WO2015059465A1

WO2015059465A1 - Vascular biomarkers

Info

Publication number: WO2015059465A1
Application number: PCT/GB2014/053141
Authority: WO
Inventors: Richard Harley Grenville Owen; Joseph Peter Corrigan; Stephen Blatcher; Nicholas Edward John WEST
Original assignee: Plaquetec Ltd
Current assignee: Plaquetec Ltd
Priority date: 2013-10-24
Filing date: 2014-10-22
Publication date: 2015-04-30
Anticipated expiration: 2016-04-24
Also published as: GB201318793D0

Abstract

The present invention relates to the detection, prognosis and treatment of Atherosclerotic vascular disease (ASVD) using biomarker gradients within blood vessels. In particular, the present invention relates to the detection, prognosis and treatment of coronary artery disease (CAD).

Description

VASCULAR B!O ARKERS

Technical field

The present invention relates to the detection, prognosis and treatment of Atherosclerotic vascular disease (ASVD). In particular, the present invention relates to the detection, prognosis and treatment of coronary artery disease (CAD).

Introduction

Atherosclerotic vascular disease (ASVD) and its complications, such as coronary artery disease (CAD), are the major cause of morbidity and mortality in the industrialised world [1]. Coronary arteries become diseased through atherosclerosis which is a destructive inflammatory condition that can occur throughout the arterial system.

The ultimate cause of death through CAD is the generation of a clot within a coronary artery that reduces the blood supply to the heart to the point the heart stops beating [12, 13, plus and references cited therein]. The coincidence of the local occurrence of atherosclerotic disease in the coronary arteries and local clotting suggests a simple link between these two factors. However, research has shown that, in fact, ASVD is a very complex disease with numerous significant factors interpiaying. For this reason CAD remains the primary cause of death in the world, despite many years of intensive research.

Atherosclerosis is a process within the coronary arteries in which there is a build-up of cholesterol within the arterial wall which provokes an immune response. This two pronged process leads to development of "vulnerable plaques" within the coronary arteries. The rate of development of atheromatous plaques is also complex and is determined by genetic and environmental factors such as diet, smoking and lifestyle. The current theory for how atherosclerosis leads to vulnerable plaques is summarised below:

Cholesterol in blood is absorbed into the coronary artery, which causes the local release of cytokines that lead to inflammation

The cytokines are part of a cascade of molecular processes that, amongst other outcomes, make the vascular smooth muscle cells in the artery wall proliferate which causes the artery to distend and to "remodel" its shape and size

· The soluble molecules released also make the endothelial ceils attract

immune-system cells from the blood. The cells enter the artery wall, form a plaque and begin to release enzymes that breakdown the structure of the artery wail.

The plaque remodelling continues, increasing in size and becomes

"vulnerable" to rupture

If the plaque continues to develop and grow in size it may become calcified and cause a significant obstruction to the blood flow within the artery and cause angina due to myocardial ischaemia - in which case stenting can be used to open up the stenosis

Prior to the calcification stage, some of these vulnerable plaques have a potential to rupture which depending on the factors described above may lead to the formation of a blood clot / thrombus large enough to block the artery which could lead to heart attack and possibly death.

The overwhelming majority of drugs that are used to decrease the risk from CAD target cholesterol metabolism by the liver in an attempt to decrease the supply of cholesterol to the plaque. However, these drugs typically take a long time to have an effect and are not effective in everyone.

Studies aimed at understanding how vascular lesions such as plaques develop within the coronary arteries have revealed that, rather than being distributed more or less throughout the coronary 'tree' of arteries (consisting of the Left Circumflex Artery (LCx), the Left Anterior Descending artery (LAD), and the Right Coronary Artery (RCA)), plaques that have the potential to cause heart attacks are concentrated at one or a few "hot spots". For example the authors of [ 3] reported that, in LCx arteries blocked by a thrombotic occlusion, 100% of the occlusions were found within 80mm of the artery ostium; for LAD arteries 100% of the occlusions were found within 80mm of the artery ostium, whilst for RCAs 100% of the occlusions were found within 130mm of the artery ostium. The identification of these thrombotic "hot spots" is consistent with the findings in [12], where the authors reported that the majority of thin-cap fibroatheromas (TCFA, also known as "vulnerable plaques") and ruptured plaques localized in the proximal third of the major coronary arteries, and in 92% of cases these lesions clustered within 2 or fewer nonoveriapping 20 mm segments. More specifically, 100% of TCFAs and ruptured plaques were found within 35 mm of the artery ostium for the LAD, within 70 mm of the artery ostium for the LCx, and within 100 mm of the artery ostium for the RCA. Accordingly, reliable plaque detection, coupled with accurate prediction of the risk of a critical event (such as plaque rupture), within these relatively short stretches of coronary artery is critical to reducing the incidence of thrombotic occlusion of coronary arteries in CAD. The potential of a number of different techniques for detecting plaques and assessing their status is reviewed in [14]. In this study the authors report the results of coronary angiography and radiofrequency intravascular ultrasonographic imaging (IVUS) on 897 patients exhibiting signs of ACS. 595 thin cap plaques and 1005 thick cap plaques were detected in the study; however, over the 3.5 year follow-up period only 5% of the thin-cap plaques and 2% of the thick-cap plaques resulted in a major adverse cardiac event. Moreover, of the major adverse cardiac events which were recorded in the patient group over the 3.5 year follow-up period, 50% occurred at sites where no vessel abnormality was detected in the original study. This study indicates that techniques based on assessing features such as plaque shape, consistency, and cap thickness are limited to giving 'snap shots' of plaque distribution and have little predictive potential in regard to the location or likelihood future plaque rupture.

One possible reason for the failure of the above studies to usefully predict those plaques that would lead to major cardiac events is that they did not take into account the interplay of ail of the important factors that determining CAD outcome. These factors include: the state of the blood and its potential to form a thrombus (white dot) or to coagulate (red clot); the level of atherosclerotic disease in the artery (i.e. the thrombogenic state of the endothelial surface and its potential to leak clotting factors into the circulation); the inflammatory status of the person (i.e. the type and intensity of the immune system's response to environmental factors); the geometry of the arteries (i.e. the size of the lumen through which blood can flow); the state of the myocardial tissue (i.e. its ability to respond to and tolerate decreases in oxygenation); the overall metabolic status of the patient (i.e. whether they are diabetic, obese, high in cholesterol, inactive or a combination of all four); and the medication that person is taking, and their reaction to it. The overall risk of a major cardiac event leading to death is the product of all these factors.

For example, a minor local rupture within a diseased portion of a major coronary artery would not lead to death if the blood was relatively insensitive to the clotting factors released. in contrast a minor rupture within a coronary artery could cause a major clot or thrombus if the blood was in a hyper-clottable or hyper-inflammatory state and, unless the myocardial tissue had sufficient flow reserve or the ability to maintain pumping despite reduced oxygen, the person would die. Thus, although the atherosclerotic disease state of coronary arterial wail is an important factor, how the diseased vessel interacts with the surrounding blood and the heart tissue are critically important to the final clinical outcome (such as mortality).

Typically, each of these factors is assessed separately: Factor Typica! assessment method

The state of the blood Testing of peripheral venous blood for

individual markers of platelet and

coagulation activity

The level of atherosclerotic disease in the Electromagnetic and ultrasound scanning of artery the arteries by medical devices

The inflammatory state of the person Testing of peripheral venous blood for

individual markers of inflammatory activity

The geometry of the arteries Electromagnetic and ultrasound scanning of the arteries by medical devices

The state of the myocardial tissue Functional tests assessing blood flow

reserve and testing of peripheral venous blood for markers of damage (troponin)

The overall metabolic status of the patient Testing of peripheral venous blood for

individual markers (e.g. cholesterol and markers of diabetes)

Medication, and patients' individual Testing of peripheral venous blood for the response to it changes caused by medications ideally, however, clinicians would have access to markers that reflect the interaction of these factors and not just each factor separately. What is needed is to assess the critical zone of the patient (the coronary artery) and to find markers that give information on how the blood is affected once in the coronary artery, how the plaque causes changes in the blood, and how the interactions of the blood and the artery then affect the myocardial tissue. This is what has been provided by the present inventors.

It will be appreciated from the forgoing that the provision of methods for reliably identifying patients having plaques at risk of imminent rupture would be of considerable value to the field. Such methods would enable the stratification of patients into groups based on disease severity and/or prognosis, and so allow for much improved targeting of medical treatment. Moreover, the ability to monitor ASVD and CAD would enable medical practitioners to accurately monitor the efficacy of particular treatments, as well as enable the discovery and testing of new treatments.

Disclosure of the invention

The present inventors have reasoned that blood vessels with ASVD in general, and atherosclerotic plaques in particular, will release molecules into their local environment. Therefore, by measuring the concentrations of molecules in the critical proximal regions of the coronary artery will and comparing them to concentrations found in healthy "control" arteries, or another suitable reference point, it will be possible to identify biomarkers specifically associated with the atherosclerotic plaques in the coronary arteries. To this end the present inventors have developed a methodology that allows for the association of specific biological molecules (or "biomarkers") with, for example, the status of an

atherosclerotic lesion on the wall of a coronary artery.

The inflammatory model of ASVD

An "inflammatory model" of ASVD in coronary arteries has been proposed to explain its development and impact on health. In this theoretical model, coronary endothelial cells, vascular smooth muscle cells and inflammatory ceils have crucial roles in the process of atherosclerosis. The mechanisms by which these cells contribute to atherosclerosis include augmented expression of molecules such as adhesion molecules as well as the secretion of proinflammatory cytokines, interleukins, enzymes such as matrix metailoproteinases and tissue factor within human plaques [2]. The changing concentrations of these biological molecules that underlie ail biological inflammatory processes are believed to drive the progression of CAD. There has been speculation that studying these biological inflammatory molecules could be a way of understanding ASVD and CAD process and developing new therapies to prevent them. [3] [4],

An example of one drug in development that targets inflammation in the plaque directly is Darapiadib from GSK (although the mechanism of action is again through cholesterol metabolism). Darapiadib is an inhibitor of lipoprotein-associated phosphoiipase A2

(LpPLA2), the enzyme that is carried on ail cholesterol particles that circulate around the bloodstream and is thought to oxidise the cholesterol it is linked to when it is inside plaques and make it more likely to stimulate an inflammatory response.

Variability in biomarker levels

Despite the theoretical knowledge implied by the inflammatory model described above there has been as yet poor correlation between potential biomarkers and individual outcome [4] [5]; inflammatory disease is a very large and complex area and there is little, if any, data indicating which specific inflammatory processes are active in and around the plaque. This greatly hinders the development of targeted anti-inflammatory therapies that can stop the inflammatory processes in and around the plaques in the critical proximal regions of the coronary arteries that increase a person's risk from CAD.

One of the main reasons for this lack of specific data is that individuals show significant variation in their levels of circulating biomarkers and this tends to mask any genuine differences caused by different CAD states [10]. Thus, whilst some weak associations between disease state and the levels of some specific inflammatory biomarkers have been identified, the correlations are not strong enough to be able to use the biomarkers as truly predictive, actionable, clinical tools [6], [7], [8], [9], [10], Local assessment of biomarkers

The difficulty caused by person to person variability in biomarkers is compounded by the limitations imposed by sample collection method: inflammatory biomarkers detected in a peripheral blood sample could have originated from any site in the body, for example, from an acute infection (e.g. an inflamed tooth), from an inflammatory disorder (e.g. rheumatoid arthritis) or from a diseased artery. Even if a specific vascular inflammatory marker were known, its detection in a peripheral sample would not reveal the location of the inflamed blood vessel. Accordingly, a peripheral reading cannot give a direct indication of the status of the proximal regions of the cardiac arteries which are crucial in CAD. The presence or absence of an inflammation biomarker in a peripheral sample also cannot be used as a reliable indicator, or proxy, of the level of that biomarker in the crucial proximal regions of the cardiac arteries. As demonstrated herein, the peripheral level of a biomarker does not show any significant correlation with the release of that biomarker in proximal regions of the cardiac arteries (see Figures 3 and 4). Accordingly, the assessment of biomarker concentration local to the plaque, suspected plaque, or key region of blood vessel (such as the proximal regions of the coronary arteries) is key for the accurate identification and detection of biomarkers related to ASVD and CAD. It follows from this that such local assessment is also critical for the diagnosis and prognosis of ASVD, CAD, and related cardiac events as discussed herein.

