U.S. patent application number 16/505788 was filed with the patent office on 2019-12-05 for a b cell depleting agent for the treatment of atherosclerosis.
The applicant listed for this patent is Hafid Ait-Oufella, Ziad Mallat, Thomas Tedder, Alain Tedgui. Invention is credited to Hafid Ait-Oufella, Ziad Mallat, Thomas Tedder, Alain Tedgui.
Application Number | 20190365890 16/505788 |
Document ID | / |
Family ID | 41651536 |
Filed Date | 2019-12-05 |
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United States Patent
Application |
20190365890 |
Kind Code |
A1 |
Mallat; Ziad ; et
al. |
December 5, 2019 |
A B Cell Depleting Agent for the Treatment of Atherosclerosis
Abstract
The present invention relates to the prevention or treatment of
atherosclerosis, in particular to a B cell depleting agent for the
prevention or treatment of atherosclerosis.
Inventors: |
Mallat; Ziad; (Paris,
FR) ; Ait-Oufella; Hafid; (Paris, FR) ;
Tedgui; Alain; (Paris, FR) ; Tedder; Thomas;
(Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mallat; Ziad
Ait-Oufella; Hafid
Tedgui; Alain
Tedder; Thomas |
Paris
Paris
Paris
Durham |
NC |
FR
FR
FR
US |
|
|
Family ID: |
41651536 |
Appl. No.: |
16/505788 |
Filed: |
July 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13790771 |
Mar 8, 2013 |
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16505788 |
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13143440 |
Jul 6, 2011 |
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PCT/EP2010/050048 |
Jan 5, 2010 |
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13790771 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/244 20130101;
A61P 9/00 20180101; A61K 2039/505 20130101; C07K 16/2887 20130101;
A61K 39/3955 20130101; A61K 47/6849 20170801; C07K 2317/24
20130101; A61P 9/10 20180101 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 6, 2009 |
EP |
09290005.9 |
Jan 7, 2009 |
EP |
9305013.6 |
Claims
1. A method for treating myocardial infarction in a subject in need
thereof, comprising the step of administering a sufficient quantity
of an anti-human CD20 antibody to said subject within one hour of
the myocardial infarction, wherein said anti-human CD20 antibody
depletes or destroys mature B cells or interferes with B cell
functions in said subject and increases fractional shortening.
2. The method of claim 1, wherein said anti-human CD20 antibody is
a full length anti-CD20 antibody.
3. The method of claim 1, wherein the myocardial infarction is a ST
segment elevation myocardial infarction.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the prevention or treatment
of atherosclerosis, in particular to a B cell depleting agent for
the prevention or treatment of atherosclerosis.
BACKGROUND OF THE INVENTION
[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 contributes 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). 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.
[0004] 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. 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.
[0005] 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. 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. 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.
[0006] 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). 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.
[0007] So, there is a recognized and permanent need in the art for
new reliable methods for treating atherosclerosis.
[0008] An existing approach for the treatment of atherosclerosis 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 (Treg) generation such as Tr1 cells, CD4+CD25+
cells or Th3 cells. In that context, it has been show that
naturally arising CD4(+)CD25(+) regulatory T cells, which actively
maintain immunological tolerance to self and nonself antigens, are
powerful inhibitors of atherosclerosis in several mouse models.
[0009] On the other hand, a recent study suggests that B cells
deficiency increases atherosclerosis in a mouse model (Major al.
2002). Another recent study has shown that protection against
atherosclerosis was conferred by B cells (Caligiuri et al., 2002).
Accordingly, the prior art suggests that depletion of B cells is
not a promising method for the treatment of atherosclerosis,
contrary to what is disclosed, but not demonstrated, in the
following patent applications US2004/202658, US2005/186206,
US2008/260641, WO2007/053661 and US2004/167619.
SUMMARY OF THE INVENTION
[0010] The present invention relates to a B cell depleting agent
for the treatment or prevention of atherosclerosis.
[0011] The present invention also relates to a pharmaceutical
composition for the treatment or prevention of atherosclerosis.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The inventors have demonstrated that administration of a B
cell depleting agent (i.e. an anti-CD20 antibody) allows
significant reduction of atherosclerotic plaque size in a mouse
model.
[0013] B cell-dependent responses are involved in the pathogenesis
of (auto)-immune disorders and B cell depletion significantly
reduces the burden of several immune-mediated diseases. However, B
cell activation has been until now associated with a protection
against atherosclerosis (Caligiuri et al., 2002; Major et al.,
2002; Binder et al., 2004; Miller et al., 2008), suggesting that B
cell depleting therapies would enhance cardiovascular risk.
[0014] Here, inventors unexpectedly show that mature B cell
depletion using a CD20 monoclonal antibody induces a significant
reduction of atherosclerosis in various mouse models of the
disease. This treatment preserves the production of natural and
potentially protective anti-oxidized low-density lipoprotein
(oxLDL) IgM autoantibodies over IgG type anti-oxLDL antibodies, and
markedly reduces pathogenic T cell activation. The atheroprotective
mechanisms of B cell depletion involve a switch of the immune
response towards diminished T cell-derived interferon-gamma
secretion and enhanced production of interleukin-17, whose
neutralization abrogates CD20 antibody-mediated
atheroprotection.
[0015] These results challenge the current paradigm that B cell
activation play an overall protective role in atherogenesis,
identify new anti-atherogenic strategies based on B cell
modulation, and suggest that patients currently treated with B
cells depleting agents such as CD20 antibodies for other
immune-mediated diseases might also benefit from a reduction of
cardiovascular risk through limitation of atherosclerotic lesion
development or inflammation.
[0016] Inventors also showed that B cell depletion is beneficial in
myocardial infarction.
Definitions
[0017] The term "B cell" has its general meaning in the art. B
cells are lymphocytes that play a large role in the humoral immune
response (as opposed to the cell-mediated immune response, which is
governed by T cells).
