TITLE OF THE INVENTION METHOD FOR PREVENTING OR TREATING ATHEROSCLEROSIS

FIELD OF THE INVENTION

[0001] The present invention relates to a new method for preventing or treating atherosclerosis, in particular a method for preventing or treating atherosclerosis using an anti-ΜΙΡ-Ιβ antibody.

BACKGROUND OF THE INVENTION

[0002] Atherosclerosis is a chronic inflammatory disorder of artery leading to

cardiovascular morbidity and mortality. Inflammatory cytokines and chemokines play important roles in the pathogenesis and complications of atherosclerosis. Endothelial dysfunction caused by various risk factors including hyperglycemia, hypertension, low density lipoprotein (LDL) and others are regarded as the key mechanism for atherogenesis. Then, circulating LDL could enter the sub- endothelial layer where it may be oxidized to oxidized LDL (ox- LDL) as one of the key components of atheroma. On the other hand, upon stimuli, endothelial cells, together with other vascular cells, may produce various inflammatory mediators, including adhesion molecules and cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-l, IL-6, and so on. They could promote endothelial adhesion of circulating leukocytes, direct the migration of bound leukocytes into intima, mature the monocytes to macrophages, and enhance the lipid uptake of macrophage to form the lipid core in atheroma plaques. Importantly, atheroma with a thin fibrous cap, a large necrotic core, and a high content of leucocyte are more inflammatory and vulnerable to rupture, suggesting a high-risk phenotype for acute cardiovascular events. It was suggested to identify novel anti- inflammatory strategy to stabilize atheroma plaques for the prevention of clinical events.

[0003] Although the cause of atherosclerosis is unknown, atherosclerosis may be treated with the heart-healthy lifestyle changes, medicines, and medical procedures or surgery. Normally, the goals of treatment include lowering the risk of blood clots forming, preventing atherosclerosis-related diseases, relieving symptoms and widening or by passing plaque-clogged arteries. Treatment of established disease may include medications to lower cholesterol such as statins, blood pressure medication, or medications that decrease clotting, such as aspirin. A number of procedures may also be carried out such as percutaneous coronary intervention, coronary artery bypass graft, or carotid endarterectomy.

[0004] It is still desirable to develop an effective method for treating atherosclerosis.

SUMMARY OF THE INVENTION

[0005] It is unexpectedly found in the present invention that a macrophage inflammatory protein 1 beta (MIP-Ιβ) inhibitor, such as a specific MIP-Ιβ antibody, could retard the progression and promote the stabilization of atheroma plaques in a mice model of atherosclerosis. Accordingly, the present invention provides a new approach for preventing, arresting, reversing or treating atherosclerosis through the inhibition of ΜΙΡ-1β.

[0006] In one aspect, the invention provides a method for preventing, arresting, reversing or treating atherosclerosis, comprising a step of administering to a subject in need thereof an therapeutically effective amount of an anti-ΜΙΡ-Ι β inhibitor.

[0007] In one further aspect, the invention provides a method for preventing or treating a inflammatory cardiovascular disease or disorder, comprising a step of administering to a subject in need thereof a therapeutically effective amount of a MIP-Ιβ inhibitor, wherein the cardiovascular disease or disorder is selected from the group consisting hyperlipidaemia, hypercholesterolaemia, heart attack, stroke, and coronary heart disease.

[0008] In another aspect, the invention provides a method for treating or preventing atherosclerosis, the method comprising the steps of:

(1) providing a sample of the subject and determining the level of MIP-Ιβ in the sample, and

(2) administering to said subject, if the subject is found to have a higher level of MIP- 1 β in the sample than a normal level of a healthy population, a therapeutically effective amount ofa MIP-Ιβ inhibitor.

[0009] In one embodiment of the invention, the therapeutically effective amount of anti- MIP-Ιβ inhibitor is the amount sufficient to reduce atherosclerotic lesions or plaques. [0010] In one embodiment of the invention, the therapeutically effective amount of anti- MIP-lp inhibitor is the amount sufficient to retard the progression and promote the stabilization of atheroma plaques.

[0011] In one embodiment of the invention, the therapeutically effective amount of anti- ΜΙΡ-1β inhibitor is the amount sufficient to lower blood lipids, triglyceride, cholesterol and non-high-density lipoprotein.