An important aspect of the methodology developed by the present inventors is the ability to reliably detect a local increase in the concentration of a biomarker in, or immediately downstream of, a vascular lesion (such as a vulnerable plaque) on the wall of a blood vessel. This local increase may, for example, take the form of a gradient of biomarker concentration along the length of, across, or immediately downstream of, the lesion.

Detection of such gradients can be made using specialised assessment devices, such as the one devised by the present inventors and disclosed in WO2009/090390; a catheter for taking a plurality of samples from with the length of a blood vessel.

Verification of candidate biomarkers

in addition to the biochemical pathways normally associated with vascular tissue, a blood vessel with ASVD is also expected to have activated inflammatory pathways. Each of these pathways contains multiple individual proteins, many of which themselves interact with other cellular constituents as, for example, substrates, products, binding partners etc. Accordingly, there are hundreds of molecules which could, in principle, act as biomarkers of the various stages of ASVD, up to and including failure of the cap zone and plaque rupture.

Determining which molecules are released locally to a vascular lesion, suspected vascular lesion, or key region of blood vessel (such as the proximal regions of the cardiac arteries) is a crucial step in identifying useful biomarkers. However, this information by itself cannot be translated into an absolute risk of a major cardiac event (such as a myocardial infarction) or indeed, the likely clinical outcome of such an event (such as death). In order to make this further step, marker identity and levels must be linked to clinical outcome.

This is illustrated by the study described in [14], where only 5% of thin-cap atherosclerotic plaques led to a major cardiac event within 3.5 years. Through the local assessment of biomarkers coupled with clinical monitoring of individuals, it is in principle possible to identify a biomarker, or combinations of biomarkers, whose local release is indicative of key stages in ASVD and CAD such as the development of 'unstable' vulnerable plaques (that is, a plaque with a high probability of rupture within, say, a 1 , 3 or 5 year window), the failure of the cap zone prior to rupture, or even actual plaque rupture. Once verified, these markers can be used as valuable assessment, diagnostic and prognostic tools both in current and developing therapeutic applications.

Utility of ASVD biomarkers

The biomarker proteins identified by this methodology are of particular use inter alia as diagnostic and prognostic markers of ASVD and CAD, in particular as diagnostic and prognostic markers of vascular lesions such 'vulnerable' atherosclerotic plaques. They may be used for example to assist diagnosing the presence of ASVD at an early stage in the progression of the disease and predicting the likelihood of clinically successful outcome, particularly with regard to the sensitivity or resistance of a particular individual's ASVD to a therapeutic agent or combinations of therapeutic agents, in addition the biomarkers may themselves be targets for clinical intervention, that is serve as targets against which therapeutics can be used and/or developed with a view to treating ASVD and the

inflammatory processes associated with it. The biomarker targets may also be used for therapeutic intervention in ASVD e.g. to specifically identify and target atherosclerotic plaques which have a high risk of causing a major cardiac event. Finally, they allow for more reliable and meaningful methods of evaluating of the ability of candidate therapeutic compounds to treat ASVD, Thus the present invention relates inter alia to the diagnosis and treatment of ASVD:

specifically, to the detection of ASVD and discrimination of the precise pathological state and prognosis of a vascular lesion based on the presence of specific molecules in and around the lesion. Thus, the invention relates to the detection of one or more molecules

("biomarkers") that are over-expressed in ASVD vascular lesions as compared with 'healthy' regions of the same blood vessels. Depending on their role in ASVD lesion development, these biomarkers may be used, for example, as a target per se for intervention and treatment of ASVD, as an ASVD marker useful in diagnosing or predicting the onset of ASVD, or in monitoring the efficacy of ASVD therapy and/or as a target of such a therapy.

It is contemplated that the skilled artisan may produce novel therapeutics for treating ASVD which include, for example: antibodies which can be administered to an individual that bind to and reduce or eliminate the biological activity of the biomarker in vivo (for example by inhibiting the association between the biomarker and its receptor); nucleic acid or pepfidyl nucleic acid sequences which hybridize with genes or gene transcripts encoding the biomarkers thereby to reduce expression of the biomarker in vivo; or small molecules, for example, organic molecules which interact with the biomarkers or other cellular moieties, for example, receptors for the biomarker, thereby to reduce or eliminate the biological activity of the biomarker or pathway of which the biomarker forms a constituent part.

The present invention provides a wide range of methods for the diagnosis, prognosis and treatment of ASVD, on the basis of the differential expression of the one or more biomarkers. In particular, the present invention provides methods for identifying and monitoring vascular lesions liable to be the cause of a major cardiac event, such as thin-capped atheromatous plaques in the proximal coronary arteries with a high-risk of rupture. These and other numerous additional aspects and advantages of the invention will become apparent to the skilled artisan upon consideration of the following detailed description of the invention. Diagnostic and prognostic applications

By measuring and identifying the biomarkers released by blood vessels at various stages of ASVD, the presence of those markers can be used to identify or diagnose the presence or progression of ASVD.

Accordingly, the present invention provides a method for assessing the risk of ASVD in an individual, which method comprises assessing a local concentration of one or more pre- specified biomarkers wherein a measure exceeding the threshold level is associated with a higher likelihood of ASVD. in some embodiments a measure exceeding the threshold level is associated with a higher likelihood of a major cardiac event such as coronary thrombosis, myocardial infarction, or plaque rupture. In some embodiments a measure exceeding the threshold level is associated with a higher likelihood of death resulting from a major cardiac event such as coronary thrombosis, myocardial infarction, or plaque rupture.

ASVD

As used herein, the term "ASVD" encompasses atherosclerotic disease and related clinical conditions. For example the presence and/or development of vascular lesions, such as atherosclerotic lesions (including initial lesions with foam cells, fatty streaks, intermediate lesions, atheromas, fibroatheromas, complicated lesions, fibrotic plaques, fibrocaicific plaques, pathological intimai thickening (PIT), Thick-cap fibroatheroma (ThCFA), and Thin- cap fibroatheroma (TCFA)), cap-failure prior to plaque rupture, plaque rupture, thrombosis, coagulation, infarction, and/or stroke.

The present invention is particularly concerned with ASVD in the coronaryarteries (i.e. CAD). Thus, the term ASVD also encompasses atherosclerotic disease of the coronary arteries and related clinical conditions. For example, the presence and/or development of vascular lesions as described above in the coronary arteries; in some embodiments ASVD refers to the presence and/or development of vascular lesions within 60mm of the LCx artery ostium, within 80mm of the LAD artery ostium, or 130mm of the RCA ostium, in some embodiments ASVD refers to coronary thrombosis, myocardial infarction (STEMI or NSTEM!), or angina (stable or unstable), in a preferred embodiment ASVD refers to the rupture of atherosclerotic plaque (such as a THCFA or TCFA) in a coronary artery, for example within 80mm of the LCx artery ostium, within 80mm of the LAD artery ostium, or 130mm of the RCA ostium.

Higher likelihood of a major cardiac event

As used herein a "higher likelihood of a major cardiac event" means an increased risk of having a major cardiac event (such as the rupture of a vulnerable plaque in a coronary artery, coronary thrombosis, or myocardial infarction), in some embodiments a "higher likelihood of a major cardiac event" means an increased risk of dying from a major cardiac event (such as the rupture of a vulnerable plaque in a coronary artery, coronary thrombosis, or myocardial infarction). In some embodiments an increased risk of a major coronary event means in the three years following assessment of the biomarker, an untreated individual whose one or more pre-specified biomarker exceeded the threshold level has at least a 10% greater chance of having or dying from major cardiac event as compared to an individual whose one or more pre-specifiecl biomarker did not exceed the threshold level, for example by at least 20%, at least 30%, at least 40%, or at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, or at least 300%. In some embodiments an increased risk of a major coronary event means in the five years following assessment of the biomarker, an untreated individual whose one or more pre- specified biomarker exceeded the threshold level has at least a 10% greater chance of having or dying from a major cardiac event as compared to individual an whose one or more pre-specified biomarker did not exceed the threshold level, for example by at least 20%, at least 30%, at least 40%, or at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, or at least 300%. in some embodiments an increased risk of a major coronary event means in the ten years following assessment of the biomarker, an untreated individual whose one or more pre- specified biomarker exceeded the threshold level has at least a 10% greater chance of having or dying from a major cardiac event as compared to an individual whose one or more pre-specified biomarker did not exceed the threshold level, for example by at least 20%, at least 30%, at least 40%, or at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, or at least 300%. individual

An "individual" as used herein may be symptomatic - that is, the individual may have symptoms of ASVD - or may be non-symptomatic. In some embodiments the individual has symptoms of, mild, moderate or severe ASVD. For example, in some embodiments the individual has symptoms of, or has been diagnosed with, at least one vascular lesion (such as THCFA or TCFA), coronary thrombosis, myocardial infarction, angina or stroke, in some embodiments the individual does not have symptoms of, and/or has not been diagnosed with, mild, moderate or severe ASVD. in some embodiments the individual is an individual that has previously been selected for treatment for ASVD, for example, by a method disclosed herein. in some embodiments the individual has previously been selected to take part in a clinical trial for a specified drug, for example, one of the treatments for ASVD disclosed herein. in some embodiments the individual has previously been selected as part of a group or cohort that possesses one or more aspect of ASVD, For example, in some embodiments the individual has at least one vascular lesion (such as THCFA or TCFA) in one or more of their coronary arteries. In some embodiments the individual has at least one vascular lesion (such as THCFA or TCFA) within 80mm of the LCx artery ostium, within 80mm of the LAD artery ostium, and/or 130mm of the RCA ostium.

Assessing local concentration /Assessing a concentration gradient

As used herein, the term "assessing a local concentration" means qualitative and/or quantitative determinations of the level of the pre-specified biomarker(s) in the region local to a vascular lesion or suspected vascular lesion (such as a ThCFA or TCFA plaque) in order to determine which biomarkers the lesion is releasing into the blood vessel. That is, in some embodiments "assessing a local concentration" comprises assessing the biomarkers / molecules released locally from a vascular lesion into the bloodstream. in some embodiments the concentration is assessed at one or more points downstream of a vascular lesion or suspected vascular lesion and at least one or more points upstream of the lesion or suspected lesion, in some embodiments the one or more points upstream of the vascular lesion and the one or more points downstream of the vascular lesion are in the same blood vessel (for example are in the coronary artery, such as the LCx, LAD or RCA). In some embodiments a systemic concentration is assessed (e.g. by taking a venous or peripheral blood sample) and compared to the concentration as assessed at one or more points downstream of a vascular lesion or suspected vascular lesion. Accordingly, in some embodiments the local concentration is assessed by comparing the concentration at a point downstream of a coronary artery lesion or suspected coronary artery lesion to the concentration in a peripheral (for example, non-coronary) sample).

