CORPORATION) diffusing phosphate buffer saline (PBS) as control or
10 .mu.g per day of P210, MDA-P210 or P240, for 14 days, at a rate
of 0.25 .mu.L/l. An additional group of mice receiving ovalbumin
(OVA) peptide (323-339) served as control.
[0145] Then, the mice were kept for another 6 or 10 weeks and
killed at 19 or 23 weeks of age.
[0146] 3. Purification and Culture of Spleen and Lymph Node
Cells
[0147] CD4+ cells were purified from spleen and draining lymph
nodes by negative selection with anti-CD11b (M1/70), anti B220,
anti CD8 and anti-NK cells (DX5) followed by depletion with a
mixture of magnetic beads coated with anti-rat Ig (DYNAL). CD11c+
dendritic cells were purified by positive selection with anti-CD11c
using directly conjugated anti-CD11c beads (clone N418; MILTENYI
BIOTEC).
[0148] For cytokine measurements, purified T cells were cultured in
96-well plates in the presence of anti-CD3 (5 .mu.g/mL)+anti-CD28
(1 .mu.G/mL) antibodies. Supernatants were collected at 24 hours
(for IL-4 measurements) and at 48 hours (for IL-5, IL-10 and
IFN-.gamma. measurements) and assayed for cytokine levels by
ELISA.
[0149] For the cell proliferation assay, purified CD4+ cells (50
000) were mixed with CD11c+ dendritic cells (10 000) and anti-CD3
antibodies (3 .mu.g/ml) for 72 hours. [3H]-Thymidine (1 .mu.Ci;
PERKIN ELMER) was added for the last 18 hours of cell culture.
Co-culture experiments were performed to evaluate regulatory T cell
function. Isolated dendritic cells were also assessed for their
cytokine production.
[0150] 4. Analysis of Atherosclerotic Plaque Size and
Composition
[0151] Total Plasma and HDL cholesterol were measured with a
commercially available cholesterol kit (SIGMA) according to the
manufacturer's instructions. Morphometric and immuno-histochemical
studies were performed in the aortic sinus and in the thoracic
aorta (spanning from the brachiocephalic artery to the renal
arteries). Collagen fibers were stained with Sirius red.
Immuno-histochemical analysis were then performed. The following
primary antibodies were used : MOMA-2 (BIOSOURCE INTERNATIONAL) as
a specific marker for macrophages; anti-mouse CD3- (SANTA CRUZ);
anti-smooth muscle actin, alkaline phosphatase conjugate, clone 1A4
(SIGMA); and anti-IL-10 antibody (SANTA CRUZ). Morphometric
analysis was performed with an automated image processor (HISTOLAB,
MICROVISION).
[0152] 5. Results
[0153] Male apoE-/- mice (11-week old) were implanted
subcutaneously with mini-osmotic pumps (ALZET1002) diffusing
phosphate buffer saline (PBS), as control, or 10 .mu.g per day of
P210, MDA-P210 or P240, for 14 days at a rate of 0.25 .mu.l/h. An
additional group of mice receiving ovalbumin (OVA) peptide
(323-339) served as control. Mice were kept for another 6 weeks and
killed at 19 weeks of age.
[0154] Total plasma cholesterol levels were not different in the
different groups: 5.09.+-.0.78 g/L in control mice receiving PBS,
4.94.+-.0.39 g/L in mice receiving P210, 5.49.+-.0.50 g/L in mice
receiving MDA-P210, 4.81.+-.0.51 g/L in mice receiving P240, and
5.51.+-.0.78 in mice receiving OVA.
[0155] Lesion size in the control group receiving PBS was 67
001.+-.12 194 .mu.m2 (n=9). it was markedly reduced in mice
receiving P210 (27 428.+-.7 735 .mu.m2, n=9, p<0.0015), MDA-P210
(31 791.+-.4 284 .mu.m2, n=11, p<0.0029) or P240 (38 080.+-.7
546 .mu.m2, n=8, p<0.02), but was not significantly different in
mice receiving OVA (52 720.+-.10 227, n=5, p=0.31).