[0012] In one yet aspect, the invention provides a method for lowering blood lipids, triglyceride, cholesterol or non-high-density lipoprotein, comprising a step of administering to a subject in need thereof a therapeutically effective amount of a MIP-Ιβ inhibitor.

[0013] In one further yet aspect, the invention provides a use of an anti-ΜΙΡ-Ιβ antibody for manufacturing a medicament for preventing, arresting, reversing or treating

atherosclerosis.

[0014] In one further aspect, the invention provides a pharmaceutical composition for preventing, arresting, reversing or treating atherosclerosis comprising a therapeutically effect amount of anti-ΜΙΡ-Ιβ antibody, a binding protein or peptide or a fragment thereof which is capable of binding to ΜΙΡ-Ιβ, and a pharmaceutically acceptable carrier.

[0015] In one embodiment of the invention, the MIP-Ιβ inhibitor is an anti- ΜΙΡ-1β antibody, or a fragment thereof.

[0016] In one particular example of the invention, the MIP-Ιβ inhibitor is a binding protein or peptide capable of binding to a MIP-Ιβ or a fragment thereof, such as a peptide binding to an amino acid sequence of SFVMDYYET (SEQ ID NO: 1), or

AVVFLTK G QIC (SEQ ID NO: 2).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0017] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings.

For the purpose of illustrating the invention, there are shown in the drawings embodiment which is presently preferred. It should be understood, however, that the invention is not limited to this embodiment.

[0018] In the drawings: [0019] Figure 1 : The effects of anti-ΜΙΡ-Ιβ antibody on cytokines, including the levels of ΜΙΡ-1β (Figures 1A, IB and 1C), IL-6 (Figure ID), TNF-a (Figure IE) in serum (n=6 in each group); wherein the Western blotting of aorta tissue and quantification of relative expression of MIP-Ιβ in aorta were obtained respectively(#l represents upper aorta; #2 represents lower aorta, quantification of relative expression of ΜΙΡ-1β = ΜΙΡ-1β expression / β-actin expression; #P<0.05, ##P<0.01 compared with the IgG _2a isotype control group).

[0020] Figure 2: The effects of anti-ΜΙΡ-Ιβ antibody on the metabolic parameters including blood glucose levels (Figure 2A), total cholesterol levels (Figure 2B), triglyceride levels (Figure 2C), non-HDL levels (Figure 2D) in serum and body weight (Figure 2E) (n=6 in each group), wherein the Westerb blotting of LX expression in liver and statistical analyses for the Western blotting were obtained (quantification of relative expression of LXR = LXR expression / β-actin expression (Figure 2F; n=3).(# <0.05, ##P<0.01 compared with the IgG ₂ a isotype control group).

[0021] Figure 3: Anti-ΜΙΡ-Ιβ antibody reduced the atherosclerosis lesion size, reduced the necrotic area, and increased the fibrous cap thickness; wherein the quantification of the plaque area (μιη ² ) (Figure 3 A, n=6 in each group). Quantification of fibrous cap thickness (μιη) (Figure 3B, n=6 in each group). Quantification of necrotic area / plaque area (%) (Figure 3C, n=6 in each group). (# <0.05, ##P<0.01 compared with the IgG _2a isotype control group).

[0022] Figure 4: Anti-ΜΙΡ-Ιβ antibody reduced the number of macrophage and ΜΙΡ-1β expressions in plaques; wherein Quantification of average F4/80 signal/DAPI (Figure 4A, n=6 in each group). Quantification of average MIP-Ιβ signal/DAPI (Figure 4B, n=6 in each group) (#P<0.05, ##P<0.01 compared with the IgG _2a isotype control group).

[0023] Figure 5: Anti-ΜΙΡ-Ιβ antibody reduced MMP2 and MMP9 expressions in plaques; wherein Quantification of MMP2 positive area / plaque area (%) (Figure 5A, n=6 in each group). Quantification of MMP9 positive area / plaque area (%) (Figure 5B, n=6 in each group) (# <0.05, ##P<0.01 compared with the IgG ₂ A isotype control group). DETAILED DESCRIPTION OF THE INVENTION

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[0025] As used herein, the singular forms "a", "an", and "the" include plural referents unless the contest clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and equivalents thereof known to those skilled in the art.