Assessing the local concentration in this way enables the determination, or assessment, of biomarkers released by the lesion. In some embodiments the released biomarkers are detected as concentration gradients across the vascular lesion or suspected vascular lesion. in some embodiments "assessing a local concentration" will comprise "assessing a concentration gradient". As used herein, a "concentration gradient" is assessed by comparing the biomarker concentrations in the downstream sampie(s) with either the upstream sample(s) and/or the systemic sample. This allows it to be determined if there is a concentration gradient for one or more biomarkers; that is, a gradient in pre-specified biomarker concentration observed from a lower concentration at the upstream or systemic point(s) to a higher concentration at one or more downstream points. The presence of a gradient indicates that the biomarker in question is being released by a vascular lesion or suspected vascular lesion. In some embodiments the concentration gradient is detected by measuring the concentration of the pre-specified biomarker(s) at a point downstream of a vascular lesion or suspected vascular lesion and comparing that concentration to the systemic concentration of the pre-specified biomarker(s) (for example, the concentration in a venous, or peripheral, blood sample).

In this context the term "local" should be understood to mean in the immediate vicinity of a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion. For example, no more than 200mm upstream or downstream of a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion, such as no more than 150mm, no more than 120mm, no more than 70mm, no more than 50mm, no more than 30mm or no more than 10mm. in some embodiments where a lesion or suspected lesion extends along a blood vessel, the measurements are made from the most upstream point of the vascular lesion or suspected vascular lesion. Thus, in one embodiment a "downstream" measurement is taken no more than 130mm downstream of the most upstream point of the lesion or suspected lesion, and that measurement compared with either (i) an "upstream" measurement is taken no more than 130mm upstream of the most upstream point of the lesion or suspected lesion, and/or (ii) a systemic concentration, in other embodiments where a lesion or suspected lesion extends along the length of blood vessel, the measurements are made from the centre of the lesion or suspected lesion. in some embodiments where the vascular lesion or suspected vascular lesion is in the LCx cardiac artery, the "downstream" assessment is made no more than 60mm downstream of the most upstream point, or centre, of the lesion or suspected lesion, in some of these embodiments, the "upstream" assessment is made within the LCx (i.e. downstream of the LCx ostium). in some embodiments where the vascular lesion or suspected vascular lesion is in the LAD cardiac artery, the "downstream" assessment is made no more than 80mm downstream of the most upstream point, or centre, of the lesion or suspected lesion, in some of these embodiments, the "upstream" assessment is made within the LAD (i.e. downstream of the LAD ostium). in some embodiments where the vascular lesion or suspected vascular lesion is in the RCA, the "downstream" assessment is made no more than 130mm downstream of the most upstream point, or centre, of the lesion or suspected lesion. In some of these embodiments, the "upstream" assessment is made within the RCA (i.e. downstream of the RCA ostium).

In some embodiments the gradient in the concentration of the pre-specified biomarker(s) is assessed using a catheter for taking a plurality of samples from within a length of the blood vessel, such as the catheter disclosed in WO2009/090390.

Systemic concentration

As used herein the term "systemic concentration" is used to mean the concentration of the one or more biomarker in general (bulk) circulation i.e. concentration that is NOT local to the vascular lesion, suspected vascular lesion, or vessel region of interest, in some

embodiments, the systemic concentration is measured by taking a sample from blood vessel in the peripheral circulation, for example a sample peripheral to the coronary circulation. In some embodiments the systemic concentration is measured from a sample drawn from a vein (a venous sample), such as a vein in the peripheral circulation. In other embodiments the systemic concentration is measured from a sample drawn from an artery peripheral to the coronary artery (an arterial sample), such as the femoral or radial artery. For the purposes of the present invention "venous sample", peripheral sample", "sample peripheral to the coronary circulation" are interchangeable, providing the sample is representative of the bulk circulation NOT local to the vascular lesion, suspected vascular lesion, or vessel region of interest (for example, NOT local to the coronary arteries).

Assessment of biomarkers

in one aspect of the present invention, the biomarker concentration in a sample is assessed using a binding moiety capable of specifically binding the biomarker. By way of example, the binding moiety may comprise a member of a iigand-receptor pair, i.e. a pair of molecules capable of having a specific binding interaction. The binding moiety may comprise, for example, a member of a specific binding pair, such as antibody-antigen, enzyme-substrate, nucleic acid-nucleic acid, protein-nucleic acid, protein-protein, or other specific binding pair known in the art. Binding proteins may be designed which have enhanced affinity for the biomarker of the invention. Optionally, the binding moiety may be linked with a detectable label, such as an enzymatic, fluorescent, radioactive, phosphorescent, coloured particle label or spin label. The labelled complex may be detected, for example, visually or with the aid of a spectrophotometer or other detector.

A preferred embodiment of the present invention involves the use of a recognition agent, for example an antibody recognising the biomarker of the invention, to contact a sample of tissues, cells, blood or body product, or samples derived therefrom, and screening for a positive response. The positive response may for example be indicated by an agglutination reaction or by a visualisable change such as a colour change or fluorescence, e.g.

immunostaining, or by a quantitative method such as in use of radio-immunological methods or enzyme-linked antibody methods.

Where the recognition agent is an antibody, an immunoassay can be used to detect binding of the antibody to the biomarker. Examples of immunoassays are antibody capture assays, two-antibody sandwich assays, and antigen capture assays. In a sandwich immunoassay, two antibodies capable of binding the marker protein generally are used, e.g. one

immobilised onto a solid support, and one free in solution and labelled with a detectable chemical compound. Examples of chemical labels that may be used for the second antibody include radioisotopes, fluorescent compounds, spin labels, coloured particles such as colloidal gold and coloured latex, and enzymes or other molecules that generate coloured or electrochemically active products when exposed to a reactant or enzyme substrate. When a sample containing the marker protein is placed in this system, the marker protein binds to both the immobilised antibody and the labelled antibody, to form a "sandwich" immune complex on the support's surface. The complexed protein is detected by washing away non- bound sample components and excess Iabelled antibody, and measuring the amount of labelled antibody complexed to protein on the support's surface. Alternatively, the antibody free in solution, which can be labelled with a chemical moiety, for example, a hapten, may be detected by a third antibody labelled with a detectable moiety which binds the free antibody or, for example, the hapten coupled thereto. Preferably, the immunoassay is a solid support- based immunoassay. Alternatively, the immunoassay may be one of the

immunoprecipitation techniques known in the art, such as, for example, a nephelometric immunoassay or a turbidimetric immunoassay. When Western blot analysis or an immunoassay is used, preferably it includes a conjugated enzyme labelling technique.

Although the recognition agent will conveniently be an antibody, other recognition agents are known or may become available, and can be used in the present invention. For example, antigen binding domain fragments of antibodies, such as Fab fragments, can be used. Also, so-called RNA aptamers may be used. Therefore, unless the context specifically indicates otherwise, the term "antibody" as used herein is intended to include other recognition agents. Where antibodies are used, they may be polyclonal or monoclonal. Optionally, the antibody can be produced by a method such that it recognizes a preselected epitope from a biomarker. In one aspect of the present invention, the local concentration is assessed by non-invasive imaging. For example, by administering the above described binding moiety directly to the individual, preferable by a non-invasive method, and then assessing the levels of the binding moiety by non-invasive imaging. In some embodiments, the local concentration is assessed by non-invasively imaging an individual to whom a biomarker-specific binding moiety has been administered. For example, in one embodiment the binding moiety is linked to a radioactive label detectable by PET/CT scanning. The location and amount of the biomarker bound by the moiety can then be determined using a PET/CT scanner. One or more pre-specified biomarkers

As ASVD develops, the developing vascular lesion is expected to release a 'fingerprint' of biomarkers characteristic of each stage of its development. The identification of any one of these biomarkers from any one of the 'fingerprints' allows for the possibility of detecting / predicting ASVD in an otherwise asymptomatic individual based on the presence of the biomarker.

However, it can be envisioned that not every vascular lesion at a given stage of development will release a particular biomarker. Moreover, even if a biomarker was released by every vascular lesion at a given stage of development, the natural variability to be expected between individuals, and between assay conditions, means that the biomarker may not be detected at significant levels in every individual. in view of this, the identification of multiple biomarkers characteristic of each stage of plaque development is desirable, since it increase the chances of detecting at a significant level at least one characteristic biomarker in an individual. This is of particular use in diagnostic applications, and applications where the individual is asymptomatic or as yet undiagnosed with ASVD.

Thresholds

As used herein, the "threshold level" for an individual biomarker is when the concentration of the biomarker downstream of the vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion is significantly greater than the concentration of the biomarker upstream of the vascular lesion or suspected vascular lesion, or the systemic (e.g. venous)

concentration, in some embodiments, significance is measured using a t-test, such as Student's t-test or Welch's t-test with a significance level of p<0.001 meeting the threshold, in other embodiments, a significance level of p<0.05, such as p<0.01 or p<0.005 meeting the threshold. In some embodiments, the threshold level for an individual biomarker is met if the

concentration of the biomarker measured downstream of the vascular lesion or suspected vascular lesion is at least 10% higher than the concentration of the biomarker upstream of the vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion, or the systemic (e.g. venous) concentration. For example, in some embodiments the concentration of the biomarker measured downstream is at least 10%, is at least 115%, is at least 120%, is at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 90%, at least 195%, or at least 200% of the concentration of the biomarker upstream of the vascular lesion or suspected vascular lesion, or the systemic (e.g. venous) concentration. in some embodiments the threshold level is met if the individual has a positive gradient of the biomarker as measured and defined in Example 1. in some embodiments where the individual has been: (i) previously selected for treatment, for example, by a method disclosed herein, (ii) previously been selected to take part in a clinical trial for a specified drug, or (iii) previously been selected as part of a group or cohort that possesses one or more aspect of ASVD (for example, the presence of a ThCFA or TCA in one or more of the coronary arteries), at least 15% of individuals in that group or meet the threshold level, in some embodiments at least 20%, such as at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of individuals in that group or meet the threshold level. in some embodiments where a panel of biomarkers is assessed, the overall "threshold level" is met if at least one of the individual biomarkers meets its individual threshold level, as defined above, in some embodiments where a panel of biomarkers is assessed, the overall "threshold level" is met if at least two of the individual biomarkers meet their individual threshold levels, as defined above, for example, if at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15 or at least 20 of the individual biomarkers meet their individual threshold levels, as defined above. Specific assessment methods

in one embodiment, the present invention provides a method used for assessing the likelihood of rupture of a vulnerable plaque (such as a TCFA), which method comprises assessing the concentration of one or more pre-specified biomarkers local to the plaque, wherein a gradient exceeding the threshold level is associated with a higher likelihood of plaque rupture. In some embodiments the vulnerable plaque is situated within 80mm of the LCx artery ostium, in some embodiments the vulnerable plaque is situated within 80mm of the LAD artery ostium. In some embodiments the vulnerable plaque is situated within 130mm of the RCA ostium. in one embodiment, the present invention provides a method used for detecting cap failure in a vulnerable plaque (such as a TCFA), which method comprises assessing the concentration of one or more pre-specified biomarkers local to the plaque,

wherein a gradient exceeding the threshold level is associated with cap failure. In some embodiments the vulnerable plaque is situated within 60mm of the LCx artery ostium, in some embodiments the vulnerable plaque is situated within 80mm of the LAD artery ostium, in some embodiments the vulnerable plaque is situated within 130mm of the RCA ostium. In another embodiment, the present invention provides a method used for assessing the likelihood of thrombosis (such as coronary thrombosis), which method comprises assessing the concentration of one or more pre-specified biomarkers local to a vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque),

wherein a gradient exceeding the threshold level is associated with a higher likelihood of thrombosis, in some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque) is situated within 60mm of the LCx artery ostium, in some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque) is situated within 80mm of the LAD artery ostium. In some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque) is situated within 130mm of the RCA ostium. in some embodiments thrombosis comprises formation of a 'white clot' composed largely or exclusively of activated platelets (that is, with no or little fibrin formation and erythrocyte adherence to the thrombus). in another embodiment, the present invention provides a method used for assessing the likelihood of coagulation, which method comprises assessing the concentration of one or more pre- specified biomarkers iocal to a vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque),

wherein a gradient exceeding the threshold level is associated with a higher likelihood of coagulation. In some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque), is situated within 60mm of the LCx artery ostium, in some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque), is situated within 80mm of the LAD artery ostium, in some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque), is situated within 130mm of the RCA ostium. in some embodiments coagulation comprises formation of a 'red dot' comprising activated platelets, fibrin fibres and adhered erythrocytes. in another embodiment, the present invention provides a method used for assessing the likelihood of infarction (such as myocardial infarction), which method comprises assessing the concentration of one or more pre-specified biomarkers local to a vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque),

wherein a gradient exceeding the threshold level is associated with a higher likelihood of infarction. In some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque) is situated within 60mm of the LCx artery ostium, in some embodiments the vascular lesion or suspected vascular lesion (such as a