[0018] A "B cell depleting agent" is a molecule which depletes or
destroys B cells in a patient and/or interferes with one or more B
cell functions, e.g. by reducing or preventing a humoral response
elicited by the B cell. The B cell depleting agent preferably binds
to a B cell surface marker. The B cell depleting agent preferably
is able to deplete B cells (i.e. reduce circulating B cell levels)
in a patient treated therewith. Such depletion may be achieved via
various mechanisms such antibody-dependent cell mediated
cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC),
inhibition of B cell proliferation and/or induction of B cell death
(e.g. via apoptosis). B cell depleting agents include but are not
limited to antibodies, synthetic or native sequence peptides and
small molecule antagonists which preferably bind to the B cell
surface marker, optionally conjugated with or fused to a cytotoxic
agent. The preferred B cell depleting agent comprises an antibody,
more preferably a B cell depleting antibody.
[0019] In a preferred embodiment, the B cell depleting agent has
not the capability to deplete plasma cells. In another preferred
embodiment, the B cell depleting agent has not the capability to
deplete B10 cells (or Breg cells). In another preferred embodiment,
the B cell depleting agent has not the capability to deplete B1
cells. Accordingly, in a particular preferred embodiment the B cell
depleting agent has not the capability to deplete plasma cells and
B10 cells. Accordingly, in a particular preferred embodiment the B
cell depleting agent has not the capability to deplete plasma
cells, B10 cells and B1 cells.
[0020] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system to antibodies which are
bound to their cognate antigen. To assess complement activation, a
CDC assay, e.g. as described in Gazzano-Santoro et al. (1997) may
be performed.
[0021] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted antibodies bound
onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g.
Natural Killer (NK) cells, neutrophils, monocytes and macrophages)
enable these cytotoxic effector cells to bind specifically to an
antigen-bearing target cell and subsequently kill the target cell.
To assess ADCC activity of a molecule of interest, an in vitro ADCC
assay, such as that described in U.S. Pat. Nos. 5,500,362 or
5,821,337 may be performed.
[0022] A "B cell surface marker" or "B cell target" or "B cell
antigen" herein is an antigen expressed on the surface of a B cell
which can be targeted with a B cell depleting agent which binds
thereto. Exemplary B cell surface markers include but arc not
limited to the CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37,
CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b,
CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 leukocyte surface
markers. The B cell surface marker of particular interest is
preferentially expressed on B cells compared to other non-B cell
tissues of a mammal and may be expressed on both precursor B cells
and mature B cells. In one embodiment, the marker is one like CD20
or CD19, which is found on B cells throughout differentiation of
the lineage from the stem cell stage up to a point just prior to
terminal differentiation into plasma cells.
[0023] A "CD20" antigen is a 35 kDa, non-glycosylated
phosphoprotein found on the surface of greater than 90% of B cells
from peripheral blood or lymphoid organs. CD20 is expressed during
early pre-B cell development and remains until plasma cell
differentiation. CD20 is present on both normal B cells as well as
malignant B cells. Other names for CD20 in the literature include
"B-lymphocyte -restricted antigen" and "Bp35". The CD20 antigen is
described in, e.g., Clark et al. PNAS (USA) 82:1766 (1985).
[0024] According to the present invention, "antibody" or
"immunoglobulin" have the same meaning, and will be used equally in
the present invention. The term "antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site that immunospecifically binds an antigen. As such, the
term antibody encompasses not only whole antibody molecules, but
also antibody fragments as well as variants (including derivatives)
of antibodies and antibody fragments. In natural antibodies, two
heavy chains are linked to each other by disulfide bonds and each
heavy chain is linked to a light chain by a disulfide bond. There
are two types of light chain, lambda (l) and kappa (k). There are
five main heavy chain classes (or isotypes) which determine the
functional activity of an antibody molecule: IgM, IgD, IgG, IgA and
IgE. Each chain contains distinct sequence domains. The light chain
includes two domains, a variable domain (VL) and a constant domain
(CL). The heavy chain includes four domains, a variable domain (VH)
and three constant domains (CH1, CH2 and CH3, collectively referred
to as CH). The variable regions of both light (VL) and heavy (VH)
chains determine binding recognition and specificity to the
antigen. The constant region domains of the light (CL) and heavy
(CH) chains confer important biological properties such as antibody
chain association, secretion, trans-placental mobility, complement
binding, and binding to Fe receptors (FcR). The Fv fragment is the
N-terminal part of the Fab fragment of an immunoglobulin and
consists of the variable portions of one light chain and one heavy
chain. The specificity of the antibody resides in the structural
complementarity between the antibody combining site and the
antigenic determinant. Antibody combining sites arc made up of
residues that are primarily from the hypervariable or
complementarity determining regions (CDRs). Occasionally, residues
from nonhypervariable or framework regions (FR) influence the
overall domain structure and hence the combining site.
Complementarity Determining Regions or CDRs refer to amino acid
sequences which together define the binding affinity and
specificity of the natural Fv region of a native immunoglobulin
binding site. The light and heavy chains of an immunoglobulin each
have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,
H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore,
includes six CDRs, comprising the CDR set from each of a heavy and
a light chain V region. Framework Regions (FRs) refer to amino acid
sequences interposed between CDRs.
[0025] The term "chimeric antibody" refers to an antibody which
comprises a VH domain and a VL domain of an antibody of the
invention, and a CH domain and a CL domain of a human antibody.
[0026] According to the invention, the term "humanized antibody"
refers to an antibody having variable region framework and constant
regions from a human antibody but retains the CDRs of the antibody
of the invention.
[0027] The term "Fab" denotes an antibody fragment having a
molecular weight of about 50,000 and antigen binding activity, in
which about a half of the N-terminal side of H chain and the entire
L chain, among fragments obtained by treating IgG with a protease,
papaine, arc bound together through a disulfide bond.
[0028] The term "F(ab')2" refers to an antibody fragment having a
molecular weight of about 100,000 and antigen binding activity,
which is slightly larger than the Fab bound via a disulfide bond of
the hinge region, among fragments obtained by treating IgG with a
protease, pepsin.