[0156] To evaluate whether the anti-atherogenic effect of
subcutaneous administration of low doses of apoB peptides for 14
days persists at long term, male apoE-/- mice (11-week old) were
implanted with mini-osmotic pumps diffusing PBS, P210, MDA-P210 or
P240 for 14 days were kept for another 10 weeks and killed at 23
weeks of age.
[0157] Total plasma cholesterol levels were not different in the
different groups: 6.38.+-.0.87 g/L in mice receiving PBS,
5.40.+-.0.67 g/L in mice receiving P210, 4.67.+-.0.89 g/L in mice
receiving MDA-P210, 6.64.+-.0.64 g/L in mice receiving P240.
[0158] Lesion size in the control group receiving PBS was 96
203.+-.13 498 .mu.m2 (n=6). It was markedly reduced in mice
receiving P210 (58 543.+-.16 735 .mu.m2, n=6, p<0.05) or P240
(53 920.+-.8 045 .mu.m2, n=8, p<0.02), but it was not different
in mice receiving MDA-P210 (69 026.+-.12 443 .mu.m2, n=4,
p=0.19)
[0159] The continuous administration of p210 peptide and p210 MDA
to apoE null mice results in a diminution of the atherosclerotic
plaques and of associated inflammation, together with a change in
the composition of the plaque toward a more stable phenotype. No
significant modification was observed with the administration of
the control peptide.
Sequence CWU 1
1
40120PRTArtificial sequencepeptide derived from apolipoprotein B
1Phe Leu Asp Thr Val Tyr Gly Asn Cys Ser Thr His Phe Thr Val Lys 1
5 10 15 Thr Arg Lys Gly 20 220PRTArtificial sequencepeptide derived
from apolipoprotein B 2Pro Gln Cys Ser Thr His Ile Leu Gln Trp Leu
Lys Arg Val His Ala 1 5 10 15 Asn Pro Leu Leu 20 320PRTArtificial
sequencepeptide derived from apolipoprotein B 3Val Ile Ser Ile Pro
Arg Leu Gln Ala Glu Ala Arg Ser Glu Ile Leu 1 5 10 15 Ala His Trp
Ser 20 420PRTArtificial sequencepeptide derived from apolipoprotein
B 4Lys Leu Val Lys Glu Ala Leu Lys Glu Ser Gln Leu Pro Thr Val Met
1 5 10 15 Asp Phe Arg Lys 20 519PRTArtificial sequencepeptide
derived from apolipoprotein B 5Leu Phe Val Thr Gln Ala Glu Gly Ala
Lys Gln Thr Glu Ala Thr Met 1 5 10 15 Thr Phe Lys 620PRTArtificial
sequencepeptide derived from apolipoprotein B 6Asp Gly Ser Leu Arg
His Lys Phe Leu Asp Ser Asn Ile Lys Phe Ser 1 5 10 15 His Val Glu
Lys 20 720PRTArtificialpeptide derived from apolipoprotein B 7Lys
Gly Thr Tyr Gly Leu Ser Cys Gln Arg Asp Pro Asn Thr Gly Arg 1 5 10
15 Leu Asn Gly Glu 20 820PRTArtificial sequencepeptide derived from
apolipoprotein B 8Arg Leu Asn Gly Glu Ser Asn Leu Arg Phe Asn Ser
Ser Tyr Leu Gln 1 5 10 15 Gly Thr Asn Gln 20 920PRTArtificial