TCFA or vulnerable plaque) is situated within 80mm of the LAD artery ostium, in some embodiments the vascular lesion or suspected vascular lesion (such as a TCFA or vulnerable plaque) is situated within 130mm of the RCA ostium. in one embodiment, the present invention provides a method used for assessing the likelihood of death of an individual from a major cardiac event (such as vulnerable plaque (such as a TCFA) rupture, coronary thrombosis, myocardial infarction, or stable / unstable angina),

Wherein the individual has at least one vascular lesion or suspected vascular lesion

(such as a TCFA or vulnerable plaque),

which method comprises assessing the concentration of one or more pre-specified biomarkers Iocal to the vascular lesion or suspected vascular lesion,

wherein a gradient exceeding the threshold level is associated with a higher likelihood of death. In some embodiments the vascular lesion or suspected vascular lesion is situated within 60mm of the LCx artery ostium. In some embodiments the vascular lesion or suspected vascular lesion is situated within 80mm of the LAD artery ostium, in some embodiments the vascular lesion or suspected vascular lesion is situated within 130mm of the RCA ostium.

Individual selection / companion diagnostic; treatment selection / companion diagnostic Specific treatments usually target, or are most effective at treating, a sub-range of disease states or symptoms. Thus, once one or more pre-specified biomarkers has been identified as being associated with a particular disease state, the presence of that biomarker(s) can be used to identify individuals who will benefit from treatment with a particular drug. Accordingly, in one aspect the present invention provides a method selecting an individual for treatment of ASVD, which method comprises assessing the concentration of one or more pre-specified biomarkers local to a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion,

wherein individuals having a measure exceeding the threshold level are selected for treatment, and wherein treatment inhibits or reduces one or more aspects of ASVD.

Treatment

The term "treatment," as used herein pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a preventative / prophylactic measure (i.e., prophylaxis) is also included. For example, use with individuals who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment." Treatment of ASVD also includes reducing the incidence of ASVD, alleviating the symptoms of ASVD, etc.

In some embodiments "treatment of ASVD" or "reducing one or more aspects of ASVD" means reducing the risk of a major cardiac event (such as the rupture of a vulnerable plaque in a coronary artery, coronary thrombosis, or myocardial infarction). In some embodiments a reduced risk of a major cardiac event means in the three years following the onset of treatment, an treated individual has no more than 90% of the risk of a major cardiac event as compared to an otherwise comparable untreated individual, for example no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, or no more than 5% of the risk. In some embodiments a reduced risk of a major cardiac event means in the five years following the onset of treatment, an treated individual has no more than 90% of the risk of a major cardiac event as compared to an otherwise comparable untreated individual, for example no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, or no more than 5% of the risk. in some embodiments a reduced risk of a major cardiac event means in the ten years following the onset of treatment, a treated individual has no more than 90% of the risk of a major cardiac event, or dying from a major cardiac event, as compared to an otherwise comparable untreated individual, for example no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 0%, or no more than 5% of the risk. The term "prevent," as used herein, refers to prophylactic treatment or treatment that prevents one or more symptoms or features of a disease, disorder, or conditions described herein. Preventative treatment can be initiated, for example, prior to ("pre-exposure prophylaxis") or following ("post-exposure prophylaxis") an event that precedes the onset of the disease, disorder, or conditions

Example treatments of ASVD include lifestyle modification (such as change in diet, increase in exercise), the administration of one or more drugs (such as aspirin, statins, ACE inhibitors, CETP inhibitors, PCSK9 inhibitors. ApoB-100 inhibitors, diuretics, beta blockers, TR-beta agonists, GPR119 receptor agonists, HDL stimulants, PPAR-aipha, beta or delta agonists, A P kinase inhibitors, Thyroid hormone receptor beta agonists, and folic acid) angioplasty (such as stenting), and bypass surgery.

Examples of specific drugs encompassed as treatments of ASVD include Advicor

(Lovastatin with Niacin), Aitocor (Lovastatin), Altoprev (Lovastatin), Atoriip (Atorvastatin), Baycol (Cerivastatin), Caduet (Atorvastatin with Amiodipine), Canef (Fiuvastatin), Crestor (Rosuvastatin), inegy (Simvastatin with Ezetimibe), Lescol (Fiuvastatin), Lipex (Simvastatin), Lipitor (Atorvastatin), Lipobay (Cerivastatin), Lipostat (Pravastatin), Lipvas (Atorvastatin), Livalo (Pitavastatin), Mevacor (Lovastatin), Pitava (Pitavastatin), Pravachoi (Pravastatin), Selektine (Pravastatin), Simcard (Simvastatin), Simcor (Simvastatin with Niacin), Simlup (Simvastatin), Sortis (Atorvastatin), Torvacard (Atorvastatin), Torvast (Atorvastatin), Totaiip (Atorvastatin), Tulip (Atorvastatin), Vytorin (Simvastatin with Ezetimibe), Zocor (Simvastatin), Juvisync (sitagliptin/simvastatin), Liptruze (ezetimibe/atorvastatin), Mevastatin, Anacetrapib Evacetrapib, Torcetrapib, Dalcetrapib, AMG 145 (Amgen), SAR236553/REGN727

Sanofi/Regeneron Pharmaceuticals, RN318 (Pfizer), RG7652 (Anti-PCSK9, PSK3169A), lomitapide (Juxtapid, Aegerion Pharmaceuticals), ipomersen sodium (Kynamro, Isis Pharmaceuticals/Genzyme), SPC-4955, REGN-728, PF-053358 0, LY-3015014, BMS- 962476, ALN-PCS, TA-8995, DRL-21995, LY-3015014, TAP31 1 , CJ-30039, ZYT-1 , KT6- 971 , RG-7652, GSK-1292263, CER-001 , GFT-505, SLx-4090, BMS-823778, RVX-208, VIA- 3196 / GL-3 96, SPC-5001 , AMG-145, K-877, alirocumab (REGN-727 / SAR-236553), dalcetrapib, JTT-302, DRL-17822, THVC:CBD (GW42003 + GW42004), RN-316 / PF- 04950615, MBX-8025 / RWJ-800025, ETC-1 QQ2, ZYH-7, LGT-209, mipomersen sodium, lomitapide, pioglitazone, anacetrapib, and evacetrapib.

Further examples of treatments of ASVD include drugs that treat the inflammatory aspects of ASVD, for examples antagonists of cytokines, IL-6 (Uniprot ref: P05231 ), MCP-1 (Uniprot ref: Q6UZ82), TNF-a (Uniprot ref: P01375), IL-18 (Uniprot ref: Q141 16), IL-10 (Uniprot ref: P22301), CRP (Uniprot ref: P02741), serum amyloid A (SAA; Uniprot ref: P0DJI8, P0DJI9, P35542), ICA (intercellular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9U F0), VCAM (vascular cell adhesion molecule; Uniprot ref: P19320), E- selectin (Uniprot ref: P18581), von Wiilebrand factor (vWF; Uniprot ref: P04275),

myeloperoxidase (MPO; Uniprot ref: P05164), secretory phospholipase A2 (sPLA2; Uniprot ref:), lipoprotein-associated phospholipase A2 (Lp-PLA2; Uniprot ref: P14555), Vascular endothelial growth factor (VEGF; Uniprot ref: P15692, P49765, P49767, 043915), placental growth factor (PIGF; Uniprot ref: P49763), hepatocyte growth factor (HGF; Uniprot ref:

P14210), matrix metailoproteinases (MMPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29965) and/or P-selectin (Uniprot ref: P16109). in some

embodiments, the antagonist is a monoclonal or polyclonal antibody that specifically binds the named molecule.

As used herein, the term "aspect of ASVD" encompasses the symptoms and clinical features associated with ASVD. Example aspects of ASVD include: the presence and/or level of a gradient in the concentration of a, or the, one or more pre-specified biomarkers; the number, size and/or status of atherosclerotic plaques in a blood vessel; the likelihood of rupture of a vulnerable plaque; the presence and/or increased concentration of inflammatory markers, such as cytokines, IL-1 (Uniprot ref: P01583, P01584), IL-6 (Uniprot ref: P05231), IL-7 (Uniprot ref: P13232), Monocyte chemo attractant protein- 1 (MCP-1 ; Uniprot ref: Q6UZ82), TNF-a (Uniprot ref: P01375), IL-18 (Uniprot ref: Q14116), IL-10 (Uniprot ref: P22301), CRP (Uniprot ref: P02741), serum amyloid A (SAA; Uniprot ref: P0DJI8, P0DJI9, P35542), ICAM (intercellular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9U F0), VCAM (vascular cell adhesion molecule; Uniprot ref: P19320), E-seiectin (Uniprot ref:

P16581), E-seiectin (Uniprot ref: P16581), von Wiilebrand factor (vWF; Uniprot ref: P04275), myeloperoxidase (MPO; Uniprot ref: P05164), secretory phospholipase A2 (sPLA2; Uniprot ref:), lipoprotein-associated phospholipase A2 (Lp-PLA2; Uniprot ref: P14555), Vascular endothelial growth factor (VEGF; Uniprot ref: P15692, P49765, P49767, 043915), placental growth factor (PIGF; Uniprot ref: P49783), hepatocyte growth factor (HGF; Uniprot ref:

P14210), matrix meta!ioproteinases (MMPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29985), P-selectin (Uniprot ref: P18109), pregnancy-associated plasma peptide A (PAPP-A), Neutrophil eiastase (Uniprot ref:P08246), Tissue Factor (Uniprot ref: P13726), Protein-bound-lnsulin-like growth factor (Uniprot ref: P05019, P01344), Neopterin, Choline, Heat Shock Proteins (Uniprot ref: Q00613, Q03933, Q9ULV5), Chlamydia pneumonia iipopolysaccharides, Degraded interstitial collagen from plaque (Type 1+1 11), Macrophage colony stimulating factor (Uniprot ref: P09603), and Cathepsin S (Uniprot ref: P25774). in some embodiments the "aspect of ASVD" which is reduced is the concentration of a, or the, one or more pre-specified biomarkers. Consequently, the present invention provides for the reduction of the expression level of the one or more pre-specified biomarkers, for example by the use of suicide inhibitors or by using antisense RNA methods to decrease the synthesis or expression of the biomarker. Similarly, this reduction in expression levels could also be achieved by down-regulation of the corresponding gene promoter, A preferred method comprises the step of administering to a patient diagnosed as having ASVD, a therapeuficaliy-effective amount of a compound which reduces in vivo the expression of the biomarker. In a preferred embodiment, the compound is a polynucleotide, for example, an anti-sense nucleic acid sequence or a peptidy! nucleic acid (PNA), more preferably from 10 to 100 nucleotides in length, capable of binding to and reducing the expression (for example, transcription or translation) of a nucleic acid encoding at least a portion of the biomarker of the invention. After administration, the anti-sense nucleic acid sequence or the anti-sense PNA molecule binds to the nucleic acid sequences encoding, at least in part, the one or more pre-specified biomarkers thereby to reduce in vivo expression of the biomarker. By way of further example, constructs of the present invention capable of reducing expression of the biomarker can be administered to the subject either as a naked polynucleotide or formulated with a carrier, such as a liposome, to facilitate incorporation into a ceil. Such constructs can also be incorporated into appropriate vaccines, such as in viral vectors (e.g. vaccinia), bacterial constructs, such as variants of the well-known BCG vaccine, and so forth.