[0026] As used herein, the term "macrophage inflammatory protein -1 beta" or "MIP- 1β," also known as chemokine (C-C motif) ligand 4 (CCL4) refers to one of the ligands of chemokine (C-C motif) receptor 5 (CCR5), id major factor produced by macrophages after they are stimulated with bacterial endotoxin, and crucial for immune responses towards infection and inflammation, and can induce the synthesis and release of other proinflammatory cytokines such as mterleukm 1 (IL- 1), IL-6 and TNF-a from fibroblasts and macrophages.

[§027] As used herein, the term "MIP- 1β inhibitor" refers to an agent or a molecule that decreases/regulates the level of ΜΙΡ-Ιβ, and/or directly or indirectly decreases o inhibits th activity of MIP-lp. Examples of the MIP- 1 β-inhibitor include (1) a ΜΊΡ- Ιβ modulating a esii c rii oisiid thai decreases the level of ΜΙΡ-Ι β, or homoiogs thereof. (2) a ΜΙΡ~1β _. agent/compound that suppresses the expression ofMIP-Ιβ, such as a siR A, _* ¾i inbse se nucleic acid, or a ribozym targeted to the ΜΙΡ~1β; (3) an agent feat inhibits transcription of MIF-lp: and (3) an agent that modulates the transcription of genes encoding MIP- iB, such as an agent destabil izing the mR As. In an exemplary embodiment, a ΜΙΡ-Ιβ-inhibitor may be compound that decreases at least one biological activity of MlP-l by at least about 10%, 25%, 50%, 75%, 100%, or more.

[0028] in a particular embodiment of the invention, th MIP- 1 β-inhibitor is a molecule which is capable of binding to MlP- lji F«r instance, according to ome embodiments of tbe present invention, an agernVmoleeuie capable of inhibiting t e activity of MIP- IB, such as an 3ΐιΐη ί "Ιβ antibody, [0029] As used. erein, fee term- "aati ody" means . irnrmMogiob Im molecule or a f agment of an immmioglobi in molecule having th ability to specifically bind to a particular antigen. The term "antibody" herein is used in the broadest sense and specifically includes a bieag h monoclonal antibody, a polyclonal antibody, a nnudspeclfic antibody (e.g., a bispecifie antibody), asid antibody fragments thereof, as long as they exhibit fee desired biological activity,

[0030] As used herein., the term "antibody fragment" refers to a portion of a fail-length antibody, preferably antigen-binding or variable regions thereof Examples of antibody fragments rnclnde fab. Fab; F(ab) _¾ W≠>% F(ab)i, Fv (typically the VL nd VH, domains of a single a m of an antibody), single-chain Fv (scFv), dsfy, Fd ifa nents (typically the VFi and CHI domain), and dAb (typically _: a VF1 domain) fragments; VH, v L. and VhH domains; minibodies, diabodies, triafsodies, tetrabodies., and kappa bodies; camel ;g ⁽ .f and ntnltispeciffc antibody fragments formed from antibody fragments, and one or mnre isolated GDRs or a functional paratope, where, isolated CDR or antigen -binding residues or polypeptides caa be associated or linked together so as to form a functional antibody fragment

[0031] In th present invention, it was confirmed that a macrophage inflammatory protein -1 beta (MIP- 1 p) inhibitor, such as a specific MIP- 1 β antibody, could retard the progression and promote the stabilization of atheroma plaques in a mice model of atherosclerosis.

[0032] Accordingly, the invention provides a method for preventing, arresting, reversing or treating atherosclerosis, comprising a step of administering to a subject in need thereof a therapeutically effective amount of an anti-MIP-l p inhibitor.

[0033] Further, the invention provides a method for preventing or treating an

inflammatory cardiovascular disease or disorder, comprising a step of administering to a subject in need thereof a therapeutically effective amount of a ΜΙΡ-Ιβ ^' inhibitor.

[0034] In addition, the invention provides a method for treating or preventing

atherosclerosis, the method comprising the steps of:

(1) providing a sample of the subj ect and determining the level of ΜΐΡ-1β in the sample, and (2) administering to said subject, if the subject is found to have a higher level of MIP- Ι β in the sample than a normal level of a healthy population, a therapeutically effective amount ofa MIP- lp inhibitor.

