WO2014064192A1 - Method and pharmaceutical composition for use in the treatment and prediction of myocardial infraction - Google Patents

Abstract

The present invention relates to a method for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction comprising the steps consisting of i) determining the expression level of MCP-3 in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis of the survival time or of the recurrence of a myocardial infarction when the expression level is lower than the predetermined reference value and a poor prognosis of the survival time or of the recurrence of a myocardial infarction when the expression level is higher than the predetermined reference value. The invention also relates to a compound which inhibits the binding of MCP-3 to CCR2; CCR1 or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCR1 or CCR3 gene expression or signalling pathway for use in the treatment of myocardial infarction.

Description

METHOD AND PHARMACEUTICAL COMPOSITION FOR USE IN THE TREATMENT AND PREDICTION OF MYOCARDIAL INFRACTION

FIELD OF THE INVENTION:

The invention relates to a method for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction comprising the steps consisting of i) determining the expression level of MCP-3 in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis of the survival time or a low risk of the recurrence of a myocardial infarction when the expression level is lower than the predetermined reference value and a poor prognosis of the survival time or a high risk of the recurrence of a myocardial infarction when the expression level is higher than the predetermined reference value.

The invention also relates to a compound which inhibits the binding of MCP-3 to CCR2; CCR1 or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCR1 or CCR3 gene expression or signalling pathway for use in the treatment of myocardial infarction.

BACKGROUND OF THE INVENTION:

Acute thrombotic obstruction of the blood flow in a coronary artery precipitates myocardial infarction. The loss of heart muscle in the necrotic zone and the compromised function of the remaining viable cardiomyocytes of the peri-necrotic region initiate a series of events that, if unopposed, frequently lead to adverse remodeling of the heart chamber, precipitating heart failure. The mainstay of treatment of acute myocardial infraction (MI) associates rapid restoration of a patent coronary artery either mechanically or through thrombolytic and anti-platelet therapies, and administration of agents that reduce oxygen consumption and unload the heart muscle. The wide use of this therapeutic strategy has led to significant reductions in both morbidity and mortality after acute MI [White, H.D et al, 2008]. Still, the clinical and social burden of ischemic heart disease is unacceptably high and the efficacy of additional anti-thrombotic therapies is often mitigated by the increased risk of hemorrhagic events. Thus, efforts are being directed towards targeting other pathophysiological pathways, particularly those involved in post-ischemic cardiac remodelling [Shah, A.M et al, 2011].

The immune system becomes activated in response to myocardial damage. Shortly after ischemia, the damaged tissue exposes ligands that are recognized by components of the innate immune system, which leads to its activation. For example, non-myosin heavy chain type II A and C are exposed following ischemia/reperfusion injury and recognized by natural IgM antibodies, leading to activation of mannan binding lectin and serum complement, which aggravates tissue injury. C-reactive protein (CRP), a short pentraxin acute-phase protein, also binds to damaged tissue and activates the complement, leading to aggravation of tissue injury in the setting of acute MI 13. In contrast, long pentraxin 3, a molecule that limits complement activation plays a cardioprotective role in this setting. The acute inflammatory response also leads to the mobilization and recruitment of innate immune cells. Few hours after the ischemic insult, neutrophils are actively recruited into the ischemic tissue and contribute to tissue inflammation and cardiovascular injury through the production of inflammatory mediators, reactive oxygen species and various proteases [Granger, D.N. et al, 1995 and Vinten-Johansen, J 2004]. The wave of neutrophil infiltration is followed by the mobilization and recruitment of monocytes. Recent studies have shed new light on the mechanisms of monocytes recruitment and life cycle in the setting of acute MI, and suggested differential pathogenic or protective roles for Ly6Chi and Ly6Clo monocytes, respectively, in cardiac remodeling and preservation of heart function [Nahrendorf, M., et al, 2007 and Leuschner, F., et al, 2012]. Despite this increasing knowledge, the utility of targeting the immune response in this setting is still uncertain as revealed by the lack of efficacy of complement inhibition in patients with acute MI [Mahaffey, K.W., et al, 2003; Granger, C.B., et al, 2003; Armstrong, P.W., et al, 2007 and Eikelboom, J.W et al, 2007]. Thus, a better characterization of the determinants of the immune response following ischemic injury and the mechanisms by which they contribute to tissue damage is required in order to fill the existing gap of knowledge that limits clinical translation, and design efficient therapeutic strategies for future use in humans.

SUMMARY OF THE INVENTION: Here, the inventors addressed the role of mature B lymphocytes, a subset of immune cells that orchestrates a variety of adaptive immune responses relevant to human diseases, but that intriguingly, has been relatively neglected in the setting of ischemic injury. They show that following acute myocardial infarction in mice, mature B cells produce MCP-3, a chemokine that induces monocyte mobilization from the bone marrow, leading to enhanced myocardial inflammation, tissue injury and deterioration of myocardial function. Moreover, they show that depletion of mature B cells using a CD20-specific monoclonal antibody impedes MCP-3 production and monocyte mobilization, limits post-ischemic myocardial injury and improves heart function. At last, they also show that the detection of circulating MCP-3 levels at the acute phase of myocardial infarction in humans is independently associated with a significant increase in the risk of death or recurrent myocardial infarction at follow-up.

Thus, the invention relates to a method for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction comprising the steps consisting of i) determining the expression level of MCP-3 in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis of the survival time or of the recurrence of a myocardial infarction when the expression level is lower than the predetermined reference value and a poor prognosis of the survival time or of the recurrence of a myocardial infarction when the expression level is higher than the predetermined reference value.

The invention also relates to a compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signalling pathway for use in the treatment of myocardial infarction.

DETAILED DESCRIPTION OF THE INVENTION:

Definitions:

Throughout the specification, several terms are employed and are defined following paragraphs.

Prognostic method The invention relates to a method for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction comprising the steps consisting of i) determining the expression level of MCP-3 in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis of the survival time or a low risk of the recurrence of a myocardial infarction when the expression level is lower than the predetermined reference value and a poor prognosis of the survival time or a high risk of the recurrence of a myocardial infarction when the expression level is higher than the predetermined reference value.

In one embodiment, the myocardial infarction may be an acute myocardial infarction.

Typically, the sample according to the invention may be a blood, plasma, serum, lymph and cardiac tissue.

As used herein, the term "MCP-3" for "Monocyte-chemoattactant protein 3" also known as "CCL7" for "Chemokine (C-C motif) ligand 7" has its general meaning in the art and refers to a small cytokine known as a chemokine. An exemplary sequence for human MCP-3 gene is deposited in the database under accession number NM_006273.2. An exemplary sequence for human MCP-3 protein is deposited in the database under accession number NP 006264.2.

As used herein, the term "CCR2" for "chemokine (C-C motif) receptor 2" has its general meaning in the art and refers to a chemokine receptor. CCR2 is one of the receptors of MCP-3. An exemplary sequence for human CCR2 gene is deposited in the database under accession number NM 001123041.2 (NM 001123396.1). An exemplary sequence for human CCR2 protein is deposited in the database under accession number NP 001116513.2 (NP 00116868.1).

As used herein, the term 'CCR1" for "C-C chemokine receptor type 1" has its general meaning in the art and refers to a chemokine receptor. CCR1 is one of the receptors of MCP- 3. An exemplary sequence for human CCR1 gene is deposited in the database under accession number NM 001295.2. An exemplary sequence for human CCR2 protein is deposited in the database under accession number NP 001286.1.

As used herein, the term 'CCR3" for "C-C chemokine receptor type 3" has its general meaning in the art and refers to a chemokine receptor. CCR3 is one of the receptors of MCP- 3. An exemplary sequence for human CCR3 gene is deposited in the database under accession numbers NM_001837.3, 178328.1, 001164680.1, 178329.2. An exemplary sequence for human CCR3 protein is deposited in the database under accession numbers NP 001828.1, 847898.1, 001158152.1, 847899.1.