A particularly useful therapeutic embodiment of the present invention provides an

oligonucleotide or peptidyl nucleic acid sequence complementary and capable of hybridizing under physiological conditions to part, or ail, of the gene encoding the one or more pre- specified biomarkers or to part, or all, of the transcript encoding the one or more pre- specified biomarkers thereby to reduce or inhibit transcription and/or translation of the gene encoding the biomarker (in cases where the biomarker is an encoded protein / nucleic acid).

Anti-sense oligonucleotides have been used extensively to inhibit gene expression in normal and abnormal ceils. For a recent review, see Phillips, ed., Antisense Technology, in Methods in Enzymoiogy, vols. 313-314, Academic Press; Hartmann, ed., 1999. In addition, the synthesis and use of peptidyl nucleic acids as anti-sense-based therapeutics are described in PCT publications PCT/EP92/01219, PCT/US92/1092, and PCT/US94/013523. Accordingly, the anti-sense-based therapeutics may be used as part of chemotherapy, either alone or in combination with other therapies.

Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire A. et al Nature, Vol 391 , (1998)). dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) (See also Fire (1999) Trends Genet. 15: 358-363, Sharp (2001) Genes Dev. 15: 485-490, Hammond et al. (2001) Nature Rev. Genes 2: 1 110-1 119 and Tuschl (2001) Chem, Biochem. 2: 239-245). RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt) The siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001) Thus in one embodiment, the invention provides double stranded RNA comprising a sequence encoding a biomarker of the present invention, which may for example be a "long" double stranded RNA (which will be processed to siRNA, e.g., as described above). These RNA products may be synthesised in vitro, e.g., by conventional chemical synthesis methods.

RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'-overhang ends (Zamore PD et al Cell, 101 , 25-33, (2000)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir SM. et al. Nature, 411 , 494-498, (2001)). Thus siRNA duplexes containing between 20 and 25 bps, more preferably between 21 and 23 bps, of the sequence encoding a biomarker of the present invention form one aspect of the invention e.g. as produced synthetically, optionally in protected form to prevent degradation. Alternatively siRNA may be produced from a vector, in vitro (for recovery and use) or in vivo.

Accordingly, the vector may comprise a nucleic acid sequence encoding a biomarker of the present invention (including a nucleic acid sequence encoding a variant or fragment thereof), suitable for introducing an siRNA into the cell in any of the ways known in the art, for example, as described in any of references cited herein, which references are specifically incorporated herein by reference. In one embodiment, the vector may comprise a nucleic acid sequence according to the invention in both the sense and antisense orientation, such that when expressed as RNA the sense and antisense sections will associate to form a double stranded RNA. This may for example be a long double stranded RNA (e.g., more than 23nts) which may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21 :324- 328).

Alternatively, the double stranded RNA may directly encode the sequences which form the siRNA duplex, as described above. In another embodiment, the sense and antisense sequences are provided on different vectors.

These vectors and RNA products may be useful for example to inhibit de novo production of the biomarker of the present invention in a cell. They may be used analogously to the expression vectors in the various embodiments of the invention discussed herein. in particular there is provided double-stranded RNA which comprises an RNA sequence encoding a biomarker of the present invention or a fragment thereof, which may be an siRNA duplex consisting of between 20 and 25 bps. Also provided are vectors encoding said dsRNA or siRNA duplexes. Also provided are methods of producing said siRNA duplexes comprising introducing such vectors into a host ceil and causing or allowing transcription from the vector in the ceil. Separate vectors may encode: (i) the sense sequence of the siRNA duplex, and (ii) the anti-sense sequence of the siRNA duplex. An additional DNA based therapeutic approach provided by the present invention is the use of a vector which comprises one or more nucleotide sequences, preferably a plurality of these, each of which encodes an immunoreactive peptide derived from the biomarker of the invention. Alternatively, a further method of the invention involves combining one or more of these nucleotide sequences encoding peptides derived from the biomarker of the invention in combination with nucleotide sequences encoding peptides derived from other ASVD markers, and encompasses inclusion of such sequences in all possible variations, such as one from each protein, several from one or more protein and one from each of one or more additional proteins, and so forth.

The present invention also provides a method of selecting individuals for treatment with an antibody which specifically binds a protein selected form the group consisting of:

IL-1 (Uniprot ref: P01583, P01584), IL-6 (Uniprot ref: P05231), IL-7 (Uniprot ref: P13232), Monocyte chemo attractant protein-1 (MCP-1 ; Uniprot ref: Q8UZ82), TNF-a (Uniprot ref: P01375), IL-18 (Uniprot ref: Q141 18), IL-10 (Uniprot ref: P22301), CRP (Uniprot ref: P02741), serum amyloid A (SAA; Uniprot ref: P0DJI8, P0DJI9, P35542), ICA

(intercellular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9U F0), VCAM (vascular cell adhesion molecule; Uniprot ref: P19320), E-seiectin (Uniprot ref:

P14210), matrix metalloproteinases (MMPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29985), P-selectin (Uniprot ref: P18109), pregnancy-associated plasma peptide A (PAPP-A), Neutrophil eiastase (Uniprot ref:P08246), Tissue Factor (Uniprot ref: P13726), Protein-bound-lnsulin-like growth factor (Uniprot ref: P05019, P01344), Neopterin, Choline, Heat Shock Proteins (Uniprot ref: Q00613, Q03933, Q9ULV5), which method comprises assessing a local concentration of one or more pre- specified biomarkers

wherein individuals having a measure exceeding the threshold level are selected for treatment, and wherein the treatment inhibits or reduces one or more aspect of ASVD. The present invention also provides a method of selecting or stratifying individuals for a clinical trial with an antibody which specifically binds a protein selected form the group consisting of: IL-1 (Uniprot ref: P01583, P01584), IL-6 (Uniprot ref: P05231), IL-7 (Uniprot ref: P13232), Monocyte cherno attractant protein-1 (MCP-1 ; Uniprot ref: Q6UZ82), TNF-a (Uniprot ref: PQ1375), !L-18 (Uniprot ref: Q141 16), !L-10 (Uniprot ref: P22301), CRP (Uniprot ref: P02741), serum amyloid A (SAA; Uniprot ref: PGDJI8, PGDJI9, P35542), I CAM

(interceiiular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9UMF0), VCA (vascular cell adhesion molecule; Uniprot ref: P19320), E-seiectin (Uniprot ref:

P16581), E-seiectin (Uniprot ref: P16581), von Willebrand factor (vWF; Uniprot ref: PQ4275), myeloperoxidase (MPO; Uniprot ref: P05164), secretory phospholipase A2 (sPLA2; Uniprot ref:), lipoprotein-associated phospholipase A2 (Lp-PLA2; Uniprot ref: P14555), Vascular endothelial growth factor (VEGF; Uniprot ref: P15692, P49785, P49787, 043915), placental growth factor (PIGF; Uniprot ref: P49763), hepatocyte growth factor (HGF; Uniprot ref:

P14210), matrix metalloproteinases ( MPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29965), P-selectin (Uniprot ref: P16109), pregnancy-associated plasma peptide A (PAPP-A), Neutrophil eiastase (Uniprot ref:P08248), Tissue Factor (Uniprot ref: P13726), Protein-bound-lnsulin-like growth factor (Uniprot ref: P05019, P01344), Neopterin, Choline, Heat Shock Proteins (Uniprot ref: Q00613, Q03933, Q9ULV5), which method comprises assessing a local concentration of one or more pre- specified biomarkers

wherein individuals having a measure exceeding the threshold level are selected for treatment, and wherein the treatment inhibits or reduces one or more aspect of ASVD.

The present invention also provides a method of selecting for an individual treatment with an antibody which specifically binds a protein selected form the group consisting of:

IL-1 (Uniprot ref: P01583, P01584), IL-6 (Uniprot ref: P05231), IL-7 (Uniprot ref:

P13232), Monocyte cherno attractant protein-1 (MCP-1 ; Uniprot ref: Q6UZ82), TNF-a (Uniprot ref: P01375), !L-18 (Uniprot ref: Q141 16), IL-10 (Uniprot ref: P22301), CRP (Uniprot ref: P02741), serum amyloid A (SAA; Uniprot ref: P0DJI8, P0DJI9, P35542), ICAM

(intercellular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9UMF0), VCAM (vascular cell adhesion molecule; Uniprot ref: P19320), E-seiectin (Uniprot ref:

P18581), E-seiectin (Uniprot ref: P18581), von Wiilebrand factor (vWF; Uniprot ref: P04275), myeloperoxidase (MPO; Uniprot ref: P05184), secretory phospholipase A2 (sPLA2; Uniprot ref:), lipoprotein-associated phospholipase A2 (Lp-PLA2; Uniprot ref: P 14555), Vascular endothelial growth factor (VEGF; Uniprot ref: P15692, P49785, P49787, 043915), placental growth factor (PIGF; Uniprot ref: P49763), hepatocyte growth factor (HGF; Uniprot ref:

P14210), matrix metalloproteinases (MMPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29965), P-selectin (Uniprot ref: P16109), pregnancy-associated plasma peptide A (PAPP-A), Neutrophil elastase (Uniprot ref:P08246), Tissue Factor (Uniprot ref: P13726), Protein-bound-insulin-like growth factor (Uniprot ref: P05019, P01344), Neopterin, Choline, Heat Shock Proteins (Uniprot ref: Q00613, Q03933, Q9ULV5), which method comprises assessing a local concentration of one or more pre- specified biomarkers

wherein the treatment is selected if the individual has a measure exceeding the threshold level, and wherein the treatment inhibits or reduces one or more aspect of ASVD. The present invention also provides a method of selecting individuals for treatment with a compound selected from the group consisting of:

Advicor (Lovastatin with Niacin), Aitocor (Lovastatin), Altoprev (Lovastatin), Atorlip (Atorvastatin), Baycoi (Cerivastatin), Caduet (Atorvastatin with Amlodipine), Canef

(Fluvastatin), Crestor (Rosuvastatin), Inegy (Simvastatin with Ezetimibe), Lescoi

(Fluvastatin), Lipex (Simvastatin), Lipitor (Atorvastatin), Lipobay (Cerivastatin), Lipostat (Pravastatin), Lipvas (Atorvastatin), Livaio (Pravastatin), Mevacor (Lovastatin), Pitava (Pitavastatin), Pravachol (Pravastatin), Selektine (Pravastatin), Simcard (Simvastatin), Simcor (Simvastatin with Niacin), Simiup (Simvastatin), Sortis (Atorvastatin), Torvacard (Atorvastatin), Torvast (Atorvastatin), Totalip (Atorvastatin), Tulip (Atorvastatin), Vytorin (Simvastatin with Ezetimibe), Zocor (Simvastatin), Juvisync (sitagliptin/simvastatin), Liptruze (ezetimibe/atorvastatin), Mevastatin, Anacetrapib, Evacetrapib, Torcetrapib, Dalcetrapib, A G 145 (Amgen), SAR236553/REGN727 Sanofi/Regeneron Pharmaceuticals, RN316 (Pfizer), RG7652 (Anti-PCSK9, MPSK3169A), iomifapide (Juxfapid, Aegerion