[0035] On the other hand, the invention provides a method for lowering blood lipids, triglyceride, cholesterol or non-high-density lipoprotein, comprising a step of administering to a subject in need thereof a therapeutically effective amount ofa MIP- lp inhibitor.

[0036] In one embodiment of the invention, the MIP- 1β inhibitor is an anti- ΜΙΡ-1β antibody, or a fragment thereof In certain ejBbodknetrtx,- the MlP- ! p-antibody is a monoclonal antibody specifically binding io MIP- l p, called as "'anii-MDVi a;ilibody. ^! in one embodiment, the riii- _. ΓΡ- Ι β monoclonal .antibody has the binding specificity for a functional fragment of MIF~ 11 ·.

[0037] In one particular example of the resent invention, the MlP-T -in ibjtor is a monoclonal antibody that binds to the antigen determinant fragment οί ΜΪΡ-Ι β, which is a peptide having a amino acid, sequence of SFV DYYET (SEQ ID I O: 1 ) _s the 46th. io 54th. amino acid, residues of ^' Μ ^' ΙΙ β; or a peptide having an ^' amino acid sequence of

AVVFLTKRORQIC (SEQ H) ^" O:2), the 62nd to 74ih amino acid redisure oF MIP- 1|i

[0038] In one particular embodiment of the present invention, the MIP p--mhibitor ts a nmnoelonal antibody or a functional fragment thereof, preferably a hiinmmzed anubodv or a ^■' human antibody.

[0039] Pharmaceutical compositions for us in accordanc with the present invention may be formiilated in a conventional maimer using one or more physiologically acceptable carriers comprising excipieuis and auxil aries which facilitate processing of the active compound into preparations which can be used pharmaceutically. Proper fomentations, including (but not limited to) oral compositions such a tablets, capsules, powders and the like, parenteral compositions such a aqueous solutions for subcutaneous, nitrasrmscular or intraperitoneal injection, and lyophilized powders combined with a physiological buffer solution just before administration, are formulated depending upon tlie chosen: route of administration,

[0040] According to the present invention, the method may com rise further administering a second therapeutically active agent, such, as proteins, peptides, polysaccharides, lipids, nucleic acid molecule, synthetic organic molecules, hormones, antibiotics, antivirals, antifungals, vasoactive compounds., inimunoffiod latory compounds _* vaccines, local anesthetics, antiangiogenie agents, and antibodies,

[0041] The term "atherosclerosis" is given its ordinary meaning in the art and refers to a disease of the arterial wall in which the layer thickens, causing narrowing of the channel and thus, impairing blood flow. Atherosclerosis may occur in any area of the body, but can be most damaging to a subj ect when it occurs in the heart, brain or blood vessels leading to the brain stem. Atherosclerosis includes thickening and hardening of artery walls or the accumulation of fat, cholesterol and other substances that form atheromas or plaques.

[0042] As used herein, the term "inflammatory cardiovascular disease or disorder" refers to a cardiovascular disease or disorder caused by inflammation. In the present invention, an inflammatory cardiovascular disease or disorder is selected from the group consisting of hyperlipidaemia, hyperch ol esterolaemia, heart attack, stroke, coronary h eart disease, and a cardiovascular disorder

[0043] As used herein, _: a "subject" refers to. any mammal (e.g., a human), sueli as a mammal that comprises at least one tissue lumen or hollow organ. Examples include a human, a non-hum-an primate, a cow, a horse, a pig, a sheep, a goat a dog, cat, or a rodent such as a, moose, -a. rat, a hamster, or -a. guinea, pig. Generally, or course, the invention is directed toward use with humans. A subject may be a subject diagnosed with the disease or condition or otherwise knowa t© have the disease or condition (e.g., atherosclerosis), in some embodiments, a subject may be diagnosed as, or known to be, at risk of developing a disease or condition.

[0044] The "therapeutically effective amount" as used herein means: that amount of a compound, material, or composition comprising a compound of the present invention which is effective for prod cing some desired therapeutic effect in u subject at a reasonable benefit/risk ratio applicable to any medical treatment. Accordingly, a therapeutically effective amount prevents, minimizes, or reverses disease progression assoc ated with, a disease or condition. Disease progression can be .monitored by clinical observations, laboratory and imaging investigations apparent t a perso skilled in the art, A

tlierapeutieaily effective amount can b an amount that is effective in a single dose or an amoua that is effective as part of a multi-dose therapy, for example an amount that is administered in two or more doses or an amount that is administered chronically.