As used herein, the term "patient", is intended for a human affected or likely to be affected with a myocardial infarction, preferably an acute myocardial infarction.

The term "determining the expression level of MCP-3" as used above includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control. Typically MCP-3 expression may be measured for example by RT-PCR, immunohistochemistry or ELISA performed on the sample.

The "control" or the "reference value" may be a healthy subject, i.e. a subject who does not suffer from any myocardial infarction. The control may also be a subject suffering from myocardial infarction. Preferably, said control is a healthy subject.

As used herein the term "good prognosis of survival time" denotes a long survival time for a patient which had myocardial infarction.

As used herein, the term "poor prognosis of survival time" denotes a short survival time for a patient which had myocardial infarction.

In another embodiment, the invention relates to a method for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction comprising the steps consisting of i) determining the expression level of MCP-3 in a sample from said patient, ii) providing a good prognosis of the survival time or a low risk of the recurrence of a myocardial infarction when the expression level of MCP-3 is not detectable and a poor prognosis of the survival time or a high risk of the recurrence of a myocardial infarction when the expression level of MCP-3 is detectable.

In another embodiment, the invention relates to a method for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction comprising the steps consisting of i) determining the expression level of MCP-3 in a sample from said patient, ii) providing a low risk of death and low risk of recurrent myocardial infarction when the expression level of MCP-3 is not detectable and a high risk of death and high risk of recurrent myocardial infarction when the expression level of MCP-3 is detectable.

For example determining the expression level of MCP-3 in sample may be performed by measuring the expression level of MCP-3 gene.

Typically, the detection comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptides or nucleic acids of interest originally present in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column... In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody-antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.

In a particular embodiment, the expression level of MCP-3 gene may be determined by determining the quantity of mR A of MCP-3 gene. Such method may be suitable to measure the expression level of MCP-3 gene in the sample.

Methods for measuring the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA may be then detected by hybridization (e. g., Northern blot analysis).

Alternatively, the extracted mRNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that enable amplification of a region in the MCP-3 gene. Preferably quantitative or semi-quantitative RT-PCR is used. Realtime quantitative or semi-quantitative RT-PCR is particularly advantageous. Extracted mRNA may be reverse-transcribed and amplified, after which amplified sequences may be detected by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.

Other methods of amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably at least 85% identical and even more preferably at least 90%, preferably at least 95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i. e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

In a particular embodiment, the method of the invention comprises the steps of providing total RNAs obtained from the sample of the patient, and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi-quantitative RT-PCR. Total R As can be easily extracted from the sample. For instance, the sample may be treated prior to its use, e.g. in order to render nucleic acids available. Techniques of cell or protein lysis, concentration or dilution of nucleic acids, are known by the skilled person.

In another embodiment, the expression level of MCP-3 gene may be measured by DNA microarray analysis. Such DNA microarray or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To measure the expression level of MCP-3 gene, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210). Detection of MCP-3 expression in the sample may also be performed by measuring the level of MCP-3 protein. In the present application, the "level of MCP-3 protein" means the quantity or concentration of said MCP-3 protein or the quantity of cells which express MCP-3.

Such methods comprise contacting a sample with a binding partner capable of selectively interacting with MCP-3 protein present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immuno diagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. More preferably, determination of the concentrations of MCP-3 is performed with a fluorescence- activated cell sorter (FACS). Said fluorescence- activated cell sorter is a machine that can rapidly separate the cells in a suspension on the basis of size and the color of their fluorescence.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

One particular method utilizes immunohistochemistry, a staining method based on immuno enzymatic reactions using monoclonal or polyclonal antibodies to detect cells or specific proteins such as tissue antigens. Typically, immunohistochemistry protocols involve at least some of the following steps:

1) antigen retrieval (eg., by pressure cooking, protease treatment, micro waving, heating in appropriate buffers, etc.);

2) application of primary antibody (i.e. anti-MCP-3 protein antibody) and washing;

3) application of a labeled secondary antibody that binds to primary antibody (often a second antibody conjugate that enables the detection in step 5) and wash;

4) an amplification step may be included;

5) application of a detection reagent (e.g. chromagen, f uorescently tagged molecule or any molecule having an appropriate dynamic range to achieve the level of or sensitivity required for the assay);

6) counterstaining may be used and 7) detection using a detection system that makes the presence of the proteins visible (to either the human eye or an automated analysis system), for qualitative or quantitative analyses.

Various immuno enzymatic staining methods are known in the art for detecting a protein of interest. For example, immunoenzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC, or Fast Red; or fluorescent labels such as FITC, Cy3, Cy5, Cy7, Alexafluors, etc. Counterstains may include H&E, DAPI, Hoechst, so long as such stains are compatable with other detection reagents and the visualization strategy used. As known in the art, amplification reagents may be used to intensify staining signal. For example, tyramide reagents may be used. The staining methods of the present invention may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated or manual systems.

The method of the invention comprises a step consisting of comparing MCP-3 expression with a reference value or a control reference.

Predetermined reference values used for comparison may consist of "cut-off values that may be determined as described hereunder. Reference ("cut-off) value for MCP-3 expression may be determined by carrying out a method comprising the steps of:

a) providing a collection of samples from patients suffering of myocardial infarction; b) determining the MCP-3 expression level for each sample contained in the collection provided at step a);

c) ranking the samples according to said expression level

d) classifying said samples in subsets of increasing, respectively decreasing, number of members ranked according to their expression level,

e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding patient (i.e. the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction or both);

f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve;

g) for each subsets of samples calculating the statistical significance (p value) between both subsets h) selecting as reference value for the expression level, the value of expression level for which the p value is the smallest.

A confidence interval may be constructed around the value of expression level thus obtained, for example ± 5 or 10%.

For example the expression level of MCP-3 has been assessed for 100 samples of 100 patients. The 100 samples are ranked according to the expression level of MCP-3. Sample 1 has the highest expression level and sample 100 has the lowest expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.

The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that according to the experiments made by the inventors, the reference value is not necessarily the median value of expression levels.

In routine work, the reference value (cut-off value) may be used in the present method to discriminate samples and therefore the corresponding patients.

Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art. P value is conventionally used in statistical significance testing.

The man skilled in the art also understands that the same technique of assessment of the expression level of MCP-3 should preferably be used for obtaining the reference value and thereafter for assessment of the expression level of MCP-3 of a patient subjected to the method of the invention.

If for example, the expression level of MCP-3 is higher than the reference value, the patient is considered as a poor prognosis of the survival time or as a high risk of the recurrence of a myocardial infarction. Similarly, the expression level of MCP-3 is lower than the reference value, the patient is considered as a good prognosis of the survival time or as a low risk of the recurrence of a myocardial infarction. The setting of a single "cut-off value allows discrimination between a poor and a good prognosis with respect to survival time and a low risk and a high risk of the recurrence of a myocardial infarction for a patient. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in one alternative embodiment of the invention, instead of using a definite reference value, a range of values is provided.

Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. According to this specific embodiment of a "cut-off value, poor, good prognosis and recurrence can be determined by comparing the expression level with the range of values which are identified. In certain embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum P value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6.

Therefore, a patient may be assessed by comparing values obtained by measuring the expression level of MCP-3, where values less than 5 reveal a good prognosis and values greater than 5 reveal a poor prognosis. In a another embodiment, a patient may be assessed by comparing values obtained by measuring the expression level of MCP-3 and comparing the values on a scale, where values below the range of 4-6 indicate a good prognosis and values above the range of 4-6 indicate a poor prognosis, with values falling within the range of 4-6 indicating an intermediate prognosis.