Pharmaceuticals), Mipomersen sodium (Kynamro, Isis Pharmaceuticals/Genzyme), SPC- 4955, REGN-728, PF-05335810, LY-3015014, BMS-962476, ALN-PCS, TA-8995, DRL- 21995, LY-3015014, TAP31 1 , CJ-30039, ZYT-1 , KT6-971 , RG-7652, GSK-1292283, CER- 001 , GFT-505, SLx-4090, BMS-823778, RVX-208, VIA-3196 / MGL-3196, SPC-5001 , AMG- 145, K-877, alirocumab (REGN-727 / SAR-236553), dalcetrapib, JTT-302, DRL-17822, THVC:CBD (GW42003 + GW42004), RN-316 / PF-04950615, MBX-8025 / RWJ-800025, ETC-1002, ZYH-7, LGT-209, mipomersen sodium, iomitapide, pioglitazone, anacetrapib, evacetrapib, JI-101 , Orantinib (also known as TSU-68; SU6668), Vatalanib, Pazopanib/ Votrienf, Motesanib / AMG 708, Trapidil (triazolopyrimidine), Axitinib (AG013736) Inlyta, AMG 706 (motesanib), BIBF 1120 (nintedanib), Sorafenib / Nexavar, X-82, Sunitinib / Sutent / SU11248, Crenolanib / CP-868,596; ARO 002, niiotinib, Imatinib / Gieevec / Glivec, Olaratumab (IMC-3G3, see [19]), MEDi-575, Fovista E10030, Vinpocetine, BIBF-1120, Tandutinib, Piperlongumine, and Eupatoiide, which method comprises assessing a iocai concentration of one or more pre- specified biomarkers

wherein individuals having a measure exceeding the threshold level are selected for treatment, and wherein the treatment inhibits or reduces one or more aspect of ASVD,

The present invention also provides a method of selecting or stratifying individuals for a clinical trial with a compound selected from the group consisting of:

Advicor (Lovastatin with Niacin), Altocor (Lovastatin), Aitoprev (Lovastatin), Atorlip (Atorvastatin), Baycoi (Cerivastatin), Caduet (Atorvasfatin with Amlodipine), Canef

(Fluvastatin), Creator (Rosuvastatin), inegy (Simvastatin with Ezetimibe), Lescoi

(Fluvastatin), Lipex (Simvastatin), Lipitor (Atorvastatin), Lipobay (Cerivastatin), Lipostat (Pravastatin), Lipvas (Atorvastatin), Livalo (Pitavastatin), Mevacor (Lovastatin), Pitava (Pitavastatin), Pravachol (Pravastatin), Selektine (Pravastatin), Simcard (Simvastatin), Simcor (Simvastatin with Niacin), Simiup (Simvastatin), Sortis (Atorvastatin), Torvacard (Atorvastatin), Torvast (Atorvastatin), Totalip (Atorvastatin), Tulip (Atorvastatin), Vytorin

(Simvastatin with Ezetimibe), Zocor (Simvastatin), Juvisync (sitagliptin/simvastatin), Liptruze (ezetimibe/atorvastatin), evastatin, Anacetrapib, Evacetrapib, Torcetrapib, Dalcetrapib, AMG 145 (Amgen), SAR236553/REGN727 Sanofi/Regeneron Pharmaceuticals, RN316 (Pfizer), RG7652 (Anti-PCSK9, PSK3189A), iomitapide (Juxtapid, Aegerion

Pharmaceuticals), Mipomersen sodium (Kynamro, !sis Pharmaceuticals/Genzyme), SPC- 4955, REGN-728, PF-05335810, LY-3015014, BMS-962476, ALN-PCS, TA-8995, DRL- 21995, LY-3015014, TAP31 1 , CJ-30039, ZYT-1 , KT6-971 , RG-7652, GSK-1292263, CER- 001 , GFT-505, SLx-4090, BMS-823778, RVX-208, VIA-3196 / MGL-3196, SPC-5001 , AMG- 145, K-877, alirocumab (REGN-727 / SAR-238553), dalcetrapib, JTT-302, DRL-17822, THVC:CBD (GW42003 + GW42004), RN-316 / PF-04950615, MBX-8025 / RWJ-800025, ETC-1002, ZYH-7, LGT-209, mipomersen sodium, Iomitapide, pioglitazone, anacetrapib, evacetrapib, JI-101 , Oranfinib (also known as TSU-68; SU6668), Vatalanib, Pazopanib/ Votrient, Motesanib / AMG 706, Trapidil (triazolopyrimidine), Axitinib (AG013736) Inlyta, AMG 706 (motesanib), BIBF 1120 (nintedanib), Sorafenib / Nexavar, X-82, Sunitinib / Sufent / SU11248, Crenolanib / CP-868,596; ARO 002, niiotinib, Imatinib / Gieevec / Glivec, Olaratumab (IMC-3G3, see [19]), MED!-575, Fovista E10030, Vinpocetine, BIBF-1120, Tandutinib, Piperlongumine, and Eupatoiide,

which method comprises assessing a local concentration of one or more pre- specified biomarkers

wherein individuals having a measure exceeding the threshold level are selected for treatment and wherein the treatment inhibits or reduces one or more aspect of ASVD. The present invention also provides a method of selecting for an individual treatment with a compound selected from the group consisting of:

Advicor (Lovasfatin with Niacin), Aitocor (Lovastatin), Altoprev (Lovastatin), Atorlip (Atorvastatin), Baycol (Cerivastatin), Caduet (Atorvastatin with Amlodipine), Canef

(Fluvastatin), Lipex (Simvastatin), Lipitor (Atorvastatin), Lipobay (Cerivastatin), Lipostat (Pravastatin), Lipvas (Atorvastatin), Livaio (Pitavastatin), Mevacor (Lovastatin), Pitava (Pitavastatin), Pravachol (Pravastatin), Selektine (Pravastatin), Simcard (Simvastatin), Simcor (Simvastatin with Niacin), Simiup (Simvastatin), Sortis (Atorvastatin), Torvacard (Atorvastatin), Torvast (Atorvastatin), Totalip (Atorvastatin), Tulip (Atorvastatin), Vytorin

(Simvastatin with Ezetimibe), Zocor (Simvastatin), Juvisync (sitagliptin/simvastatin), Liptruze (ezetimibe/atorvastatin), Mevastatin, Anacetrapib, Evacetrapib, Torcetrapib, Dalcetrapib, A G 145 (Amgen), SAR236553/REGN727 Sanofi/Regeneron Pharmaceuticals, RN316 (Pfizer), RG7852 (Anti-PCSK9, PSK3169A), iomitapide (juxtapid, Aegerion

Pharmaceuticals), Mipomersen sodium (Kynamro, isis Pharmaceuticals/Genzyme), SPC- 4955, REGN-728, PF-05335810, LY-3015014, BMS-962478, ALN-PCS, TA-8995, DRL- 21995, LY-3015014, TAP31 1 , CJ-30Q39, ZYT-1 , KT6-971 , RG-7652, GSK-1292263, CER- 001 , GFT-505, SLx-4090, BIV1S-823778, RVX-208, VIA-3196 / MGL-3196, SPC-5001 , A G- 145, K-877, alirocumab (REGN-727 / SAR-236553), dalcetrapib, JTT-302, DRL-17822, THVC:CBD (GW42003 + GW42004), RN-316 / PF-04950615, MBX-8025 / RWJ-800025, ETC-1002, ZYH-7, LGT-209, mipomersen sodium, Iomitapide, pioglitazone, anacetrapib, evacetrapib, Ji-101 , Orantinib (also known as TSU-68; SU6668), Vatalanib, Pazopanib/ Votrienf, Motesanib / AMG 708, Trapidil (triazolopyrimidine), Axitinib (AG013736) Inlyta, AMG 706 (motesanib), BIBF 1120 (nintedanib), Sorafenib / Nexavar, X-82, Sunitinib / Sutent / SU11248, Crenolanib / CP-868,596; ARO 002, nilotinib, Imatinib / Gieevec / G!ivec, Olaratumab (IMC-3G3, see [19]), MEDi-575, Fovista E10030, Vinpocetine, B1BF-1120, Tandutinib, Piperlongumine, and Eupatoiide,

wherein the treatment is selected if the individual has a measure exceeding the threshold level, and wherein the treatment inhibits or reduces one or more aspect of ASVD.

As ASVD progresses from fatty streaks on vessel walls, through intermediate lesions, atheroma, and fibroatheroma to complicated lesions and vulnerable plaques, the profile of biomarkers released by the lesion / plaque is expected to change, as is the type of treatment which is required / most effective. Accordingly, the present invention also provides a method of timing the application of treatment of an individual with an antibody which specifically binds a protein selected form the group consisting of:

IL-1 (Uniprot ref: P01583, P01584), IL-6 (Uniprot ref: P05231), IL-7 (Uniprot ref: P13232), Monocyte chemo aftractant protein-1 (MCP-1 ; Uniprot ref: Q6UZ82), TNF-a

(Uniprot ref: P01375), IL-18 (Uniprot ref: Q141 16), IL-10 (Uniprot ref: P22301), CRP (Uniprot ref: P02741), serum amyloid A (SAA; Uniprot ref: P0DJI8, P0DJI9, P35542), ICA

(intercellular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9U F0), VCA (vascular cell adhesion molecule; Uniprot ref: P19320), E-selectin (Uniprot ref:

P16581), E-seiectin (Uniprot ref: P16581 ), von Wiliebrand factor (vWF; Uniprot ref: P04275), myeloperoxidase (MPO; Uniprot ref: P05164), secretory phospholipase A2 (sPLA2; Uniprot ref:), lipoprotein-associated phospholipase A2 (Lp-PLA2; Uniprot ref: P14555), Vascular endothelial growth factor (VEGF; Uniprot ref: P15692, P49765, P49767, 043915), placental growth factor (PIGF; Uniprot ref: P49783), hepatocyte growth factor (HGF; Uniprot ref:

P14210), matrix metailoproteinases (MMPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29965), P-selectin (Uniprot ref: P16109), pregnancy-associated plasma peptide A (PAPP-A), Neutrophil eiastase (Uniprot ref:P08246), Tissue Factor (Uniprot ref: P13726), Protein-bound-insulin-like growth factor (Uniprot ref: P05019, P01344), Neopterin, Choline, Heat Shock Proteins (Uniprot ref: Q00613, Q03933, Q9ULV5), which method comprises assessing a local concentration of one or more pre- specified biomarkers

wherein the treatment is applied if the individual has a measure exceeding the threshold level, and wherein the treatment inhibits or reduces one or more aspect of ASVD.