[0045] The present invention will now be described more specifically with reference to the following examples, which are provided for the purpose of demonstration rather than limitation.

[0046] Examples

[0047] MATERIALS AND METHODS

[0048] Animal Model

[0049] Apolipoprotein E-deficient (ApoE EO) mice are well validated model of atherosclerosis that follows a pattern of progression similar to that of human disease. Wild- type (WT) and ApoE KO mice on a C57BL 6 background were purchased from the Jackson Laboratories (ME, U.S.A.). The mice were fed with standard chow diet or Western diet. 'H e water was given ad libitum. The mice were maintained on a 12-h light and dark cycle.

[0050] From 5 weeks of age. male control C57BL/6 mice were fed a standard chow and mal ApoE EO mice wer fed a Western diet (20.% fat, 0.15% cholesterol; AIN-76A) for a given period of time (5-16 weeks). After 12 weeks on standard chow or the Western diet, mice were sacrificed. Additionally, ApoE KO mice fed a Western diet were treated with a mouse anti-ΜΙΡ-Ιβ monoclonal antibody (#46907) [MAB451](1 or 10 ig per mouse, i.p. R&D Systems) or IgG2a isotype control [MAB006] 3 times per week up to 4 weeks. The animal study project was approved by the Institutional Animal Care and Use Committee of School of National Yang-Ming University, Taipei, Taiwan. All experiments conformed to the relevant regulatory standards.

[0051] Tissue Harvesting

[0052] Mice were anesthetized and left ventricle was perfused with PBS ( 10 ml) with an exit through the severed right femoral artery. The heart and aorta (section between the heart and the bifurcation of an iliac artery) were harvested, cleaned of adventitial fat and fixed in 4% paraformaldehyde solution overnight. The heart and aorta were embedded into Paraffin.

[0053] Histologic Staining

[0054] After the heart and aorta were embedded into Paraffin, for the quantitation of atherosclerosis, 8 μπι serial sections of aortic sinus or arch were stained with hematoxylin and eosin to determine lesion size. Elastica van Gieson staining was used for the

visualization of vascular elastic fibers and collagen. Furthermore, it was used to determine necrotic core area as well as fibrous cap thickness. Quantification analysis of plaques areas was assessed with Motic Images Plus 2.0 software. The necrotic core area and fibrous cap thickness were quantitated by Image J software.

[0055] Immunohistochemical Staining

[0056] Immunohistochemical assays were performed with the following primary antibodies: rat F4/80 antibody (C1-A3-1) [NB600-404] (1:50 dilutions; Novus), rabbit MMP-9 antibody [PA5- 13199] (1 :50 dilution; Thermo scientific), rabbit MMP-2 antibody [PA1- 16667] (4 μg/ml dilution; Thermo scientific), goat MIP-Ιβ antibody (M20) [sc-1387]

(1:50 dilution; Santa Cruz Biotechnologies). Secondary antibodies used in these assays were purchased from Jackson ImmunoResearch Laboratories, Inc. The reaction was visualized by staining with 3, 3-diaminobenzidine (DAB) or fluorescence (FITC). Quantification analysis of fluorescent was assessed with Metamorph software [Immunohistochemical analysis was quantitated by Image J software].

[0057] Biochemical Indexes

[0058] Blood samples from mice were harvested at time points of 12, 14, 16 weeks old mice after a 5-hour fast. Placed blood samples at room for 2 hours. Then blood samples were centrifuged for 25 minutes at 2100 rpm, and sera were transferred and stored at -80°C. Levels of total cholesterol (TC), triglycerides (TGs), and non-high-density lipoprotein (non-

HDL) were determined by using Automated Clinical Chemistry Analyzer (FUJI DRI- CHEM 4000i) ; blood glucose was measured by Optium Xceed.

[0059] Enzyme Linked Immunosorbant Assay

[0060] Levels of IL-6, TNF-a, and MIP-Ιβ in serum were measured by R&D systems ELISA kits. The assay employs the quantitative sandwich enzyme immunoassay technique.