In a particular embodiment, the method of the invention comprises comparison steps which include a classification of the quantification values measured for the expression level of MCP-3 into two possibilities, respectively: (i) a first possibility when the quantification value for the expression level is higher than the predetermined corresponding reference value (the first possibility is named "Hi" for example) and (ii) a second possibility when the quantification value for the expression level is lower than the predetermined corresponding reference value (the second possibility is named " Lo " for example). As used herein, "the expression level of MCP-3" refers to an amount or a concentration of a transcription product, for instance mRNA coding for MCP-3, or of a translation product, for instance the protein MCP-3 or of percentage of cells which express MCP-3 or of mean fluorescence intensity of MCP-3 (by FACS). Typically, a level of mRNA expression can be expressed in units such as transcripts per cell or nanograms per microgram of tissue. A level of a polypeptide can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe an expression level.

In a particular embodiment, when the measure of MCP-3 gene expression is performed by rt qPCR, the expression level of MCP-3 gene in a patient with a poor prognosis of the survival time or of the recurrence of myocardial infarction is increased by at least 35%, preferably by at least 40%>, preferably by at least 50%>; preferably by at least 60 %, preferably by at least 70%, preferably by at least 80%, more preferably by at least 90%, even more at least 100% compared to a control reference. In other words, preferably, when MCP-3 gene expression is measured by rt qPCR, the quantity of mRNA encoding MCP-3 gene in a patient with a poor prognosis of the survival time or of the recurrence of myocardial infarction is increased by at least 35%, preferably by at least 40%>, preferably by at least 50%>; preferably by at least 60 %, preferably by at least 70%>, preferably by at least 80%>, more preferably by at least 90%, even more at least 100% compared to a control reference.

The present invention also relates to kits for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction, comprising means for detecting MCP-3 expression.

According to the invention, the kits of the invention may comprise an anti-MCP-3 protein antibody; and another molecule coupled with a signalling system which binds to said

MCP-3 protein antibody.

Typically, the antibodies or combination of antibodies are in the form of solutions ready for use. In one embodiment, the kit comprises containers with the solutions ready for use. Any other forms are encompassed by the present invention and the man skilled in the art can routinely adapt the form to the use in immunohistochemistry.

The present invention also relates to MCP-3 gene or protein as a biomarker for the prediction of the survival time of a patient suffering from myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction.

In another embodiment, the invention relates to an in vitro method for monitoring a patient's response to myocardial infarction treatment which comprises a step of measuring the expression level of MCP-3 gene, or a step of measuring the level of MCP-3 protein, in a sample from a patient.

Thus, the present invention relates to the use of MCP-3 gene or protein as a biomarker for the monitoring of anti myocardial infarction therapies.

According to the invention, the expression level of MCP-3 gene or the level of MCP-3 protein may be determined to monitor a patient's response to myocardial infarction treatment.

Compounds and uses thereof

A second object of the invention relates to a compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signalling pathway for use in the treatment of myocardial infarction.

In particular embodiment, the compound according to the invention inhibits the binding of MCP-3 to CCR2.

In particular embodiment, the compound according to the invention inhibits the binding of MCP-3 to CCRl .

In particular embodiment, the compound according to the invention inhibits the binding of MCP-3 to CCR3. In particular embodiment, the compound according to the invention is an inhibitor of

MCP-3 gene expression.

In particular embodiment, the compound according to the invention is an inhibitor of CCR2 gene expression.

In particular embodiment, the compound according to the invention is an inhibitor of CCRl gene expression.

In particular embodiment, the compound according to the invention is an inhibitor of CCR3 gene expression. In another particular embodiment, the compound according to the invention is an inhibitor of MCP-3, CCR2, CCRl or CCR3 signalling pathway.

In one embodiment, the myocardial infarction is an acute myocardial infarction.

In one embodiment, the compound according to the invention may bind to MCP-3, CCR2, CCRl or CCR3 and block the binding of MCP-3 on CCR2, CCRl or CCR3 and block its effect on the monocyte mobilization or infiltration. To identify a compound able to block the interaction between MCP-3 and its receptors, a test which demonstrates the effect of the compound on cell migration or signalling pathway may be used (see for example Chou CC et al. 2002; Loetscher P et al, 2001 and Blanpain C et al, 1999.

As used herein, the term "inhibitor of the signaling pathway" denotes a compound which blocks the signaling cascade of a receptor that is to say the activation of molecules implicated in this pathway.

Typically, the compound according to the invention includes but is not limited to a small organic molecule, an antibody, and a polypeptide. In one embodiment, the compound according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

In one embodiment, the compound according to the invention is an antagonist of CCRl like MIP-lbeta (see for example Chou CC et al, 2002).

In one embodiment, the compound according to the invention is an antibody. Antibodies directed against MCP-3, CCR2, CCRl or CCR3.can be raised according to known methods by administering the appropriate antigen or epitope 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. Monoclonal antibodies against MCP-3, CCR2, CCRl or CCR3 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 originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al, 1983); and the EBV-hybridoma technique (Cole et al. 1985). 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 anti-MCP-3, anti-CCR2, anti-CCRl or anti-CCR2 single chain antibodies. Coumpounds useful in practicing the present invention also include anti-MCP-3, anti-CCR2, anti-CCRl or anti-CCR2 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 MCP- 3, CCR2, CCRl or CCR3.

Humanized anti-MCP-3, anti-CCR2, anti-CCRl or anti-CCR2 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). Then, for this invention, neutralizing antibodies of MCP-3, CCR2, CCRl or CCR3 are selected.

In one embodiment, the compound according to the invention is an anti-MCP-3 antibody which neutralizes MCP-3 or an anti-MCP-3 fragment thereof which neutralizes MCP-3.

In a particular embodiment, the antibody according to the invention is the AB-282-NA antibody sold by R&D Systems.

In a particular embodiment, the antibody according to the invention is the MAI -25125 antibody sold by pierce-antibodies.

In a particular embodiment, the antibody according to the invention is the ab 18694 antibo dy so Id by abeam.

In another embodiment, the compound according to the invention is an anti-CCR2 antibody which neutralizes CCR2 or an anti-CCR2 fragment thereof which neutralizes CCR2. In a particular embodiment, the antibody according to the invention may be an antibody according to the patent applications WO0005265 or US2010233155.

In a particular embodiment, the antibody according to the invention may be an antibody according to the patent application US2004151721.

In a particular embodiment, the antibody according to the invention may be an antibody according to the patent application W09731949.

In another embodiment, the compound according to the invention is an anti-CCRl antibody which neutralizes CCRl or an anti-CCRl fragment thereof which neutralizes CCRl .

In a particular embodiment, the antibody according to the invention may be an antibody according to the patent application US2004265304. In a particular embodiment, the antibody according to the invention may be an antibody according to the patent application WO0044790.

In another embodiment, the compound according to the invention is an anti-CCR3 antibody which neutralizes CCR3 or an anti-CCR3 fragment thereof which neutralizes CCR3.

In a particular embodiment, the antibody according to the invention may be an antibody according to the patent application US6207155. In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996).

Then, for this invention, neutralizing aptamers of MCP-3, CCR2, CCRl or CCR3 are selected. In one embodiment, the compound according to the invention is a polypeptide.

In a particular embodiment the polypeptide is a functional equivalent of CCR2, CCRl or CCR3. As used herein, a "functional equivalent" of CCR2, CCRl or CCR3 is a compound which is capable of binding to MCP-3, thereby preventing its interaction with CCR2, CCRl or CCR3. The term "functional equivalent" includes fragments, mutants, and muteins of CCR2, CCRl or CCR3. The term "functionally equivalent" thus includes any equivalent of CCR2, CCRl or CCR3 obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to MCP-3. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence. Functional equivalents include molecules that bind MCP-3 and comprise all or a portion of the extracellular domains of CCR2, CCR1 or CCR3.

In one embodiment, the polypeptide according to the invention is able to treat myocardial infarction through its properties of decoy receptor.

By "decoy receptor", is meant that the polypeptide according to the invention traps

MCP-3 and prevents its physiological effects on CCR2, CCR1 or CCR3.