The present invention also provides a method of timing the application of treatment of an individual with a compound selected from the group consisting of:

Advicor (Lovastatin with Niacin), Altocor (Lovastatin), Altoprev (Lovastatin), Atoriip

(Atorvastafin), Baycoi (Cerivastafin), Caduef (Atorvastatin with Amlodipine), Canef

(Fluvastatin), Lipex (Simvastatin), Lipitor (Atorvastatin), Lipobay (Cerivastafin), Lipostat (Pravastatin), Lipvas (Atorvastatin), Livaio (Pitavastatin), Mevacor (Lovastatin), Pitava (Pitavastatin), Pravachoi (Pravastatin), Seiektine (Pravastatin), Simcard (Simvastatin), Simcor (Simvastatin with Niacin), Simiup (Simvastatin), Sortis (Atorvastatin), Torvacard (Atorvastatin), Torvast (Atorvastatin), Totalip (Atorvastatin), Tulip (Atorvastatin), Vytorin

(Simvastatin with Ezetimibe), Zocor (Simvastatin), Juvisync (sitagliptin/simvastatin), Liptruze (ezetimibe/atorvastatin), Mevastatin, Anacetrapib, Evacetrapib, Torcetrapib, Daicetrapib, A G 145 (Amgen), SAR236553/REGN727 Sanofi/Regeneron Pharmaceuticals, RN316 (Pfizer), RG7652 (Anti-PCSK9, MPSK3169A), iomitapide (Juxtapid, Aegerion

Pharmaceuticals), Mipomersen sodium (Kynamro, isis Pharmaceuticals/Genzyme), SPC- 4955, REGN-728, PF-05335810, LY-3015014, B S-962476, ALN-PCS, TA-8995, DRL- 21995, LY-3015014, TAP31 1 , CJ-30039, ZYT-1 , KT6-971 , RG-7652, GSK-1292263, CER- 001 , GFT-505, SLx-4090, B S-823778, RVX-208, VIA-3196 / MGL-3196, SPC-5001 , AIV3G- 145, K-877, alirocumab (REGN-727 / SAR-236553), dalcetrapib, JTT-302, DRL-17822, THVC:CBD (GW42003 + GW42004), RN-318 / PF-04950615, BX-8025 / RWJ-800025, ETC- 1002, ZYH-7, LGT-209, mipomersen sodium, Iomitapide, pioglitazone, anacetrapib, evacetrapib, JI-101 , Orantinib (also known as TSU-68; SU6888), Vatalanib, Pazopanib/ Votrient, Motesanib / AMG 706, Trapidil (triazolopyrimidine), Axitinib (AG013738) Inlyta, AMG 708 (motesanib), BIBF 1 20 (nintedanib), Sorafenib / Nexavar, X-82, Sunitinib / Sutent / SU11248, Crenolanib / CP-868,596; ARO 002, nilotinib, Imatinib / Gieevec / Glivec, Olaratumab (IMC-3G3, see [19]), MEDi-575, Fovista E10030, Vinpocetine, BIBF-1120, Tandutinib, Piperlongumine, and Eupatolide,

wherein the treatment is applied if the individual has a measure exceeding the threshold level, and wherein the treatment inhibits or reduces one or more aspect of ASVD,

Clotting assay

A local increase in the concentration of a biomarker in a section of a blood vessel can, depending on the identity of the biomarker, influence the clotting properties of the blood flowing through the blood vessel. Thus, blood flowing through blood vessels having ASVD may have an increased, tendency to clot, which would in turn increase the likelihood of thrombosis or coagulation.

Thus, in one aspect the present invention provides a method of assessing risk of thrombosis or coagulation within a section of a blood vessel, the method comprising collecting a sample of blood from the section of the blood vessel and assessing the clotting ability of the blood, in some embodiments, the clotting ability of the blood is assessed by measuring the level of platelet activation in the sample by, for example, measuring the level of one or more soluble markers of thrombosis and/or coagulation such as thromboxane B2, soluble P-selectin, von Wiliebrand factor, D-dimer, platelet surface P-selectin, leukocyte surface CD11 b expression and platelet-leukocyte conjugation. In some embodiments an increase the level of the one or more soluble markers of thrombosis and/or coagulation is measured by determining the concentration of the one or more soluble marker in the sample of blood, wherein a measure exceeding the threshold level is associated with an increased risk of thrombosis or coagulation. in preferred embodiments the section of the blood vessel is known or suspected of having ASVD, In some particularly preferred embodiments the section of the blood vessel is known to have at least one TCFA lesion. Using this clotting method as an assay, the efficacy of drugs designed to treat thrombosis can be studied.

Thus, the present invention provides a method of assessing the efficacy of a drug in treating thrombosis in an individual, which method comprises assessing the effect of the drug on the clotting ability of blood sampled from a blood vessel, or the effect of the drug on the biological activity of a pathway associated with the one or more pre-specified biomarkers. In preferred embodiments the section of the blood vessel is known or suspected of having ASVD. In some particularly preferred embodiments the section of the blood vessel is known to have at least one TCFA lesion.

New medical uses

Biomarkers of ASVD may be either indicative (i.e. their levels correlate with disease outcome but the disease cannot be affected by changing their activity) or causative (i.e. if their expression or activity is modified, the disease progression will change). In the case of causative biomarkers, these markers may form targets for treatment.

Accordingly, in one aspect the invention provides a method of treating ASVD comprising administering to an individual in need of treatment a therapeutically effective amount of a drug which lowers the concentration of one or more pre-specified biomarkers local to the vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion, or inhibits the biological activity of a pathway associated with the one or more pre-specified biomarkers. in some embodiments the invention provides a method of treating a TCFA or ThFCA in a coronary artery comprising administering to an individual in need of treatment a

therapeutically effective amount of a drug which lowers the concentration of one or more pre-specified biomarkers local to the TCFA or ThCFA, or inhibits the biological activity of a pathway associated with the one or more pre-specified biomarkers. The term "therapeuticaliy-effective amount," as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen. in one aspect the present invention provides a drug which lowers the concentration of one or more pre-specified biomarkers local to a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion, or inhibits the biological activity of a pathway associated with the one or more pre-specified biomarkers for use in a method of treatment of ASVD. in some embodiments the vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion is within 80mm of the LCx artery ostium. In some embodiments the vascular lesion (such as a TCFA or ThCFA) is situated within 80mm of the LAD artery ostium. In some embodiments the vascular lesion (such as a TCFA or ThCFA) is situated within 130mm of the RCA ostium. in one aspect the present invention provides for the use of a drug which lowers the concentration of one or more pre-specified biomarkers local to a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion, or inhibits the biological activity of a pathway associated with the one or more pre-specified biomarkers, in the manufacture of a medicament for the treatment of ASVD. in some embodiments the vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion is within 60mm of the LCx artery ostium, in some embodiments the vascular lesion (such as a TCFA or ThCFA) is situated within 80mm of the LAD artery ostium. In some embodiments the vascular lesion (such as a TCFA or ThCFA) is situated within 130mm of the RCA ostium. in one aspect the present invention provides for the use of a drug which lowers the concentration of one or more pre-specified biomarkers local to a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion, or inhibits the biological activity of a pathway associated with the one or more pre-specified biomarkers, in the treatment of ASVD. in some embodiments the vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion is within 60mm of the LCx artery ostium, in some embodiments the vascular lesion (such as a TCFA or ThCFA) is situated within 80mm of the LAD artery ostium. In some embodiments the vascular lesion (such as a TCFA or ThCFA) is situated within 130mm of the RCA ostium. in some embodiments, treatment with the drug lowers the local concentration of the one or more pre-specified biomarkers from above the threshold level. In some embodiments treatment with the drug lowers the local concentration of the one or more pre-specified biomarkers to below a threshold level. In some embodiments treatment with the drug inhibits or reduces one or more aspect of ASVD. in some embodiments the method comprises the step of testing the individual for an increased concentration of one or more pre-specified biomarkers and administering the drug to those individuals having an increased concentration of the one or more pre-specified biomarkers. In some embodiments the method comprises the step of administering the drug to those individuals known to have an increased concentration of the one or more pre- specified biomarkers. in some embodiments the method comprises the step of administering the drug to those individuals that have been previously selected as having an increased concentration of the one or more pre-specified biomarkers. In some embodiments the concentration is assessed locally to a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion as hereinabove described. in some embodiments the one or more pre-specified biomarker(s) is the target per se of the drug. For example, the drug may comprise an antibody or other binding moiety which specifically binds to the one or more pre-specified biomarker(s). In some embodiments a protein associated with the one or more pre-specified biomarker(s) is the target per se of the drug. For example, the protein associated with the one or more pre-specified biomarker(s) may be a receptor or other binding partner which the one or more pre-specified biomarker(s) binds to elicit its biological effect. Thus, the drug may comprise an antibody or other binding moiety which specifically binds to the protein associated with the one or more pre-specified biomarker(s). in some embodiments the concentration is assessed locally to a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion as hereinabove described. in preferred embodiments the drug inhibits or reduces one or more aspect of ASVD.

Assessing drug efficacy

The identification of biomarkers that are indicative of a disease state provides a readily measurable proxy for monitoring the progression, or regression, of that disease state. Thus, a treatment intended to reduce or eliminate a symptom or disease state should also reduce or eliminate the biomarker associated with that symptom or disease state. Accordingly, the present invention provides a method of assessing the efficacy of a drug in treating ASVD in an individual, which method comprises assessing the effect of the drug on the concentration of one or more pre-specified biomarkers, or the effect of the drug on the biological activity of a pathway associated with the one or more pre-specified biomarkers. In some embodiments the drug reduces one or more aspect of ASVD.

The present invention also provides selecting a drug for use in treating ASVD in an individual, which method comprises assessing the effect of the drug on the concentration of one or more pre-specified biomarkers, or the effect of the drug on the biological activity of a pathway associated with the one or more pre-specified biomarkers. in some embodiments the drug reduces one or more aspect of ASVD.

Assessing drug effects

As used herein, "assessing the effect of the drug on the concentration" means assessing any changes in the concentration of the biomarker in response to treatment with the drug. In some embodiments the drug effects are assessed by measuring the concentration of the biomarker both before treatment with the drug has begun, and during or after treatment with the drug. Comparison of the concentration of one or more pre-specified biomarkers before and after treatment with the drug enables an assessment of drug efficacy in treating those disease states of symptoms associated with the one or more pre-specified biomarkers. In other embodiments the biomarker concentration is assessed after treatment has begun, or been completed, with the treatment deemed effective if the concentration is, or is reduced below, a threshold. Preferably the concentration is assessed locally to a vascular lesion (such as a TCFA or ThCFA) or suspected vascular lesion.

Biomarker associaied pathways

As used herein "a pathway associated with the one or more pre-specified biomarkers" is any biological pathway or process which is mechanistically linked to the one or more pre- specified biomarkers. For example, the one or more biomarker may be a constituent member of the pathway, in some embodiments the pathway is a signalling pathway; in these embodiments the effect of the drug can be measured, for example, as the effect on the signalling activity of the pathway, in some embodiments the pathway is a developmental pathway, for example vascular neogenesis. In some embodiments the "biological activity of a pathway associated with the one or more pre-specified biomarkers" is inhibited by inhibiting another member of the pathway of which the one or more pre-specified biomarkers is/are a member. For example, in embodiments where the one or more pre-specified biomarker is a signalling molecule, the activity of the receptor to which the signalling molecule binds may be inhibited (by a small molecule or antibody which specifically binds the receptor, for instance). in some embodiments, treatment with the drug lowers the local concentration of the one or more pre-specified biomarkers from above the threshold level. In some embodiments treatment with the drug lowers the local concentration of the one or more pre-specified biomarkers to below the threshold level, in some embodiments treatment with the drug inhibits or reduces one or more aspect of ASVD.

Detection of drug adverse side effects

in the same way that biomarkers can indicate if a treatment is reducing one or more symptom or aspect of ASVD, biomarkers can also indicate if a treatment or other factor is increasing the risk of ASVD, for example, increasing the likelihood that a vulnerable plaque will rupture. Accordingly, the present invention provides a method of assessing the increased risk of

ASVD in an individual caused by a drug, which method comprises assessing the effect of the drug on the local concentration of one or more pre-specified biomarkers (for example local to a vascular lesion or suspected vascular lesion), or the effect of the drug on the biological activity of a pathway associated with the one or more pre-specified biomarkers. In some embodiments treatment with the drug causes or increases one or more aspect of ASVD. in some embodiments, treatment with the drug raises the local concentration of the one or more pre-specified biomarkers to above the threshold level, in some embodiments treatment with the drug raises the local concentration of the one or more pre-specified biomarkers from below a threshold level. In some embodiments treatment with the drug causes or increases one or more aspect of ASVD.

Screening for new therapeutic compounds

A further aspect of the present invention provides novel methods for screening for compositions that modulate the expression or biological activity of the one or more biomarker and, therefore, have therapeutic potential in cases where the biomarker is mechanistically linked to ASVD pathology. As used herein, the term "biological activity" means any observable effect resulting from interaction between the biomarker and a ligand or binding partner.