An affinity purified polyclonal antibody specific for mouse IL-6, TNF-a or MIP-Ιβ had been pre-coated onto a microplate individually. Standard, control, and sample were pipetted into wells and any mouse IL-6, TNF-a or MIP-Ιβ present were bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for mouse IL-6, TNF-a or MIP- lpwas added to the wells. Following a wash to remove any unbound antibody- enzyme reagent, a substrate solution was added to the wells. The enzyme reaction yielded a blue product that turned yellow when the stop solution was added. The intensity of the color measured is in proportion to the amount of mouse IL-6, TNF-a or MIP-Ιβ bound in the initial step. The sample values were then read off the standard curve.

[0061] Western Blotting

[0062] There were three groups of blood vessels tissues. The blood vessels dissected into two sections for different groups. Different groups of blood vessel tissues were rinsed into RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 50 mM NaF) containing protease inhibitor cocktail

(Calbiochem) for 1-hour incubation on ice. After centrifugation, the supernatant contained whole cell lysates. Protein concentrations were measured by BCA assay (Thermo). Protein was subjected to 12% SDS-PAGE in running buffer (25 mM Tris pH 8.8, 192 mM glycine, 0.1% SDS). PVDF membrane was activated by methanol before electroblotting separated protein onto a PVDF membrane in transfer buffer (25 mM Tris pH 8.8, 192 mM glycine,

20% methanol). Membranes were probed with monoclonal antibodies directing to ΜΙΡ-1β

(R&D antibodies), and β-actin (Chemicon) at 4 °C overnight. After incubation with secondary antibody, the probed proteins were visualized by using chemiluminescence detection reagents according to the manufacturer's instructions.

[0063] Statistical Analyses

[0064] Data were presented as mean ± standard deviation (SD). Statistical differences were assessed by one-way analysis of variance ( ANOVA) and unpaired t tests in treatment groups and control group. P value less than 0.05 was regarded as significant.

[0065] EXAMPLE 1 : Elevated Levels of MIP- Ιβ in Serum and Aorta of Atherosclerotic Mice Were Reduce After 4 Weeks Antibody Treatment

[0066] This study defines the levels of MIP-Ιβ in the serum of animal model. The ELISA results indicate that the MIP-Ιβ level was elevated in IgG control group (n=6) from

46.82±22.9 to 71.87±35.79 pg/mL. (Figure 1 A). On the other hand, after a 4-week treatment, the level of MIP-Ιβ in serum in both 1 μg and 10 μg anti-ΜΙΡ-Ιβ antibody- treated groups (n=6 for each group) remain unchanged (1 μg: 47.75±10.13~46.67±27.57 pg/mL; 10 μ _β : 51.17±24.20~46.45±21.70 pg/mL) (Figure 1A).

[0067] At the time of sacrifice, this study dissected the aorta into two sections for western blot assay. The lower sections of aorta had more MIP-Ιβ expression than upper sections. Moreover, the MIP-Ιβ protein level in the whole aorta significantly decreased in

10 μg antibody-treated group (—78%) (n=6 for each group) (Figure IB and C).

[0068] EXAMPLE 2. MIP- 1 β Neutralization Attenuated Pro-Inflammatory Factors in

Circulating and Atherosclerotic Plaques

[0069] Atherosclerosis and increased risk of thromboembolic complications have been associated with increased circulating levels of IL-6 and TNF-a. To confirm the effects of anti-MIP-lp antibody on proinflammatory factors IL-6 and TNF-a level, the ELISA was performed and the results show the levels of IL-6 were reduced compared to IgG control group in anti-ΜΙΡ-Ιβ antibody-treated groups (IgG: 9.07±7.01~20.40±10.54 pg mL; 1 μg: 9.73±9.16~13.68±5.75 pg/mL; 10 μ _β : 9.06±7.17~11.47±14.31 pg/mL) (Figure ID).

Compared to IgG control group, the levels of TNF-a were also reduced in anti-ΜΙΡ-Ιβ antibody-treated groups (IgG: 1.13±0.93~1.70±0.98 pg mL; 1μ^ 1.16±0.69~0.76±0.29 pg/mL; 10 μg: 1.16±0.82~0.59±0.40 pg mL) (Figure IE).