The functional equivalents include soluble forms of CCR2, CCR1 or CCR3. A suitable soluble form of these proteins, or functional equivalents thereof, might comprise, for example, a truncated form of the protein from which the transmembrane domain has been removed by chemical, proteolytic or recombinant methods.

Preferably, the functional equivalent is at least 80% homologous to the corresponding protein. In a particular embodiment, the functional equivalent is at least 90% homologous as assessed by any conventional analysis algorithm such as for example, the Pileup sequence analysis software (Program Manual for the Wisconsin Package, 1996).

The term "a functionally equivalent fragment" as used herein also may mean any fragment or assembly of fragments of CCR2, CCR1 or CCR3 that binds to MCP-3. Accordingly the present invention provides a polypeptide capable of inhibiting binding of MCP-3 to CCR2, CCR1 or CCR3, which polypeptide comprises consecutive amino acids having a sequence which corresponds to the sequence of at least a portion of an extracellular domain of CCR2, CCR1 or CCR3, which portion binds to MCP-3. In one embodiment, the polypeptide corresponds to an extracellular domain of CCR2, CCR1 or CCR3.

Functionally equivalent fragments may belong to the same protein family as the human CCR2, CCR1 or CCR3 identified herein. By "protein family" is meant a group of proteins that share a common function and exhibit common sequence homology. Homologous proteins may be derived from non-human species. Preferably, the homology between functionally equivalent protein sequences is at least 25% across the whole of amino acid sequence of the complete protein. More preferably, the homology is at least 50%, even more preferably 75% across the whole of amino acid sequence of the protein or protein fragment. More preferably, homology is greater than 80% across the whole of the sequence. More preferably, homology is greater than 90%> across the whole of the sequence. More preferably, homology is greater than 95% across the whole of the sequence.

In one embodiment, the polypeptide according to the invention may be also a functional equivalent of MCP-3. As used herein, a "functional equivalent" of MCP-3 is a compound which is capable of binding to CCR2, CCR1 or CCR3, thereby preventing its interaction with the natural ligand MCP-3. The term "functional equivalent" includes fragments, mutants, and muteins of MCP-3. The term "functionally equivalent" thus includes any equivalent of MCP-3 obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to CCR2, CCR1 or CCR3. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence.

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptides according to the invention or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify bio distribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify bio distribution.

A strategy for improving drug viability is the utilization of water-soluble polymers.

Various water-soluble polymers have been shown to modify bio distribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydro xyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri- functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e- amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold- limiting glomerular filtration (e.g., less than 60 kDa).

In addition, to the polymer backbone being important in maintaining circulatory half- life, and bio distribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of bio distribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

In another embodiment, the compound according to the invention is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signaling pathway.

Small inhibitory RNAs (siRNAs) can also function as inhibitors of MCP-3, CCR2, CCRl or CCR3 gene expression for use in the present invention. MCP-3, CCR2, CCRl or CCR3 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that MCP-3, CCR2, CCRl or CCR3 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as inhibitors of MCP-3, CCR2, CCRl or CCR3 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of MCP-3, CCR2, CCRl or CCR3 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays. Both antisense oligonucleotides and ribozymes useful as inhibitors of MCP-3, CCR2, CCR1 or CCR3 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing MCP-3, CCR2, CCR1 or CCR3. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Particluar viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Particular viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and micro encap sulation.

In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

Another object of the invention relates to a method for treating myocardial infarction comprising administering to a subject in need thereof a therapeutically effective amount of a compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signalling pathway as described above.

In one aspect, the invention relates to a method for treating myocardial infarction comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of the binding of MCP-3 to CCR2 receptor as above described.

As used herein, the term "treating" or "treatment", denotes reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.

In another embodiment, the myocardial infarction is an acute myocardial infarction. Another object of the invention relates to a method for treating patient who has been considered as a poor prognosis for the survival time or for the recurrence of a myocardial infarction according to the above method of the invention comprising administering to a subject in need thereof a compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signaling pathway as described above or a B cell depleting agent.

In another embodiment, the invention relates to a compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signaling pathway or a B cell depleting agent for use in the treatment of patient who has been considered as a poor prognosis for the survival time or for the recurrent of a myocardial infarction according to the above method of the invention.

In another embodiment, the B cell depleting agent is a B cell depleting antibody.

In still another embodiment, the B cell depleting agent is an anti-CD20 antibody.

In a particular embodiment, the compound which inhibits the binding of MCP-3 to is an antibody.

A "B cell depleting agent" has its general meaning in the art and refers to a molecule which depletes or destroys B cells in a patient and thus reduces the humoral response elicited by the B cell. B cell depleting agents are well known in the art (Thomas Dorner, Peter E Lipsky, B-cell targeting: a novel approach to immune intervention today and tomorrow, Expert Opinion on Biological Therapy Sep 2007, Vol. 7, No. 9, Pages 1287-1299: 1287- 1299). 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 B cell depleting agent may bind to a B cell surface marker. 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 CD10, CD 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86. Alternatively, the B cell depleting agent may target a B cell survival factor or a cytokine imperative for B cell function or an effector thereof (e.g., a receptor which binds the aforementioned factor). Typically, the B cell survival factor is the B- cell activating factor (BAFF). In a particular embodiment, the B cell depleting agent depletes the B2 cells and not the Bl cells.

In a particular embodiment, the B cell depleting agent is an anti-B cell antibody, preferably a monoclonal antibody (e.g. a chimeric, humanized or human antibody). For example, a suitable anti-B cell antibody can be an antibody targeting any B cell surface marker e.g. an antiCD20 monoclonal antibody [e.g. Rituximab (Roche), Ibritumomab tiuxetan (Bayer Schering), Tositumomab (GlaxoSmithKline), AME-133v (Applied Molecular Evolution), Ocrelizumab (Roche), Ofatumumab (HuMax-CD20, Gemnab), TRU-015 (Trubion) and IMMU-106 (Immunomedics)], an anti-CD22 antibody [e.g. Epratuzumab, Leonard et al, Clinical Cancer Research (Z004) 10: 53Z7-5334], an anti-CD79a antibody, an anti-CD27 antibody, or an antiCD 19 antibody (e.g. U.S. Pat. No. 7, 109,304). Another example of anti-B cell antibody include an antibody targeting a B cell survival factor or a cytokine imperative for B cell function or an effector thereof (e.g., a receptor which binds the aforementioned factor). Such antibodies include the anti-BAFF antibody (e.g. Belimumab, GlaxoSmithKline), the anti- APRIL antibody (e.g. anti-human APRIL antibody, ProSci inc.), the anti-IL-6 antibody [previously described by De Benedetti et al, J Immunol (2001) 166: 4334-4340 and by Suzuki et al, Europ J of Immunol (1992) 22 (8) 1989-1993, fully incorporated herein by reference], the anti-IL-7 antibody (R&D Systems, Minneapolis, Minn.) or the SDF-1 antibody (R&D Systems, Minneapolis, Minn.).

Depletion of B cells may also be achieved by the use of fusion proteins which block activation of B cell receptors. For example, a fusion protein composed of the extracellular ligand binding portion of TACI which blocks activation of TACI by April and BLyS (e.g. Atacicept, Merck) or a fusion protein composed of the extracellular ligand-binding portion of BAFF-R which blocks activation of BAFF-R by BLys (e.g. BR3-Fc, Biogen and Genentech). Such fusion proteins can be generated using methods known in the art, such as recombinant DNA technology as is described in details herein below.

Typically, a "B cell depleting agent" may be an agent as described in Gullick Nicola et al. 2007.

In another embodiment, the B cell depleting agent is a B cell depleting antibody.

In still another embodiment, the B cell depleting agent is an anti-CD20 antibody.

In still another embodiment, the B cell depleting agent is an anti-BAFF antibody.