sequencepeptide derived from apolipoprotein B 9Ser Leu Thr Ser Thr
Ser Asp Leu Gln Ser Gly Ile Ile Lys Asn Thr 1 5 10 15 Ala Ser Leu
Lys 20 1020PRTArtificial sequencepeptide derived from
apolipoprotein B 10Thr Ala Ser Leu Lys Tyr Glu Asn Tyr Glu Leu Thr
Leu Lys Ser Asp 1 5 10 15 Thr Asn Gly Lys 20 1120PRTArtificial
sequencepeptide derived from apolipoprotein B 11Asp Met Thr Ser Phe
Lys Gln Asn Ala Leu Leu Arg Ser Glu Tyr Gln 1 5 10 15 Ala Asp Tyr
Glu 20 1220PRTArtificial sequencepeptide derived from
apolipoprotein B 12Met Lys Val Lys Ile Ile Arg Thr Ile Asp Gln Met
Gln Asn Ser Glu 1 5 10 15 Leu Gln Trp Pro 20 1320PRTArtificial
sequencepeptide derived from apolipoprotein B 13Ile Ala Leu Asp Asp
Ala Lys Ile Asn Phe Asn Glu Lys Leu Ser Gln 1 5 10 15 Leu Gln Thr
Tyr 20 1420PRTArtificial sequencepeptide derived from
apolipoprotein B 14Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys
Ala Gln Tyr Lys 1 5 10 15 Lys Asn Lys His 20 1520PRTArtificial
sequencepeptide derived from apolipoprotein B 15Glu Glu Glu Met Leu
Glu Asn Val Ser Leu Val Cys Pro Lys Asp Ala 1 5 10 15 Thr Arg Phe
Lys 20 1620PRTArtificial sequencepeptide derived from
apolipoprotein B 16Gly Ser Thr Ser His His Leu Val Ser Arg Lys Ser
Ile Ser Ala Ala 1 5 10 15 Leu Glu His Lys 20 1720PRTArtificial
sequencepeptide derived from apolipoprotein B 17Ile Glu Asn Ile Asp
Phe Asn Lys Ser Gly Ser Ser Thr Ala Ser Trp 1 5 10 15 Ile Gln Asn
Val 20 1820PRTArtificial sequencepeptide derived from
apolipoprotein B 18Ile Arg Glu Val Thr Gln Arg Leu Asn Gly Glu Ile
Gln Ala Leu Glu 1 5 10 15 Leu Pro Gln Lys 20 1920PRTArtificial
sequencepeptide derived from apolipoprotein B 19Glu Val Asp Val Leu
Thr Lys Tyr Ser Gln Pro Glu Asp Ser Leu Ile 1 5 10 15 Pro Phe Phe
Glu 20 2020PRTArtificial sequencepeptide derived from
apolipoprotein B 20His Thr Phe Leu Ile Tyr Ile Thr Glu Leu Leu Lys
Lys Leu Gln Ser 1 5 10 15 Thr Thr Val Met 20 2120PRTArtificial
sequencepeptide derived from apolipoprotein B 21Leu Leu Asp Ile Ala
Asn Tyr Leu Met Glu Gln Ile Gln Asp Asp Cys 1 5 10 15 Thr Gly Asp
Glu 20 2220PRTArtificial sequencepeptide derived from
apolipoprotein B 22Cys Thr Gly Asp Glu Asp Tyr Thr Tyr Lys Ile Lys
Arg Val Ile Gly 1 5 10 15 Asn Met Gly Gln 20 2320PRTArtificial
sequencepeptide derived from apolipoprotein B 23Gly Asn Met Gly Gln
Thr Met Glu Gln Leu Thr Pro Glu Leu Lys Ser 1 5 10 15 Ser Ile Leu
Lys 20 2420PRTArtificial sequencepeptide derived from
apolipoprotein B 24Ser Ser Ile Leu Lys Cys Val Gln Ser Thr Lys Pro