[0070] EXAMPLE 3 : MIP- 1 β Neutralization Effect on Metabolic Parameters

[0071] The serum lipids in ApoE KO mice fed a Western diet from 5 weeks of age until 16 weeks of age were examined. After a 4-week 10 μg anti-ΜΙΡ-Ιβ antibody treatment, there was a significant 9.7 % decrease in serum total cholesterol compared to IgG control group (Figure 2B); The serum triglyceride remains unchanged and was less than IgG control group (-20%) (Figure 2C). And non-HDL level was less than IgG control group (-10%) (Figure 2D). However, in 1 μg anti-ΜΙΡ- Ιβ antibody treatment, the effect of lower lipid profile was not significant (Figures 2B, 2C and 2D). On the other hands, there was a significant 8.7% decrease in blood glucose after 10 μg anti-ΜΙΡ-Ιβ antibody treatment compared to IgG control group (Figure 2A).

[0072] MIP-Ιβ inhibition significantly increased LX expressions in liver tissues in ApoE KO mice (Figure 2F). The above data showed that MIP-Ιβ inhibition could modify lipid profile via upregulating LX s and attenuate the elevate trend of blood sugar in atherosclerotic mice.

[0073] The metabolic data were provided in Table 1; wherein the data were means ± SD (n=6 in each group); TCHO represents total cholesterol; TG represents triglyceride; Non- HDL represents non-high-density lipoprotein (# <0.05, ##P<0.01 compared with the IgG ₂ a isotype control group).

Table 1 : Metabolic data in normal and atherosclerosis mice

[0074] EXAMPLE 4: Effect of MIP- 1 β Depletion on Atherosclerotic Plaque

Development

[0075] To examine the impact of MIP-lp on atherosclerosis in this model, 12-week-old ApoE KO mice were given anti-ΜΙΡ-Ιβ for 4 weeks. As shown in figure 1 A-C, there were elevated levels of MIP-lp in serum and aorta of IgG control group. After 4-week antibody treatment, the value of MIP-lp was reducing. To further understand the effect of the anti-

MIP-lp antibody on atherosclerotic plaques. The atherosclerotic lesion area was analyzed and quantified on cross-sectional aortic root staining with HE staining. As compared to that in IgG control group, the atherosclerotic lesion areas were significantly attenuated by 10 μg antibody treatment, for 4 weeks in ApoE KO mice (—28%) (Figure 3 A).

[0076] EXAMPLE 5 : Effect of MIP- 1 β Depletion on Atherosclerotic Plaque Quality

[0077] Rupture of the fibrous cap is considered to be the critical event that leads to thromboembolic complications in atherosclerotic coronary and carotid artery disease ³³ . The characteristic feature of ruptured plaques is a thin fibrous cap with a higher ratio of macrophages to vascular smooth muscle cells (VSMCs) covering a large, lipid-rich, collagen-poor necrotic core ³⁴ . Therefore, this study measured the thickness of the fibrous cap and the size of the lipid-rich necrotic core. Treatment with anti-MIP-lp antibody visibly increased fibrous cap thickness (-78% increase compared to IgG control) in the aorta (Figure 3B) and the anti-ΜΙΡ-Ιβ antibody group exhibited significantly smaller necrotic areas (—25% decrease compared to IgG control) (Figure3C).

[0078] Because increased numbers of inflammatory cells are implicated in plaque vulnerability ³⁵ , this study next examined macrophage infiltration into plaques. Levels of immunoreactive macrophage marker F4/80 show that macrophage content within plaques was decreased in 10 μg anti-MIP-lp antibody-treated groups (—40% decrease compared to IgG control) (Figure 4A). The MIP-lp level was also reduced in plaques (—21% decrease compared to IgG control) (Figure 4B).

[0079] In plaque vulnerability, MMPs are importance because they directly degrade

ECM components and are efficient at neutral pH ³⁴ . Among them, MMP-2 actively degrade intact fibrillar collagens and have a special role in weakening plaques. Destruction of elastin, especially by MMP-9, appears to have a role in outward remodeling and aneurysm formation ³⁶ . Therefore, this study examined MMP-2 and MMP-9 expressions within plaques and found that both of them were decreased in 10 μg anti-MIP-lp antibody-treated groups (MMP-2: -77.2%; MMP-9: -54% decrease compared to IgG control) (Figures 5 A and 5B).

[0080] The descriptions and claims as provided should be understood as of

demonstrative purpose instead of limitative in any way to the scope of the present invention.