[0029] The term "Fab' " refers to an antibody fragment having a
molecular weight of about 50,000 and antigen binding activity,
which is obtained by cutting a disulfide bond of the hinge region
of the F(ab')2.
[0030] A single chain Fv ("scFv") polypeptide is a covalently
linked VH::VL heterodimer which is usually expressed from a gene
fusion including VH and VL encoding genes linked by a
peptide-encoding linker. "dsFv" is a VH::VL heterodimer stabilised
by a disulfide bond. Divalent and multivalent antibody fragments
can form either spontaneously by association of monovalent seFvs,
or can be generated by coupling monovalent scFvs by a peptide
linker, such as divalent sc(Fv)2.
[0031] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
[0032] "B cell depleting antibodies" are defined as those
antibodies which bind to a B cell surface marker on the surface of
B cells and mediate their destruction or depletion when they bind
to said cell surface marker. The term includes antibody fragments.
Such antibodies include, but are not limited to, anti-CD20,
anti-CD19, anti-CD22, anti-CD21, anti-CD23, anti-CD28, anti-CD37,
anti-CD40, anti-CD52 antibodies. An example of an anti-CD20
antibody is RITUXAN.RTM. (rituximab). B cell depleting antibodies
also include antibodies that destroy B cells via other mechanisms.
For example, these include radiolabeled antibodies that facilitate
the destruction of B cells by binding to the B cell surface and
delivering a lethal dose of radiation. These include 131 I-Lym-1
(anti-HLA-D), 131 T-tositumomab (BEXXAR.RTM.), ibritumomab tiuxetan
(Y-90, In-I 11 ZEVALIN.RTM.) and 90 Y-cpratuzumab.
[0033] In the context of the invention, the term "treating" or
"treatment", as used herein, means reversing, alleviating, or
inhibiting the progress of the disorder or condition to which such
term applies, or one or more symptoms of such disorder or
condition. A "therapeutically effective amount" is intended for a
minimal amount of active agent which is necessary to impart
therapeutic benefit to a subject. For example, a "therapeutically
effective amount" to a patient is such an amount which induces,
ameliorates or otherwise causes an improvement in the pathological
symptoms, disease progression or physiological conditions
associated with or resistance to succumbing to a disorder.
[0034] In its broadest meaning, the term "preventing" or
"prevention" refers to preventing the disease or condition from
occurring in a subject which has not yet been diagnosed as having
it.
[0035] The term "patient" refers to any subject (preferably human)
afflicted with or susceptible to be afflicted with
atherosclerosis.
[0036] "Pharmaceutically" or "pharmaceutically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to a mammal,
especially a human, as appropriate. A pharmaceutically acceptable
carrier or excipient refers to a non-toxic solid, semi-solid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type.
Methods of Treatment
[0037] The present invention relates to a method for preventing or
treating atherosclerosis in a patient in need thereof comprising
the step of depleting the B cells population of said patient.
[0038] More particularly, the present invention relates to a method
for preventing or treating atherosclerosis in a patient in need
thereof comprising the step of administrating said patient with a B
cell depleting agent.
[0039] The method according to the present invention can be
supplied to a patient, which has been diagnosed as presenting one
of the following coronary disorders: [0040] asymptomatic coronary
artery coronary diseases with silent ischemia or without ischemia;
[0041] chronic ischemic disorders without myocardial necrosis, such
as stable or effort angina pectoris; [0042] acute ischemic
disorders without myocardial necrosis, such as unstable angina
pectoris; [0043] ischemic disorders with myocardial necrosis, such
as ST segment elevation myocardial infarction or non-ST segment
elevation myocardial infarction.
[0044] Indeed, said pathologies are atherosclerosis complications
and are thus considered as indicative of atherosclerosis.
[0045] A further aspect of the invention relates to a method for
preventing or treating a vascular or coronary disorder in a patient
in need thereof comprising the step of depleting the B cells
population of said patient.
[0046] More particularly, the invention relates to a method for
preventing or treating a vascular or coronary disorder comprising
the step of administrating a patient in need thereof with a B cell
depleting agent.
[0047] In a particular embodiment, said coronary disorder or
vascular disorders is selected from the group consisting of
atherosclerotic vascular disease, such as aneurysm or stroke,
asymptomatic coronary artery coronary diseases, chronic ischemic
disorders without myocardial necrosis, such as stable or effort
angina pectoris; acute ischemic disorders without myocardial
necrosis, such as unstable angina pectoris; and ischemic disorders
such as myocardial infarction.
[0048] In a particular embodiment, the invention relates to a
method for preventing or treating myocardial infarction in a
patient in need thereof comprising the step of depleting the B
cells population of said patient.
[0049] More particularly, the invention relates to a method for
preventing or treating myocardial infarction comprising the step of
administrating a patient in need thereof with a B cell depleting
agent.
[0050] In another particular embodiment, the invention relates to a
method for preventing or treating aneurysm in a patient in need
thereof comprising the step of depleting the B cells population of
said patient.
[0051] More particularly, the invention relates to a method for
preventing or treating aneurysm comprising the step of
administrating a patient in need thereof with a B cell depleting
agent.
[0052] In particular embodiment the B cell depleting agent may
consist in a B cell depleting antibody.
[0053] Antibodies directed against a B cell surface marker can be
raised according to known methods by administering the appropriate
antigen or cpitopc to a host animal selected, e.g., from pigs,
cows, horses, rabbits, goats, sheep, and mice, among others.
Various adjuvants known in the art can be used to enhance antibody
production. Although antibodies useful in practicing the invention
can be polyclonal, monoclonal antibodies are preferred.
[0054] Monoclonal antibodies can be prepared and isolated using any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. Techniques for production and
isolation include but are not limited to the hybridoma technique,
the human B-cell hybridoma technique and the EBV-hybridoma
technique. Alternatively, techniques described for the production
of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can
be adapted to produce single chain antibodies against a B cell
surface marker. Useful antibodies according to the invention also
include antibody fragments including but not limited to F(ab')2
fragments, which can be generated by pepsin digestion of an intact
antibody molecule, and Fab fragments, which can be generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab and/or scFv expression libraries can be
constructed to allow rapid identification of fragments having the
desired specificity to the B cell surface marker.