Ser Leu Met Ile 1 5 10 15 Gln Lys Ala Ala 20 2520PRTArtificial
sequencepeptide derived from apolipoprotein B 25Ile Gln Lys Ala Ala
Ile Gln Ala Leu Arg Lys Met Glu Pro Lys Asp 1 5 10 15 Lys Asp Gln
Glu 20 2620PRTArtificial sequencepeptide derived from
apolipoprotein B 26Arg Leu Asn Gly Glu Ser Asn Leu Arg Phe Asn Ser
Ser Tyr Leu Gln 1 5 10 15 Gly Thr Asn Gln 20 2720PRTArtificial
sequencepeptide derived from apolipoprotein B 27Ser Leu Asn Ser His
Gly Leu Glu Leu Asn Ala Asp Ile Leu Gly Thr 1 5 10 15 Asp Lys Ile
Asn 20 2820PRTArtificial sequencepeptide derived from
apolipoprotein B 28Trp Ile Gln Asn Val Asp Thr Lys Tyr Gln Ile Arg
Ile Gln Ile Gln 1 5 10 15 Glu Lys Leu Gln 20 2920PRTArtificial
sequencepeptide derived from apolipoprotein B 29Thr Tyr Ile Ser Asp
Trp Trp Thr Leu Ala Ala Lys Asn Leu Thr Asp 1 5 10 15 Phe Ala Glu
Gln 20 3020PRTArtificial sequencepeptide derived from
apolipoprotein B 30Glu Ala Thr Leu Gln Arg Ile Tyr Ser Leu Trp Glu
His Ser Thr Lys 1 5 10 15 Asn His Leu Gln 20 3120PRTArtificial
sequencepeptide derived from apolipoprotein B 31Ala Leu Leu Val Pro
Pro Glu Thr Glu Glu Ala Lys Gln Val Leu Phe 1 5 10 15 Leu Asp Thr
Val 20 3220PRTArtificial sequencepeptide derived from
apolipoprotein B 32Ile Glu Ile Gly Leu Glu Gly Lys Gly Phe Glu Pro
Thr Leu Glu Ala 1 5 10 15 Leu Phe Gly Phe 20 3320PRTArtificial
sequencepeptide derived from apolipoprotein B 33Ser Gly Ala Ser Met
Lys Leu Thr Thr Asn Gly Arg Phe Arg Glu His 1 5 10 15 Asn Ala Lys
Phe 20 3420PRTArtificial sequencepeptide derived from
apolipoprotein B 34Asn Leu Ile Gly Asp Phe Glu Val Ala Glu Lys Ile
Asn Ala Phe Arg 1 5 10 15 Ala Lys Val His 20 3520PRTArtificial
sequencepeptide derived from apolipoprotein B 35Gly His Ser Val Leu
Thr Ala Lys Gly Met Ala Leu Phe Gly Glu Gly 1 5 10 15 Lys Ala Glu
Phe 20 3620PRTArtificial sequencepeptide derived from
apolipoprotein B 36Phe Lys Ser Ser Val Ile Thr Leu Asn Thr Asn Ala
Glu Leu Phe Asn 1 5 10 15 Gln Ser Asp Ile 20 3720PRTArtificial
sequencepeptide derived from apolipoprotein B 37Phe Pro Asp Leu Gly
Gln Glu Val Ala Leu Asn Ala Asn Thr Lys Asn 1 5 10 15 Gln Lys Ile
Arg 20 3820PRTArtificial sequencepeptide derived from
apolipoprotein B 38Ala Thr Arg Phe Lys His Leu Arg Lys Tyr Thr Tyr
Asn Tyr Gln Ala 1 5 10 15 Gln Ser Ser Ser 20 3914PRTArtificial
sequencepeptide derived from apolipoprotein B 39Gln Glu Val Ala Leu
Asn Ala Asn Thr Lys Asn Gln Lys Ile 1 5 10 4017PRTArtificial
sequencepeptide derived from ovalbumin 40Ile Ser Gln Ala Val His
Ala Ala His Ala Glu Ile Asn Glu Ala Gly 1 5 10 15 Arg
* * * * *