In another embodiment, the compound which inhibits the binding of MCP-3 to CCR2; CCR1 or CCR3 or the compound which is an inhibitor of MCP-3, CCR2, CCR1 or CCR3 gene expression or signalling pathway may be used to improve heart function after acute myocardial infarction.

As used herein, the term "to improve heart function" denotes a reduction in end- systolic left ventricular (LV) dimension, a significant improvement of LV shortening fraction, an increase in LV myocardial contractility and by significant improvement in both postishemic ventricular remodeling and myocardial function.

Pharmaceutical composition

Another object of the invention relates to a therapeutic composition comprising a compound according to the invention for use in the treatment of myocardial infarction.

Preferably, said compound is an inhibitor of the binding of MCP-3 to CCR2; CCRl or CCR3 or an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signaling pathway.

In a particular embodiment, the invention relates to a therapeutic composition comprising a compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signaling pathway or a B cell depleting agent for use in the treatment of patient who has been considered as a poor prognosis for the survival time or for the recurrent of a myocardial infarction according to the above method of the invention.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers 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.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

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.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

Compounds of the invention may be administered in the form of a pharmaceutical composition, as defined below.

By a "therapeutically effective amount" is meant a sufficient amount of compound to treat and/or to prevent myocardial infarction.

It will be understood that the total daily 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 coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well 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. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: The injured myocardium trigger B lymphocytes recruitment, which is prevented by the B cell-depleting anti-CD20 antibody.

(a) Representative dot plots of B220+ IgM+ B cells (gated on CD45+ cells) in sham- operated (sham) and infarcted (MI) hearts on day 5 after surgery. Numbers of infiltrating B cells were determined per milligram of tissue at specified days following MI (n= 5 to 10 mice per time point). Mean values ± SEM are represented. p < 0.001.

(b) Representative sections of B220 immuno staining on days 3 and 5 after surgery in healthy hearts and in the border infarct area. Bars, lOOum.

(c) B cell (B220+ IgM+) depletion efficiency in infarcted hearts (gated on CD45+ cells) of C57BL/6J mice treated with or without the CD20 antibody (a-CD20) (n=5 to 15 mice per group).

(d, e) Representative examples and quantitative analysis of B220hi IgM+ B cells stainings in blood (d) and spleens (e) of anti-CD20 antibody treated mice compared to controls (n=12 to 15 per group). Mean values ± SEM are represented. Bars, ΙΟΟμιη. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2: B cell depletion using anti-CD20 antibody reduces infarct size, improves heart function and limits myocardial inflammation.

(a) Echocardiography analysis after anti-CD20 (a-CD20) therapy. We measured LV shortening fraction (SF), LV internal diameter at end systole (LVIDs) and LV posterior wall thickness at end systole (LVPWs).

(b) Representative photomicrographs and quantitative analysis of infarct size (%) and myocardial fibrosis evaluation (%), evaluated by Masson trichrome and Sirius Red stainings, respectively, in the 2 groups of mice. Data are representative of 10-14 mice per group in three independent experiments.

(c) Representative histograms of mRNA levels of the pro -inflammatory cytokines IL- 1 β, TNF-α, IL-18 and the anti- inflammatory cytokines IL-10 and TGF-β, within the injured myocardium on day 14 after MI (n=8-12 per group). Mean values ± SEM are represented. Bars, ΙΟΟμιη. *, p < 0.05; **, p < 0.01 vs PBS.

Figure 3: B lymphocyte depletion impairs monocyte mobilization.

(a) Representative examples of 7/4hi and 7/41o monocyte stainings as well as quantification of their numbers in bone marrow (a) and blood (b) of B cell-depleted mice compared to controls on day 3 after MI (n=8-15 per group). Mean values ± SEM are represented. *, p < 0.05 vs PBS. Figure 4: B lymphocyte depletion selectively reduces MCP-3 levels after acute MI and trigger MCP-3-dependent monocyte transmigration in vitro and in vivo.

(a) MCP-1 and MCP-3 protein levels in blood after CD20 mAb treatment, 1 and 3 days after MI (n=5-8 per group).

(b) Bar graphs show MCP-3 release in the supernatant of nonstimulated cultured B cells and activated B cells with anti-CD40 and anti-IgM antibodies.

(c) Representative photomicrographs and histograms of the transmigration of cultured monocytes in the presence of non-stimulated or activated B cells with or without neutralizing anti-MCP-1 or anti-MCP-3 antibodies. Data are representative of four independent experiments, each condition was performed in triplicate. Bars, 300 μιη. Mean values ± SEM are represented. *, p < 0.05; p < 0.001 vs control.

(d) Representative histograms of the number of 7/4hi monocytes in blood (left) and in the infarcted myocardium (right) of Ragl^/^ mice injected with either wild-type splenocytes, B cell-depleted splenocytes, or B cell-depleted splenocytes re-supplemented with wild type or Mcp-3^4 B cells, 3 days after MI. Data are representative of 10 to 12 mice per group in two independent experiments. Mean values ± SEM are shown. *, p < 0.05 vs WT splenocytes ; ##, p< 0.01 vs a-CD20 splenocytes; $, p < 0.05 vs a-CD20 splenocytes with WT B cells.

Figure 5: B lymphocytes trigger adverse ventricular remodeling and alter heart function through the production of MCP-3.

(a) Echocardiography analysis after 14 days of MI and assessment of LV shortening fraction (SF), LV internal diameter at end systole (LVIDs) and LV posterior wall thickness (LVPWs) of Rag 14, mice injected with either wild-type splenocytes, B cell-depleted splenocytes, or B cell-depleted splenocytes re-supplemented with wild-type or Μο -3 Άΐ· B cells.

(b, c) Representative photomicrographs and quantitative analysis of infarct size (b), fibrosis and collagen content (c) in the 4 groups of mice. Results are pooled from three independent experiments with 10 to 25 mice per group. Mean values ± SEM are shown. Bars, lOOum. *, p < 0.05 and ***, p < 0.001 vs WT splenocytes ; #, p < 0.05; and ###, p < 0.001 vs a- CD20 splenocytes; $, p < 0.05; and $$$, p < 0.001 vs a-CD20 splenocytes with WT B cells.

Figure 6: B lymphocytes and MCP-3 in patients with acute ML

(a) Representative photomicrographs of CD20-postive cells (dark brown) in human biopsies 24-48h and 6-7 days after acute MI.

(b) The probability of outcome events (death or recurrent MI) as a function of baseline circulating MCP-3 level in 1000 patients with acute MI. Detectable MCP-3 levels at the admission for acute MI was highly predictive of death and recurrent MI after one year of follow-up after multiple adjustments (see Methods).

EXAMPLE: Material & Methods

Myocardial infarction. All mice were on full C57B1/6J background. C57BL/6 (Janvier, France), Mcp-3-/- [Tsou, C.L., et al, 2007] and Ragl-/- mice (The Jackson Laboratory) were 8 weeks old. Myocardial infarction was induced by left coronary ligation 35. Mice were anesthetized using ketamine (870 mg/kg) and xylazine (140 mg/kg) via intraperitoneal injection (i.p.), then intubated and ventilated with air using a small animal respirator. The chest wall was shaved and a thoracotomy was performed in the fourth left intercostal space. The left ventricle was visualized, the pericardial sac was then removed and the left anterior descending artery was permanently ligated using a 7/0 monofilament suture (Peters surgical, France) at the site of its emergence from under the left atrium. Significant color changes at the ischemic area were considered indicative of successful coronary occlusion. The thoracotomy was closed with 6/0 monofilament sutures. The same procedure was performed for sham-operated control animals except that the ligature was left untied. The endotracheal tube was removed once spontaneous respiration resumed, and animals were placed on a warm pad maintained at 37°C until the mice were completely awake. 1 hour after myocardial infarction induction, mice were treated i.p. with a mouse monoclonal CD20 antibody (200 μ§/ηκηΐ8ε) previously validated [Uchida, J., et al. 2004 and Ait-Oufella, H., et al. 2010] or with PBS. In other sets of experiments, 7 days before myocardial infarction induction, Rag 1— >i- mice received either 2x107 wild-type splenocytes, 1.2x107 B cell- depleted splenocytes recovered from CD20 mAb-treated mice, B cell-depleted splenocytes re- supplemented with 8x106 wild-type B lymphocytes or with 8x106 Mcp-3-/- B lymphocytes. Experiments were conducted according to the French veterinary guidelines and those formulated by the European Community for experimental animal use, and were approved by the Institut National de la Sante et de la Recherche Medicale.