[0055] Humanized antibodies and antibody fragments therefrom can
also be prepared according to known techniques. "Humanized
antibodies" are forms of non-human (e.g., rodent) chimeric
antibodies that contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region (CDRs) of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. Methods for making humanized antibodies are
described, for example, by Winter (U.S. Pat. No. 5,225,539) and
Boss (Celltech, U.S. Pat. No. 4,816,397).
[0056] Then after raising antibodies directed against a B cell
surface marker as above described, the skilled man in the art can
easily select those that deplete B cells, for example those that
deplete B cells via antibody-dependent cell mediated cytotoxicity
(ADCC), complement dependent cytotoxicity (CDC), inhibition of B
cell proliferation or induction of B cell death (e.g. via
apoptosis).
[0057] In a particular embodiment, the B cell depleting antibody
may consist in an antibody directed against a B cell surface marker
which is conjugated to a cytotoxic agent or a growth inhibitory
agent.
[0058] Accordingly the invention contemplates the use of
immunoconjugates comprising an antibody against a B cell surface
marker conjugated to a cytotoxic agent or a growth inhibitory
agent.
[0059] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
B cell, either in vitro or in vivo. Examples of growth inhibitory
agents include agents that block cell cycle progression, such as
agents that induce G1 arrest and M-phase arrest. Classical M-phase
blockers include the vincas (vincristine and vinblastine), taxanes,
and topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest G1
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, and 5-fluorouracil.
[0060] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At211, I131, I125, Y90, Re186, Re188,
Sm153, Bi212, P32, and radioactive isotopes of Lu),
chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca
alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents, enzymes and fragments thereof such as
nueleolytic enzymes, antibiotics, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof,
e.g., gelonin, ricin, saporin, and the various antitumor or
anticancer agents disclosed below. Other cytotoxic agents are
described below. A tumoricidal agent causes destruction of tumor
cells.
[0061] Conjugation of the antibodies of the invention with
cytotoxic agents or growth inhibitory agents may be made using a
variety of bifunctional protein coupling agents including but not
limited to N-suceinimidyl (2-pyridyldithio) propionate (SPDP),
suceinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothio lane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediaminc), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al (1987). Carbon labeled I-isothiocyanatobenzyl
methyldicthylene triamincpentaacetic acid (MX-DTPA) is an exemplary
chelating agent for conjugation of radionucleotide to the antibody
(WO 94/11026).
[0062] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent or growth inhibitory agent may be made, by
recombinant techniques or peptide synthesis. The length of DNA may
comprise respective regions encoding the two portions of the
conjugate either adjacent one another or separated by a region
encoding a linker peptide which does not destroy the desired
properties of the conjugate.
[0063] In a particular embodiment, the preferred B cell surface
marker is CD20.
[0064] Thus, in a preferred embodiment of the invention, B cell
depleting agent is an anti-CD20 antibody.
[0065] Examples of depleting antibodies that arc contemplated by
the invention include antibodies which bind the CD20 antigen:
"C2B8" which is "rituximab" ("RITUXAN.RTM.") (U.S. Pat. No.
5,736,137, expressly incorporated herein by reference); the
yttrium-[90]-labeled 2138 murine antibody designated "Y2B8" (U.S.
Pat. No. 5,736,137, expressly incorporated herein by reference);
murine IgG2a "131" optionally labeled with 1311 to generate the
"1311-B1" antibody (BEXXARTM.RTM.) (U.S. Pat. No. 5,595,721,
expressly incorporated herein by reference); murine monoclonal
antibody "1F5" (Press et al. Blood 69(2): 584-591 (1987));
"chimeric 2H7" antibody (U.S. Pat. No. 5,677,180 expressly
incorporated herein by reference); and monoclonal antibodies L27,
G28-2, 93-1133, B-Cl or NU-B2 available from the International
Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing
III (M cMichael, Ed., p. 440, Oxford University Press (1987)).
[0066] The terms "rituximab" or "RITUXAN.RTM." herein refer to the
genetically engineered chimeric murine/human monoclonal antibody
directed against the CD20 antigen and designated "C2B8" in U.S.
Pat. No. 5,736,137, expressly incorporated herein by reference. The
antibody is an IgG, kappa immunoglobulin containing murine light
and heavy chain variable region sequences and human constant region
sequences. Rituximab has a binding affinity for the CD20 antigen of
approximately 8.0 nM. It is commercially available, e.g. from
Genentech (South San Francisco, Calif.).
[0067] The B cell depleting agent of the invention may be
administered in the form of a pharmaceutical composition, as
defined below.
[0068] Preferably, said B cell depleting agent is administered in a
therapeutically effective amount.
[0069] By a "therapeutically effective amount" is meant a
sufficient amount of the B cell depleting agent to treat or to
prevent atherosclerosis at a reasonable benefit/risk ratio
applicable to any medical treatment.
[0070] It will be understood that the total perodically usage of
the compounds and compositions of the present invention will be
decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective dose level
for any particular patient will depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; activity of the specific compound employed; the specific
composition employed, the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidential with the specific polypeptide employed; and like
factors well known in the medical arts. For example, it is well
known within the skill of the art to start doses of the compound at
levels lower than those required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved. However, the daily dosage of the products may
be varied over a wide range from 0.01 to 1,000 mg per adult per
day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5,
1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the
active ingredient for the symptomatic adjustment of the dosage to
the patient to be treated. A medicament typically contains from
about 0.01 mg to about 500 mg of the active ingredient, preferably
from 1 mg to about 100 mg of the active ingredient. An effective
amount of the drug is ordinarily supplied at a dosage level from
0.0002 mg/kg to about 20 mg/kg of body weight per day, especially
from about 0.001 mg/kg to 7 mg/kg of body weight per day.
Pharmaceutical Compositions
[0071] The B cell depleting agent of the invention may be combined
with pharmaceutically acceptable excipients, and optionally
sustained-release matrices, such as biodegradable polymers, to form
therapeutic compositions.