Echocardiographic measurements. Transthoracic echocardiography was performed 14 days after surgery using an echo cardiograph (Vivid 7, GE Medical Systems ultrasound) equipped with a 12-MHz linear transducer (12L). Animals were anesthetized by isoflurane inhalation. Two-dimensional views of the left ventricle were obtained for guided M-mode measurements of the LV internal diameter at end diastole (LVDD) and end systole (LVDS), as well as the interventricular septal wall thickness, and posterior wall thickness at the same points. Percent fractional shortening (%FS) were calculated by the following formulas: %FS = [(LVDD - LVDS)/LVDD] X 100.

Histopathological analysis. Cardiac healing following myocardial infarction was assessed at day 14. Hearts were excised, rinsed in PBS and frozen in liquid nitrogen. Hearts were cut by a cryostat (CM 3050S, Leica) into 7^m-thick sections. Masson's trichrome and Sirius Red stainings were performed for infarct size and myocardial fibrosis evaluation. Infarct size (in percent) was calculated as total infarct circumference divided by total LV circumference. The collagen volume fraction was calculated as the ratio of the total area of interstitial fibrosis to the myocyte area in the entire visual field of the section. B lymphocyte immuno staining was performed according to an indirect immunoperoxidase method using an anti-CD45R/B220 antibody (RA3-6B2, Southern Biotechnology) and AEC substrate kit (Dako). Endothelial cells forming capillaries were visualized after BS-1 lectin staining (FITC conjugated Griffonia simplicifolia, Sigma- Aldrich) and arterioles using an anti-a-actin smooth muscle antibody (Abeam). Cells. Mice were killed at 12h and on days 1, 3, 5, 7 and 14 after myocardial infarction (n=5-10 mice per time point). Peripheral blood was drawn via inferior vena cava puncture with heparin solution. Whole blood was lysed after immunofluoresence staining using the BD Facs lysing solution and total blood leukocyte numbers were determined using Trypan Blue. Spleens were removed and bone marrow cells were drawn from femur and tibia and filtered through 40 um nylon mesh (BD Biosciences). The cell suspension was centrifuged at 400g for 10 min at 4°C. Red blood cells were lysed (Sigma- Aldrich) and splenocytes and bone marrow cells were washed with PBS supplemented with 3% FBS. Infarct tissue and healthy hearts were harvested, minced with fine scissors, and placed into a cocktail of Collagenase I (450 U/ml), Collagenase XI (125 U/ml), DNase I (60 U/ml), and Hyaluronidase (60 U/ml) (sigma- Aldrich) and shaken at 37°C for lh. Cells were then triturated through nylon mesh (40 μιη) and centrifuged ( 10 min, 400 g, 4°C). Mononuclear cells were purified by density centrifugation (25 min, 400 g, room temperature). The resulting cell suspensions were washed and total leukocyte numbers were determined.

Cell purification, culture and transmigration assay. B cells were isolated from C57BL/6J spleens using a B cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. B cells were stimulated during 48h with 10 μg/ml of anti-mouse IgM (Jackson ImmunoResearch Laboratories, Inc.) and 2.5 μg/ml of anti-mouse CD40 (clone HM40-3, BioLegend). Monocytes were isolated from bone marrow. Bone marrow fraction was enriched by neutrophils depletion using autoMACS columns (Miltenyi) and anti-Ly6G magnetic beads. 7/4hi monocytes (7/4 staining is equivalent to Ly6C staining [Cochain, C, et al. 2010]) were then sorted on a FACSAria (BD Biosciences). In vitro monocytes transmigration was performed over cell culture inserts (Millicell-PCF, Millipore) with porous polycarbonate filters (8μιη pore size) in 24-well plates. Inserts were coated with rat-tail type I collagen (60 μg/ml) for 30min at 37°C, then blocked with 3% BSA in PBS for lh at 37°C. 300μί of RPMI-FBS 10% containing 2x106 of B lymphocytes were cultured in the lower compartment and 200μΙ_^ containing 9x104 of 7/4hi monocytes were added to the upper compartment. 20μg/ml of anti-MCP-3 neutralizing antibody or ^g/ml of anti- MCP-1 neutralizing antibody (R&D systems) were added in the lower compartment before migration. Monocytes were allowed to migrate for 4h at 37°C. The filters were then washed with PBS, and fixed in 1% paraformaldehyde. The upper surfaces of the filters were scraped with cotton swabs to remove the non-migrating cells. The filters were then stained with DAPI (Sigma- Aldrich). Migrating cells attached to the lower surfaces of the filters were visualized under an Axioimager Zl microscope (Zeiss). Each experiment was performed in triplicate.

Flow Cytometry. The following antibodies were used: FITC-conjugated anti-CD l ib (Ml/70, BD Pharmingen), PE-conjugated anti-Ly6G (1A8, BD Pharmingen), PEconjugated anti-NK-1.1 (PK 136, BD Pharmingen), APC-conjugated anti-Ly-6B.2 (7/4, AbD Serotec), APC-conjugated anti-CD3e (17A2, eBioscience), FITC-conjugated anti- CD4 (RM 4-5, eBioscience), PercP-conjugated anti-CD8a (53-6.7, BD Pharmingen), PEconjugated anti- CD45R/B220 (RA3-6B2, eBioscience), APC-conjugated anti-IgM (11/41, eBioscience), PE- Cy7-conjugated anti-CDl lc (N418, eBioscience). Monocytes were identified as CD 1 lbhi Ly6G- 7/4hi/lo. Neutrophils were identified as CDl lb+ Ly6Ghi 7/4hi. Macrophages/dendritic cells were identified as CD1 lchi. NK cells were identified as CD1 lb+ Ly6G- 7/4- NK1.1+. Mature B lymphocytes were identified as B220hi IgMhi. Normalization to weight of infarct was performed for total cell numbers determination in the heart. Cells were analyzed using a flow cytometer (LSR II, BD).

Antibody measurements. Circulating IgM, IgGl, and IgG2c levels were measured in plasma of treated mice at indicated time points using a chemiluminescent-based sandwich ELISA.

Quantitative real-time PCR. Quantitative real-time PCR was performed on a Step- one Plus (Applied Biosystems). GAPDH was used to normalize gene expression. Primers were used. Chemokines levels. Plasma and heart levels of MCP-1 and MCP-3 were measured using Quantikine Elisa Kits (R&D Systems and PeproThec, respectively) according to the manufacturer's instructions.

Immunohistochemistry on human samples. Apical ischemic myocardial tissue was removed as planned surgical procedure for the implantation of left ventricle assist devices in 6 patients presenting cardiogenic shock due to acute myocardial infarction (from 1 to 7 days). The clinical course of these patients was previously described and published [Cazes, A., et al. 2010]. The tissues were submitted to surgical pathology diagnosis as previously reported [Cazes, A., et al. 2010]. The remnant tissues in paraffin blocks were used for research purposes with informed consent of the patients. The samples consisted of chips of left myocardium measuring 0.5 to 2 cm of long axis and were samples of cardiac apical left ventricles removed for implantation of a left ventricle assist device. In paraffin sections, antigen retrieval was obtained with citrate buffer in a microwave oven (3X4 min. at 400 W). The anti-CD20 polyclonal antibody was revealed with a three-step technique using ABC peroxidase kit (Vector Laboratories, Burlingame, CA, USA). Negative controls consisted of incubation of sections with a non-relevant goat polyclonal antibody, anti-nephrin C-17 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Population of patients with acute ML The population and methods of the French registry of Acute ST-elevation and non-ST-elevation Myocardial Infarction (FAST-MI) have been described in detail in previous publications 38. Briefly, all patients >18 years of age were included in the registry if they had elevated serum markers of myocardial necrosis higher than twice the upper limit of normal for creatine kinase, creatine kinase- MB or elevated troponins , and either symptoms comp atib le with acute MI and/or electrocardiographic changes on at least two contiguous leads with pathologic Q waves (>0·04 sec) and/or persisting ST elevation or depression >0· 1 mV. The time from symptom onset to intensive care unit admission had to be <48 h. Patients were managed according to usual practice; treatment was not affected by participation in the registry. Of the 374 center in France that treated patients with acute MI at that time, 223 (60%) participated in the registry. Among these, 100 center recruited 1029 patients who contributed to a serum bank. For the present study, 1000 samples were available. Written informed consent was provided by each patient. Their baseline characteristics were comparable to the overall population of the registry. More than 99% of patients were Caucasians. Follow-up was collected through contacts with the patients' physicians, the patients themselves or their family, and registry offices of their birthplace. One-year follow-up was >99% complete. The study was reviewed by the Committee for the Protection of Human Subjects in Biomedical Research of Saint Antoine University Hospital and the data file was declared to the Commission Nationale Informatique et Liberie. Human MCP-3 analysis was carried out using the Bioplex Pro magnetic technology (Bio-Plex Pro™ Human Cytokine MCP-3 Set #171-B6012M, Bio-Rad), following the manufacturer's instructions. Frozen (-80°C) EDTA plasma samples from each patient were all analyzed on the same day. Samples were thawed and spun at 2500g for 15 minutes at room temperature prior to use and particle clear plasma was used at 1 :4 dilution. Data were acquired on 50 beads/patient and analyzed following a 1 :4 serial dilution standard curve (concentration in range =0.33-3472 pg/ml) using the Bio-Plex Manager 6.1 Software and 5PL logistic regression (FitProb. = 0.0000, ResVar. = 7.7585).

Statistical analysis. Results were expressed as mean ± SEM. Kruskal-Wallis ANOVA was used to compare each parameter. Post hoc Mann-Whitney U tests with Bonferroni correction were then performed to identify which group differences accounted for the significant overall Kruskal-Wallis result. Statistical analysis in FAST-MI was conducted as follows. An outcome event was defined as all-cause death or non-fatal MI during the one-year follow-up period. The primary endpoint was defined as a composite of all-cause death and non- fatal MI, and was adjudicated by a committee whose members were unaware of patients' medications, and blood measurements. Continuous variables are described as mean ± SD and categorical variables as frequencies and percentages. Baseline demographic and clinical characteristics, treatment factors, and therapeutic management during hospitalisation were compared among patients with or without detectable circulating MCP-3 levels using chi- square or Fisher' s exact tests for discrete variables, and by unpaired T tests, Wilcoxon sign- rank tests for continuous variables. Survival curves according to detectable or undetectable MCP-3 levels are estimated using the Kaplan Meier estimator. We used a multivariable Cox proportional-hazards model to assess the independent prognostic value of variables with the primary endpoint during the 1-year follow-up period. The multivariable model comprised sex, age, previous or current smoking, family history of coronary disease, history of hypertension, previous MI, heart failure, renal failure, diabetes, heart rate at admission, Killip class, left ventricular ejection fraction, hospital management (including reperfusion therapy, statins, beta blockers, clopidogrel, diuretics, digitalis, heparin), and log CRP levels. Results are expressed as hazard ratios for Cox models with 95% confidence intervals (CIs). All statistical tests were two-sided and performed using S AS software version 9.1.

Results Mature B lymphocytes are recruited to the ischemic tissue after myocardial infarction.

To identify the inflammatory cell repertoire within the injured myocardium, we induced acute MI in C57BL/6J mice by permanent coronary artery ligation and analyzed cell suspensions of digested infarcts at different time points by flow cytometry. We found that neutrophils (CD1 lb+ Ly6Ghi 7/4hi cells) peaked as early as day 1 after MI (data not shown), followed by successive waves of 7/4hi (Ly6Chi) and 7/4 lo (Ly6Clo) monocytes (CDl lbhi Ly6G-) (data not shown), and by accumulation of dendritic cells, macrophages and Natural Killer cells (data not shown). We also found that CD3+ T lymphocytes accumulated in the injured myocardium within 1 day after MI and dropped thereafter (data not shown). The results are in agreement with previous studies 17. Intriguingly, the duration and the intensity of B lymphocyte infiltration into the ischemic cardiac tissue remain largely undefined. We found that B lymphocytes, defined as B220+ IgM+ cells, accumulated in the infarct area within the first hours, remained in relatively stable numbers (~ 200 cells/mg tissue) during the first days, peaked on day 5 after MI (~ 900 cells/mg tissue) and waned thereafter to levels comparable to those in sham-operated mice (Fig. la). Immunohisto logical analysis confirmed the increased accumulation of B220-positive B lymphocytes in the border infarct area after MI compared to sham-operated animals (Fig. lb). Thus, myocardial infarction triggers the infiltration of B220+ IgM+ B cells into cardiac tissue, suggesting a potential role of mature B cells in this setting.

B lymphocyte depletion prevents adverse ventricular remodeling and improves cardiac function after acute ML

To directly assess the role of mature B cells in cardiac remodeling after MI, we depleted B lymphocytes using a CD20-specific monoclonal antibody (CD20 mAb) 24,25. As expected, we found that CD20 mAb treatment led to sustained and profound reduction of the number of mature B cells in the blood, spleen and cardiac tissue (Fig. lc-le). We next assessed cardiac function by echocardiography 14 days after MI. Of interest, CD20 mAb induced B cell depletion led to a reduction in end-systolic left ventricular (LV) dimension (p=0.016), a significant improvement of LV shortening fraction (p=0.021) and an increase in LV myocardial contractility (p=0.025) compared to control animals (Fig. 2a). This was associated with a reduction of infarct size (p=0.024) and interstitial fibrosis as assessed by collagen content (p=0.016) (Fig. 2b). In addition, B cell depletion reduced the number of apoptotic cells within the injured myocardium, as shown by TUNEL staining (p=0.005) but had no effect on neovascularization (data not shown). Overall, these results show that B cell depletion significantly reduces post-ischemic injury, prevents adverse ventricular remodeling and improves cardiac function after acute MI. B lymphocyte depletion reduces both systemic and local pro-inflammatory responses after acute ML

We next assessed the potential mechanisms involved in B cell-mediated effects on cardiac remodeling and function. Previous studies reported a pathogenic role for natural IgM antibodies in the first 24 hours following ischemia-reperfusion injury [Haas, M.S., et al. 2010 and Busche, M.N., et al, 2009]. We therefore assessed changes in antibody production after CD20 mAb treatment. B2 cell depletion did not alter IgM or IgG levels in the first three days after MI, making unlikely the hypothesis of an antibody-mediated effect. However, we observed only a moderate increase in IgM levels at day 14 after MI in CD20 mAb-treated animals (data not shown), which is consistent with the reduction of myocardial necrosis. We next assessed changes in the inflammatory response. Treatment with CD20 mAb resulted in a profound reduction of both systemic and local pro -inflammatory cytokines measured at day 14 after MI. Hence, IL-Ιβ (p=0.04), TNF-a (p=0.004) and IL-18 (p=0.041) mRN A levels were significantly lower in infarct hearts of B cell depleted-mice compared to the control group (Fig. 2c). In contrast, expression of anti-inflammatory mediators, IL-10 and TGF-β, tended to be unaffected in the B cell-depleted animals (Fig. 2c). Similar results were found in spleens and lymph nodes of B cell-depleted mice (data not shown), suggesting a limited inflammatory response. B lymphocyte depletion impairs monocyte mobilization and selectively reduces