[0072] In the pharmaceutical compositions of the present invention,
the active principle, alone or in combination with another active
principle, can be administered in a unit administration form, as a
mixture with conventional pharmaceutical supports, to animals and
human beings. Suitable unit administration forms comprise
oral-route forms such as tablets, gel capsules, powders, granules
and oral suspensions or solutions, sublingual and buccal
administration forms, aerosols, implants, subcutaneous,
transdermal, topical, intraperitoneal, intramuscular, intravenous,
subdermal, transdermal, intrathecal and intranasal administration
forms and rectal administration forms.
[0073] Preferably, the pharmaceutical compositions contain vehicles
which are pharmaceutically acceptable for a formulation capable of
being injected. These may be in particular isotonic, sterile,
saline solutions (monosodium or disodium phosphate, sodium,
potassium, calcium or magnesium chloride and the like or mixtures
of such salts), or dry, especially freeze-dried compositions which
upon addition, depending on the case, of sterilized water or
physiological saline, permit the constitution of injectable
solutions.
[0074] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and storage and
must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0075] Solutions comprising compounds of the invention as free base
or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0076] The B cell depleting agent of the invention can be
formulated into a composition in a neutral or salt form.
Pharmaceutically acceptable salts include the acid addition salts
(formed with the free amino groups of the protein) and which are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0077] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetables oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin.
[0078] Sterile injectable solutions are prepared by incorporating
the active polypeptides in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0079] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0080] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual
subject.
[0081] The B cell depleting agent of the invention may be
formulated within a therapeutic mixture to comprise about 0.0001 to
1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to
1.0 or even about 10 milligrams per dose or so. Multiple doses can
also be administered.
[0082] In addition to the compounds of the invention formulated for
parenteral administration, such as intravenous or intramuscular
injection, other pharmaceutically acceptable forms include, e.g.
tablets or other solids for oral administration; liposomal
formulations; time release capsules ; and any other form currently
used.
[0083] The invention will further be illustrated in view of the
following figures and examples.
FIGURES
[0084] FIG. 1: B cell depletion in the blood after 3 months of
anti-murine CD20 treatment, one intraperitoneal (200 .mu.g)
injection every 3 weeks.
[0085] FIG. 2: B cell depletion in spleen after 3 months of
anti-CD20 treatment, one intraperitoneal (200 .mu.g) injection
every 3 weeks. FIGS. 2A and 2B represent B cell depletion in 2
distinct experiments.
[0086] FIG. 3: B cell depletion (B220 high) in bone marrow after 3
months of anti-CD20 treatment, one intraperitoneal (200 .mu.g)
injection every 3 weeks.
[0087] FIG. 4: B cell depletion induces a decrease of CD69
expression by splenic CD4 T cell suggesting a CD4 T cell
deactivation.
[0088] FIG. 5: B cell depletion induces a decrease of CD44high
expression by splenic CD4 T cell suggesting a CD4 T cell
deactivation
[0089] FIG. 6: B cell depletion induces a decrease of BrdU
incorporation in vivo by splenic CD4 T cell indicating a decrease
of in vivo CD4 T cell proliferation.
[0090] FIG. 7: Cytokine production by purified splenic CD4+ T cell
stimulated in vitro with anti-CD3 antibody in the presence of
purified CD11c+ dendritic cells
[0091] FIG. 8: Neutrophil cell count (Ly6G+ CD11b+) and monocyte
cell count (CD11b+/Ly6G-/Ly6Chigh, low or neg) was assessed using
flow cytometry (First set of experiments). inventors found
increased neutrophil and monocyte cell count after 3 months of
anti-CD20 treatment.
[0092] FIG. 9: Neutrophil cell count (Ly6G+ CD11b+) and monocyte
cell count (CD11b+-Ly6G-/Ly6Chigh, low or neg) was assessed using
flow cytometry (Second set of experiments). Inventors found
increased neutrophil and monocyte cell count after 3 months of
anti-CD20 treatment.
[0093] FIG. 10: Weight and plasma cholesterol levels were similar
between the groups after 3 months of treatment.
[0094] FIG. 11: Significant reduction in aortic sinus lesion size
in the groups of mice treated with an anti-CD20 antibody (MB20-11)
after 6 or 12 weeks of treatment.
[0095] FIG. 12: CD20 mAb (.alpha.-CD20) treatment depletes B cells
and reduces the development of atherosclerosis. Panels A to D show
reduction of atherosclerosis development after .alpha.-CD20 therapy
in 4 different experiments using Apoe.sup.-/- or Ldlr.sup.-/- mice
fed either a chow diet (CD) or a western diet (WD). Representative
photomicrographs of Oil red O-stained aortic sinuses are shown for
each experimental setting along with quantification of intimal
lesion size. Bars indicate median values.
[0096] FIG. 13: CD20 mAb (.alpha.-CD20) treatment reduces the
development of atherosclerosis in the thoracic aorta. Quantitative
analysis of the extent of Oil red O staining in thoracic aortas
Apoe.sup.-/- mice fed a western diet for 12 weeks and treated with
.alpha.-CD20 or a control antibody. Data (mean values.+-.s.c.m.)
are representative of 9 (Ctr) to 10 mice (.alpha.-CD20) per
group.
[0097] FIG. 14: B cell depletion after myocardial infarction.
Quantitative cardiac function is shown.