MCP-3 levels after acute ML

We then addressed the mechanisms that could account for the reduced inflammatory response and the improvement in ventricular remodeling. A striking observation was that B cell-depleted mice constantly showed altered monocyte compartmentalization as revealed by enhanced accumulation of 7/4hi monocytes in the bone marrow (p=0.01 vs controls) (Fig. 3a) and the spleen (p=0.02 vs controls) (data not shown), associated with a significant decrease of their circulating blood count (p=0.015 vs PBS) (Fig. 3b), suggesting impaired monocyte mobilization. Since CCR2-mediated signals are required for monocyte mobilization from the bone marrow 17,27, we examined potential changes in the production of MCP-1 and MCP-3, the 2 major CCR2 ligands. We found that acute MI led to a significant increase of both MCP- 1 and MCP-3 mR A and protein levels, in blood and cardiac tissue (data not shown). Intriguingly, B cell depletion was associated with a significant and selective impairment of MCP-3 levels (p=0.03) compared to the control group (Fig. 4a). In contrast, MCP-1 levels were unaffected by B cell depletion (Fig. 4a). B lymphocytes produce MCP-3 and trigger monocyte migration.

These results suggested that B cells might produce MCP-3 and induce MCP-3 - dependent monocyte mobilization. To address this hypothesis, we first evaluated MCP-3 secretion by cultured B cells. Whereas MCP-1 remained undetectable in B cell supernatants, these cells produced significant amount of MCP-3, which was further increased after their activation with anti-CD40 and anti-IgM antibodies (Fig. 4b). We also found that activated cultured B cells strongly enhanced 7/4hi monocytes transmigration in vitro compared to non- stimulated B cells (p<0.0001) (Fig. 4c). Interestingly, neutralizing anti- MCP-3 antibody abrogated B cell-induced migration of cultured 7/4hi monocytes (p=0.0003; Fig. 4c). In contrast, neutralization of MCP-1 had no effects (p=0.325; Fig. 4c).

MCP3-deficient B lymphocytes fail to affect cardiac remodeling and function after acute ML

These findings prompted us to investigate the direct role of B cell-derived MCP-3 in post-ischemic cardiac remodeling. First, we unraveled a key role of MCP-3 in this setting using MCP-3 -deficient mice. We found that following acute MI, MCP-3 deficiency was associated with a reduction of 7/4hi monocyte mobilization from bone marrow (p=0.013 vs WT) to the blood (p=0.04 vs WT), and improvement of cardiac function (data nit shown). To further substantiate the role of B cell-derived MCP-3 in this setting, we injected Ragl-/- mice either with wild-type splenocytes, B cell-depleted splenocytes, or B cell-depleted splenocytes re-supplemented with wild-type or Mcp-3-/- B lymphocytes. We first verified that re-supplementation with wild-type or Mcp-3-/- B cells significantly increased B cell count in spleens of Rag 1-/- mice compared to mice injected with B cell-depleted splenocytes only (data not shown). We then evaluated circulating MCP-3 levels and found that re- supplementation with wild-type B cells was associated with an increase of MCP-3 levels compared to mice receiving B cell-depleted splenocytes only (968.5 ± 46.06 pg/ml and 201.1 ± 28.31 pg/ml, respectively, p<0.0001). In contrast, re-supplementation of B cell-depleted splenocytes with B cells isolated from Mcp-3-/- mice failed to increase MCP-3 levels (152.6 ± 39.9 pg/ml, p<0.0001 vs wild type B cells). Interestingly, we also found that re- supplementation with wild-type B cells was associated with an increase of 7/4hi monocyte count in the blood and within the injured myocardium compared to the group receiving B cell-depleted splenocytes only (Fig. 4d), a phenotype that was abrogated in mice re- supplemented with Mcp-3-/- B cells (Fig. 4d). Thus, B cell-derived MCP-3 triggers selective mobilization and tissue recruitment of 7/4hi monocytes after acute MI.

We then examined the consequences of MCP-3 deficiency in B cells on post-ischemic cardiac remodeling. We found that transfer of B cell-depleted splenocytes into Ragl-/- mice reduced end-systolic LV chamber dimension (p=0.04) and improved left ventricular shortening fraction (p=0.008) after MI compared to the transfer of non-depleted splenocytes (Fig. 5a). This effect was abrogated after re-supplementation of B cell-depleted splenocytes with B cells isolated from wild-type mice (p=0.0007 and p=0.02 respectively) but was not altered after re-supplementation with Mcp-3-/- B cells (Fig. 5a). B cell depletion also reduced infarct size (p=0.04; Fig. 5b) and collagen content (p=0.09; Fig. 5c), which was antagonized by re-supplementation with wild-type but not with Mcp- 3-/- B cells (Fig. 5b and 5c).

B lymphocytes accumulate in the ischemic human heart and detection of circulating MCP-3 at the acute phase of MI is associated with adverse cardiovascular outcome.

Finally, we addressed the relevance of these findings to the human disease. We first assessed B cell accumulation in human biopsies from patients with a diagnosis of recent MI. We found that CD20-positive B cells infiltrated cardiac tissue of patients with acute MI and were detected in biopsies obtained 24-48 hours or 6-7 days after the diagnosis of MI (Fig. 6a). We then assessed the relationship between circulating MCP-3 levels and clinical outcomes in 1000 patients admitted for acute MI. Interestingly, we found that patients with detectable circulating levels of MCP-3 at their admission for acute MI were at substantially increased risk of death and recurrent myocardial infarction after one year of follow-up compared to patients with no detectable MCP-3 levels (HR=1.63, 95%CI=1.11-2.39, p=0.012), even after adjustment for several multivariable risk factors (see Methods) (HR=1.62, 95%CI=1.08-2.43, p=0.02) (Fig. 6b).

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Claims

CLAIMS:

1. A compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signalling pathway for use in the treatment of myocardial infarction.

2. The compound for use according to the claim 3 wherein said compound is an antibody which inhibits the binding of MCP-3 to CCR2.

3. The compound for use according to the claim 4 wherein said antibody is an antibody against MCP-3.

4. The compound for use according to the claim 4 wherein said antibody is an antibody against CCR2.

5. The compound for use according to claims 3 to 6 wherein the myocardial infarction is an acute myocardial infarction.

6. A pharmaceutical composition for use in the treatment of myocardial infarction comprising a compound according to any one of claims 3 to 6 and a pharmaceutically acceptable carrier.

7. A method for treating myocardial infarction comprising administering to a subject in need thereof a therapeutically effective amount of a compound which inhibits the binding of MCP-3 to CCR2; CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 gene expression or signalling pathway.

8. A method for predicting the survival time of a patient suffering of myocardial infarction or the recurrence of a myocardial infarction of a patient who has suffered from a myocardial infarction comprising the steps consisting of i) determining the expression level of MCP-3 in a sample from said patient, ii) comparing said expression level with a predetermined reference value and iii) providing a good prognosis of the survival time or of the recurrence of a myocardial infarction when the expression level is lower than the predetermined reference value and a poor prognosis of the survival time or of the recurrence of a myocardial infarction when the expression level is higher than the predetermined reference value.

9. The method according to claim 1, wherein said sample is selected in the group consisting of blood, plasma, serum, lymph and cardiac tissue.

10. A method for treating patient who has been considered as a poor prognosis for the survival time or for the recurrence of a myocardial infarction according to claim 1 comprising administering to a subject in need thereof a compound which inhibits the binding of MCP-3 to CCR2, CCRl or CCR3 or a compound which is an inhibitor of MCP-3, CCR2, CCRl or CCR3 expression or a B cell depleting agent.