EXAMPLE 1: B CELL DEPLETION AND ATHEROSCLEROSIS
[0098] Results and Discussion
[0099] The development of atherosclerosis is associated with signs
of B cell activation, particularly manifested by enhanced
production of natural IgM type and adaptive IgG types anti-oxidized
low-density lipoprotein (ox-LDL) (auto)antibodies (Caligiuri et
al., 2002; Shaw et al., 2000). However, in contrast to other
immune-mediated diseases, i.e., rheumatoid arthritis and systemic
lupus erythematosus, B cells have been assigned a protective role
in atherosclerosis (Caligiuri et al., 2002; Major et al., 2002;
Binder et al., 2004; Miller et al., 2008). Although IgG types
anti-ox-LDL antibodies show variable association with vascular
risk, circulating levels of IgM type anti-ox-LDL antibodies have
been more frequently linked with reduced vascular risk in humans
(Karvonen et al., 2003; Tsimikas et al., 2007). In mice, IL-5- and
IL-33-mediated atheroprotective effects have been indirectly
associated with specific B1 cell activation and enhanced production
of natural IgM type anti-oxLDL antibodies (Binder C J et al, 2004;
Miller A M et al, 2005). On the other hand, splenectomy or transfer
of .mu.MT-deficient (B cell deficient) bone marrow into
lethally-irradiated atherosclerosis-susceptible mice resulted in
profound reduction of IgG or total anti-oxLDL antibody production
and was associated with acceleration of lesion development.
[0100] These studies led to the current paradigm that overall B
cell activation is atheroprotective.
[0101] Surprisingly however, whether mature B cell depletion
accelerates atherosclerotic lesion development in immuno-competent
mice, as expected from previous studies, is still unexplored. This
is a critical question given the potentially important risk of
cardiovascular complications that might arise from the clinical use
of B cell depleting CD20-targeted immune therapy in patients with
severe rheumatoid arthritis or systemic lupus erythematosus, who
are at particularly high risk of cardiovascular diseases.
[0102] In order to directly assess the role of B cells in
atherosclerosis, inventors examined lesion development in mice with
or without B cell depletion. They first used Apoe.sup.-/- mice fed
a high fat western diet, a model previously shown to be associated
with significant B cell activation and high production of
anti-ox-LDL antibodies, and previously used to assess the
protective role of B cells in atherosclerosis. To deplete B cells,
mice were treated every 3 weeks with a previously validated mouse
monoclonal CD20 antibody (Uchida et al., Int Immunol, 2004; Uchida
et al., J Exp Med, 2004) for either 6 or 12 weeks. Control mice
received a control monoclonal antibody (mAb). As expected,
treatment with CD20 mAb led to sustained and profound reduction of
the number of mature B cells in blood (FIG. 1), spleen (FIG. 2),
peritoneum and bone marrow. B220.sup.high IgM.sup.+ cells were
severely depleted (92% to 100%) at all studied sites. Spleen
B220.sup.low IgM.sup.+ cells also showed a marked reduction
(.about.80%) but, as previously observed, immature bone marrow
B220.sup.low (IgM.sup.+) cells (FIG. 3) were less sensitive to CD20
mAb-mediated depletion. Treatment with CD20 mAb for 6 weeks did not
affect plasma cholesterol levels (6.4.+-.0.9 vs 6.3.+-.0.8 g/L in
control and CD20 mAb-treated groups, respectively, P=0.88) but
unexpectedly led to a significant reduction, not acceleration, of
atherosclerotic lesion development (FIG. 12A). Inventors
subsequently analyzed the experiments of Apoe.sup.-/- mice treated
for 12 weeks under high fat diet and still found a significant
reduction in atherosclerosis at 2 different vascular sites (FIG.
12B and FIG. 13), despite similar plasma cholesterol levels
(18.7.+-.1.1 vs 17.9.+-.1.0 g/L in control IgG and
anti-CD20-treated groups, respectively, P=0.68). In order to rule
out the possibility that the athero-protective effect of CD20 mAb
treatment was due to the use of a mouse model that generates
excessive inflammation in response to a very high lipid overload,
the effect of B cell depletion was examined in Apoe.sup.-/- mice
fed a chow diet. Treatment of these mice with CD20 antibody for 12
weeks also resulted in significant reduction of lesion development
(FIG. 12C), despite similar plasma cholesterol levels (5.5.+-.0.6
vs 5.7.+-.0.8 g/L in control IgG and CD20 mAb-treated groups,
respectively, P=0.96). The elevated plasma cholesterol levels in
Apoe.sup.-/- mice are mostly of the very low-density lipoprotein
(VLDL) subtype, whereas elevated LDL is the major atherosclerosis
risk factor in humans. Thus, inventors examined the effects of B
cell depletion in the LDL receptor-deficient (LDLr.sup.-/-) mouse
model. Again, treatment of LDLr.sup.-/- mice with CD20 mAb led to
marked B cell depletion and to a significant reduction of
atherosclerosis (FIG. 12D).
[0103] Overall, these studies provide solid evidence for an
unsuspected pro-atherogenic role of B cells in three mouse models
of atherosclerosis.
[0104] Inventors next addressed the potential mechanisms
responsible for atheroprotection after B cell depletion. They found
that treatment with CD20 depleting antibody resulted in a profound
reduction of IgG type anti-oxLDL antibodies both at 6 and 12 weeks
of treatment, which was consistent with the profound depletion of
B220.sup.high cells in blood, spleen and bone marrow. It could be
argued that anti-oxLDL IgG reduction might have limited the
potentially deleterious consequences of immune complex formation on
atherosclerosis. However, in other studies and particularly after
splenectomy, profound reduction in anti-oxLDL IgG levels was
observed in association with acceleration, not reduction, of
atherosclerosis. Thus, in the absence of studies directly
addressing the role of IgG type anti-oxLDL antibodies on
atherosclerosis, changes in anti-oxLDL IgG levels following CD20
mAb treatment could not be held responsible for lesion reduction.
Levels of IgM type antibodies against either copper-oxidized or
malondialdehyde-modified LDL were also reduced after 6 or 12 weeks
of CD20-targeted therapy. IgM type antibodies are endowed with
athero-protective properties and their reduction after CD20 mAb
therapy could not account for athero-protection but instead, might
have impeded the reduction of atherosclerosis. It is interesting to
note, however, that IgM type anti-oxLDL and TI5id+ IgM antibodies
showed a much lower reduction compared to IgG type antibodies,
which might have preserved an athero-protective pathway. IgM type
antibodies dominate the humoral response to oxLDL in Apoe.sup.-/-
mice and are increased even at a young age (before the initiation
of CD20 mAb treatment in this study), which may explain, at least
in part, the persistence of a significant IgM level after CD20
immunotherapy, a treatment that does not dramatically affect
pre-existing antibodies titers. IgM persistence may also be related
to the delay required to markedly deplete peritoneal B1 cells using
CD20 mAb.
[0105] Inventors next examined atherosclerotic lesion composition
to gain more insight into the mechanisms of atheroprotection.
Interestingly, CD20 mAb treatment was associated with a significant
and specific reduction of T lymphocyte accumulation within the
lesions, suggesting a role for B cells in driving T cell-dependent
lesion inflammation. As expected at this stage of lesion formation,
very few B cells were detected within the plaques or within the
adventitial layer regardless of the treatment group, suggesting
that modulation of lesion T cell accumulation by CD20 mAb therapy
most likely occurred as a consequence of systemic modulation of T
cell function following systemic B cell depletion. In order to
address this hypothesis, inventors examined T cell activation and
proliferation. Interestingly, inventors consistently found marked
reductions in CD69 and CD44.sup.high expression on spleen-derived
CD4.sup.- T cells of mice treated with CD20 antibody compared with
controls at both 6 weeks and 12 weeks of high fat diet, indicating
reduced T cell activation. B cell depletion also led to significant
reduction of in vivo BrdU staining of effector CD4.sup.+CD25.sup.-
T cells, suggesting reduced proliferation. Reduced T cell
activation in CD20 mAb-treated mice was also consistent with the
marked reduction of CD40 expression on CD11c.sup.+ dendritic cells.
Thus, a major consequence of B cell depletion using CD20 antibody
is a marked reduction of T cell activation in vivo, which could
potentially account for its atheroprotective effect.
[0106] T cell-derived cytokines significantly alter lesion
development. Therefore, inventors examined the consequences of B
cell depletion on cytokine production by purified T cells.
Inventors found a marked reduction of pro-atherogenic IFN-.gamma.
by purified T cells recovered from CD20 mAb-treated mice compared
with controls. Of note, this was associated with a deviation of the
immune response towards a significant increase of T cell-derived
IL-17A production in CD20 mAb-treated animals. Recent studies in
inventor's laboratory identified an unexpected regulatory and
protective role for IL-17A production in atherosclerosis. IL17A has
also been shown to modulate Th1 polarization. In order to examine
whether CD20 mAb-induced changes in T cell cytokine profile
(reduced Th1 and increased IL17) could be responsible for CD20
mAb-dependent atheroprotection, CD20 mAb was administered to
Apoe.sup.-/- mice (on high fat diet for 6 weeks) in the presence of
control or anti-IL17A neutralizing antibody. IL17 neutralization
led to increased IFN-.gamma. production in the atherosclerotic
aortas and completely abrogated the athero-protective effects of
CD20 mAb therapy, despite similar circulating cholesterol levels
and despite no significant changes in anti-oxLDL antibodies
levels.
[0107] Collectively, these results identify a hitherto unsuspected
role for B cells in driving the development of atherosclerosis
through modulation of T cell activation and eytokine production.
Present results may seem in contrast with previous studies showing
that both .mu.MT deficiency and splenectomy accelerate
atherosclerosis in mice. However, these studies did not directly
address the role of mature B cell depletion on atherosclerosis in
immuno-competent mice. Several other concomitant immune cell
dysfunctions might have contributed to enhanced lesion development
in RMT deficient animals. Furthermore, the reported limitation of
atherosclerosis acceleration in splenectomized mice following
reconstitution with purified B cells could have been confounded by
the significant reduction of plasma cholesterol levels in B
cell-reconstituted mice and could not be selectively attributed to
B cells since T cell reconstitution also resulted in
atheroprotection. Finally, it should be noted that although B cell
depletion significantly limited lesion development in the present
studies, the roles of specific subtypes of B cells in driving or
controlling atherosclerosis merit further investigation. More
particularly, it will be important to address the respective roles
of regulatory versus non-regulatory B cells in these processes.
[0108] In conclusion, inventors provide strong evidence that mature
B cell depletion reduces the development of atherosclerosis in
mice. These results challenge the paradigm that overall B cell
function is atheroprotective and show that a major B cell role in
atherosclerosis is to drive T cell activation towards enhanced
pro-atherogenic Th1 immune response and limited production of
athero-protective IL-17. Although limited vascular B cell
infiltration is detectable in the early stages of atherosclerosis,
B cell accumulation substantially increases with time within and
around advanced atherosclerotic coronary lesions and
atherosclerotic abdominal aortic aneurysms, both in mice and
humans, and is even prominent in vascular inflammation associated
with other immune-mediated diseases. Inhibition of excessive B cell
activation either through depletion or immune modulation might
substantially limit vascular inflammation and atherosclerotic
lesion development.
[0109] Methods
[0110] Animals. All mice were on C57B1/6 background. Apoe mice were
10-week-old males maintained on chow diet for 12 weeks or put on
western diet (20% fat, 0.15% cholesterol, 0% cholate) for either 6
or 12 weeks. Ldlr.sup.-/- mice were 10-week-old males put on
western diet for either 6 weeks. At 10-week-old, mice were treated
intra-peritoneally (i.p.)
[0111] with a previously validated mouse monoclonal CD20 antibody
(Uchida et al., Int Immunol, 2004; Uchida et al., J Exp Med, 2004)
or a control IgG (200 .mu.g every 3 weeks), for either 6 or 12
weeks. In some experiments, mice received an i.p. injection of
either purified neutralizing anti-IL-17A specific antibody (200
.mu.g/mouse, twice per week) (Uyttenhove et al., 2006 and 2007;
Wang et al., 2009) or control IgG for 6 weeks. Experiments were
conducted according to the guidelines of the French veterinary
guidelines and those formulated by the European Community for
experimental animal use (L358-86/609EEC), and were approved by
inventor's institution Inserm.
[0112] Extent and composition of atherosclerotic lesions.
Quantification of lesion size and composition was perfotuied as
previously described (Taleb et al., 2007).
[0113] Cell recovery and purification, culture, proliferation and
cytokine assays. CD11c.sup.+ and CD4.sup.- cells were purified and
processed for cell proliferation assays and cytokine production as
previously described in detail (Taleb et al., 2007). IL-17 and
IFN-.gamma. productions in the supernatants were measured using
specific ELISAs (BD Biosciences and R&D Systems).
[0114] Flow cytometry. APC-conjugated anti-CD3.epsilon. (145-2C11),
FITC- or PE-Cy7-conjugated anti-CD4 (RM4-5), APC-conjugated
anti-CD25 (PC61.5), PE-conjugated anti-CD69 (H1.2F3),
APC-conjugated anti-IgM (II/41), FITC-conjugated anti-CD86 (GL1),
PE-conjugated anti-CD80 (16-10A1), APC-conjugated anti-CD40 (1C10),
PE-Cy7-conjugated anti-CD11c (N418), PE-Cy7-conjugated anti-CD11b
(M1/70) and PE-conjugated anti-CD45R (B220) (RA3-6B2) were from
eBioscience. FITC-conjugated anti-CD5 (53-7.3), biotin-conjugated
anti-CD44 followed by APC-conjugated Streptavidin,
APC-Cy7-conjugated anti-CD45R (B220) (RA3-6B2), APC-conjugated
anti-IFN.gamma. (XMG1.2) and PE-conjugated anti-IL17A (TC11-18H10)
were from BD Biosciences. For blood staining, erythrocytes were
lysed using BD FACS lysing solution (BD Biosciences). For
intracellular cytokine staining, lymphocytes were stimulated in
vitro with leukocyte activation cocktail (BD Biosciences) according
to manufacturer's instruction for 4 hours. Surface staining was
performed before permeabilization using intracellular staining kit
(eBioseience). Forward scatter (FSC) and side scatter (SSC) were
used to gate live cell excluding red blood cells, debris, and cell
aggregates in total splenocyte, lymph node, bone marrow and
peritoneum populations. Cells were analyzed using a BD Cantoll or
BD LSRII flow cytometer (Becton Dickinson).
[0115] Bromodeoxyuridine (BrdU) Labeling and Cell Analysis. BrdU
labeling was performed as previously described (Fisson et al.,
2007). Mini osmotic pumps (ALZET1007D; Charles River Laboratories),
delivering 1.2 mg per day of BrdU (Sigma-Aldrich) for 7 d, were
transplanted to mice subcutaneously under isoflurane anesthesia one
week before sacrifice. Lymph node cells and splenocytes were
stained with a PE-Cy7-conjugated anti-CD4 (RM4-5) and
APC-conjugated anti-CD25 (PC61.5). BrdU detection was performed
using FITC BrdU Flow kit (BD Pharmingen) according to
manufacturer's instructions. Cells were analyzed using a BD CantoII
or BD LSRII flow cytometer (Becton Dickinson).
[0116] Quantitative real time polymerase chain reaction.
Quantitative Real time PCR was performed on an ABI prizm 7700 in
triplicates. CT for GAPDH was used to nonnalize gene expression.
Quantitative Real time PCR was performed for the following
proteins: IL10, TGF-.beta. and IFN-.gamma..
[0117] Determination of circulating antibodies. Specific antibody
titers to given antigens in plasma were determined by
chemiluminescent ELISA as previously described (Friguet et al.,
1985; Binder et al., 2003; Chou et al., 2009).
[0118] Statistical analysis. Values are expressed as
means.+-.s.e.m. Differences between values were examined using
nonparametric Mann-Whitney or Kruskal-Wallis tests and were
considered significant at P<0.05.
EXAMPLE 2: B CELL DEPLETION IS ASSOCIATED WITH AN INCREASE IN
FRACTIONAL SHORTENING IN A MYOCARDIAL INFARCTION MODEL.
[0119] Myocardial infarction was induced in 8 weeks old male
C57BL6J mice by ligation of the left anterior descending coronary
artery. One hour after myocardial ischemic injury mice were treated
or not by intraperitoneal injection of a mouse monoclonal CD20
antibody (160 .mu.g). Inventors showed that B cells infiltrated the
infarct area. Blood B cells levels were analyzed by flow cytometry
at days 1, 3, 7 and 14 after MI. The percentage of IgM+ B220+-B
cells was markedly reduced after CD20 antibody treatment. Mice were
sacrificed at day 14 post-MI and cardiac function was measured by
echocardiography (FIG. 14). Such treatment was associated with an
increase in fractional shortening suggesting that said treatment
may be beneficial for the treatment of myocardial infarction.
EXAMPLE 3: EFFECTS OF B CELL DEPLETION IN ABDOMINAL AORTIC
ANEURYSM
[0120] First, inventors use a validated mouse model of aneurysm
formation. Apoe-/- mice fed a chow or high fat diet develop
abdominal aortic aneurysm when infused with angiotensin (Ang) II
for 28 days (Daugherty A et al, 2000). This model reproduces the
accumulation of inflammatory cells, including B cells, within and
around the aneurysmal vessel.
[0121] For more precise results, inventors may also use a new model
of aortic aneurysm with a high incidence of aneurysm rupture,
described in patent application WO2009056419. The model uses
Apoe+/+ or Apoe-/- mice, and associates both AngII infusion and
neutralization of TGF-.beta. activity, two factors with a high
relevance to the human disease. In this model, systemic
neutralization of TGF-.beta. activity leads to unexpected and
marked increase in the susceptibility of these mice to AngThinduced
aortic aneurysm (92.5%), and to a high level of mortality from
aortic dissection and rupture (65%).
[0122] B cell depletion using anti-CD20 mAb will be initiated at
the time of aneurysm induction in order to assess its effect on
aneurysm development. A first infusion of 200 .mu.g i.p. is done
one hour after aneurysm induction and a second two weeks after.
[0123] Mice are sacrificed after four weeks. Abdominal aortic
aneurysm and immune response are assessed, an echography is
done.
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present disclosure.
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