The term "biological activity" also encompasses both the inhibition and the induction of the expression of the biomarker of the invention. Further, the term "biological activity" encompasses any and all effects resulting from the binding of a ligand or other in vivo binding partner by a polypeptide derivative of the protein of the invention. In one

embodiment, a method of screening drug candidates comprises providing a cell that expresses the biomarker of the invention, adding a candidate therapeutic compound to said cell and determining the effect of said compound on the expression or biological activity of said protein. In a further embodiment, the method of screening candidate therapeutic compounds includes comparing the level of expression or biological activity of the protein in the absence of said candidate therapeutic compound to the level of expression or biological activity in the presence of said candidate therapeutic compound. Where said candidate therapeutic compound is present its concentration may be varied, and said comparison of expression level or biological activity may occur after addition or removal of the candidate therapeutic compound. The expression level or biological activity of said biomarker may show an increase or decrease in response to treatment with the candidate therapeutic compound.

Candidate therapeutic molecules of the present invention may include, by way of example, peptides produced by expression of an appropriate nucleic acid sequence in a host ceil or using synthetic organic chemistries, or non-peptide small molecules produced using conventional synthetic organic chemistries well known in the art. Screening assays may be automated in order to facilitate the screening of a large number of small molecules at the same time.

As used herein, the terms "candidate therapeutic compound" refers to a substance that is believed to interact with the biomarker of the invention (or a fragment thereof), and which can be subsequently evaluated for such an interaction. Representative candidate therapeutic compounds include "xenobiotics", such as drugs and other therapeutic agents, natural products and extracts, carcinogens and environmental pollutants, as well as

"endobiotics" such as steroids, fatty acids and prostaglandins. Other examples of candidate compounds that can be investigated using the methods of the present invention include, but are not restricted to, agonists and antagonists of the biomarker of the invention, toxins and venoms, viral epitopes, hormones (e. g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, co-factors, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, small molecules, and polyclonal and/or monoclonal antibodies.

In one preferred embodiment the present invention provides a method of drug screening utilising eukaryotic or prokaryotic host ceils stably transformed with recombinant

polynucleotides expressing the biomarker of the invention or a fragment thereof, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. For example, the assay may measure the formation of complexes between a biomarker and the agent being tested, or examine the degree to which the formation of a complex between the biomarker or fragment thereof and a known iigand or binding partner is interfered with by the agent being tested. Thus, the present invention provides methods of screening for drugs comprising contacting such an agent with the biomarker of the invention or a fragment thereof or a variant thereof and assaying (i) for the presence of a complex between the agent and the biomarker, fragment or variant thereof, or (ii) for the presence of a complex between the biomarker, fragment or variant and a Iigand or binding partner. In such competitive binding assays the biomarker or fragment or variant is typically labelled. Free biomarker, fragment or variant thereof is separated from that present in a protein: protein complex and the amount of free (i.e. uncomplexed) label is a measure of the binding of the agent being tested to the biomarker or its interference with binding of the biomarker to a Iigand or binding partner, respectively.

Alternatively, an assay of the invention may measure the influence of the agent being tested on a biological activity of the biomarker. Thus, the present invention provides methods of screening for drugs comprising contacting such an agent with the biomarker of the invention or a fragment thereof or a variant thereof and assaying for the influence of such an agent on a biological activity of the biomarker, by methods well known in the art. In such activity assays the biological activity of the biomarker, fragment or variant thereof is typically monitored by provision of a reporter system. For example, this may involve provision of a natural or synthetic substrate that generates a detectable signal in proportion to the degree to which it is acted upon by the biological activity of the target molecule. it is contemplated that, once candidate therapeutic compounds have been elucidated, rational drug design methodologies well known in the art may be employed to enhance their efficacy. The goal of rational drug design is to produce structural analogues of biologically active polypeptides of interest or of small molecules with which they interact (e. g. agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, for example, enhance or interfere with the function of a polypeptide in vivo. In one approach, one first determines the three-dimensional structure of a protein of interest, such as the biomarker of the invention or, for example, of the biomarker in complex with a ligand, by x-ray crystallography, by computer modelling or most typically, by a combination of approaches. For example, the skilled artisan may use a variety of computer programmes which assist in the development of quantitative structure activity relationships (QSAR) that act as a guide in the design of novel, improved candidate therapeutic molecules. Less often, useful information regarding the structure of a

polypeptide may be gained by modelling based on the structure of homologous proteins. In addition, peptides can be analysed by alanine scanning (Wells, Methods Enzymol. 202: 390-411 , 1991), in which each amino acid residue of the peptide is sequentially replaced by an alanine residue, and its effect on the peptide's activity is determined in order to determine the important regions of the peptide. It is also possible to design drugs based on a pharmacophore derived from the crystal structure of a target-specific antibody selected by a functional assay. It is further possible to avoid the use of protein crystallography by generating anti-idiotypic antibodies to such a functional, target-specific antibody, which have the same three-dimensional conformation as the original biomarker. These anti-idiotypic antibodies can subsequently be used to identify and isolate peptides from libraries, which themselves act as pharmacophores for further use in rational drug design. For use as a medicament in vivo, candidate therapeutic compounds so identified may be combined with a suitable pharmaceutically acceptable carrier, such as physiological saline or one of the many other useful carriers well characterized in the medical art. Suitable dose ranges and cell toxicity levels may be assessed using standard dose ranging methodology. Dosages administered may vary depending, for example, on the nature of the malignancy, the age, weight and health of the individual, as well as other factors.

A further aspect of the present invention provides for animals which express the biomarker of the invention and can be used as model systems to study and test for substances which have potential as therapeutic agents.

Animals for testing candidate therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. As discussed in more detail below, by way of example, such treatments can include insertion of genes encoding the biomarker of the invention in wild-type or variant form, typically from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous biomarker gene(s) of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques that are well known in the art. After test substances have been administered to the animals, the development of ASVD can be assessed. If the test substance prevents or suppresses the development of ASVD, then the test substance is a candidate therapeutic agent for the treatment of individuals with ASVD who express the biomarker of the invention. These animal models provide an extremely important testing vehicle for potential therapeutic compounds.

Thus the present invention thus provides a transgenic non-human animal, particularly a rodent, which comprises an inactive copy of the gene encoding a biomarker of the present invention.

The invention further provides a method of testing a putative therapeutic of the invention which comprises administering said therapeutic to an animal according to the invention and determining the effect of the therapeutic. For the purposes of the present invention, it will be understood that reference to an inactive copy of the gene encoding a biomarker of the present invention includes any non-wild-type variant of the gene which results in knock out or down regulation of the gene. Thus the gene may be deleted in its entirety, or mutated such that the animal produces a truncated protein, for example by introduction of a stop codon and optionally upstream coding sequences into the open reading frame of the gene encoding a biomarker of the present invention. Equally, the open reading frame may be intact and the inactive copy of the gene provided by mutations in promoter regions.

Generally, inactivation of the gene may be made by targeted homologous recombination. Techniques for this are known as such in the art. This may be achieved in a variety of ways. A typical strategy is to use targeted homologous recombination to replace, modify or delete the wild-type gene in an embryonic stem (ES) ceil. A targeting vector comprising a modified target gene is introduced into ES ceils by electroporation, lipofection or microinjection. In a few ES cells, the targeting vector pairs with the cognate chromosomal DNA sequence and transfers the desired mutation carried by the vector into the genome by homologous recombination. Screening or enrichment procedures are used to identify the transfected cells, and a transfected cell is cloned and maintained as a pure population. Next, the altered ES ceils are injected into the blastocyst of a preimplantation mouse embryo or alternatively an aggregation chimera is prepared in which the ES cells are placed between two blastocysts which, with the ES ceils, merge to form a single chimeric blastocyst. The chimeric blastocyst is surgically transferred into the uterus of a foster mother where the development is allowed to progress to term. The resulting animal will be a chimera of norma! and donor ceils. Typically the donor cells will be from an animal with a clearly distinguishable phenotype such as skin colour, so that the chimeric progeny is easily identified. The progeny is then bred and its descendants cross-bred, giving rise to heterozygotes and homozygotes for the targeted mutation. The production of transgenic animals is described further by Capecchi, , R., 1989, Science 244; 1288-1292; Valancius and Smithies, 1991 , oi. Cell. Biol. 11 ; 1402-1408; and Hasty et al, 1991 , Nature 350; 243- 246, the disclosures of which are incorporated herein by reference.

Homologous recombination in gene targeting may be used to replace the wild-type gene encoding a biomarker of the present invention with a specifically defined mutant form (e.g. truncated or containing one or more substitutions).

The inactive gene may also be one in which its expression may be selectively blocked either permanently or temporarily. Permanent blocking may be achieved by supplying means to delete the gene in response to a signal. An example of such a means is the cre-!ox-¹ system where phage lox sites are provided at either end of the transgene, or at least between a sufficient portion thereof (e.g. in two exons located either side or one or more introns).

Expression of a ere recombinase causes excision and circuiarisation of the nuclei acid between the two lox sites. Various lines of transgenic animals, particularly mice, are currently available in the art which express ere recombinase in a developmentally or tissue restricted manner, see for example Tsien, Ceil, Vol.87(7): 1317-1326, (1996) and Betz, Current Biology, Vol.6(10): 307-1316 (1996). These animals may be crossed with lox transgenic animals of the invention to examine the function of the gene encoding a biomarker of the present invention. An alternative mechanism of control is to supply a promoter from a tetracycline resistance gene, tet, to the control regions of the target gene locus such that addition of tetracycline to a cell binds to the promoter and blocks expression of the gene encoding a biomarker of the present invention. Alternatively GAL4, VP16 and other transactivators could be used to modulate gene expression including that of a transgene containing the gene encoding a biomarker of the present invention. Furthermore, the target gene could also be expressed in ectopic sites, that is in sites where the gene is not normally expressed in time or space.

Transgenic targeting techniques may also be used to delete the gene encoding a biomarker of the present invention. Methods of targeted gene deletion are described by Brenner et ai, W094/21787 (Cell Genesys), the disclosure of which is incorporated herein by reference. In a further embodiment of the invention, there is provided a non-human animal which expresses the gene encoding a biomarker of the present invention at a higher than wild-type level. Preferably this means that the gene encoding a biomarker of the present invention is expressed at least 120-200% of the level found in wild-type animals of the same species, when cells which express the gene are compared. Also, this gene could be expressed in an ectopic location where the target gene is not normally expressed in time or space.

Comparisons may be conveniently done by northern blotting and quantification of the transcript level. The higher level of expression may be due to the presence of one or more, for example two or three, additional copies of the target gene or by modification to the gene encoding a biomarker of the present inventions to provide over-expression, for example by introduction of a strong promoter or enhancer in operable linkage with the wild-type gene. The provision of animals with additional copies of genes may be achieved using the techniques described herein for the provision of "knock-out" animals. In another aspect, animals are provided in which the gene encoding a biomarker of the present invention is expressed at an ectopic location. This means that the gene is expressed in a location or at a time during development which does not occur in a wild-type animal. For example, the gene may be linked to a developmentally regulated promoter such as Wnt-1 and others (Echeiand, Y. Et a!., Development 120, 2213 - 2224, 1998;

Rinkenberger, J.C, et a!,, Dev. Genet. 21 , 6-10, 1997, or a tissue specific promoter such as HoxB ( achonochie, M.K. et ai, Genes & Dev 1 1 , 1885-1895, 1997).

Non-human mammalian animals include non-human primates, rodents, rabbits, sheep, cattle, goats, pigs. Rodents include mice, rats, and guinea pigs. Amphibians include frogs. Fish such as zebra fish, may also be used. Transgenic non-human mammals of the invention may be used for experimental purposes in studying ASVD, and in the development of therapies designed to alleviate the symptoms or progression of ASVD. By "experimental" it is meant permissible for use in animal experimentation or testing purposes under prevailing legislation applicable to the research facility where such experimentation occurs.

All patents and literature references referred to herein are, unless otherwise specified, herein explicitly incorporated by reference.

Other features of the invention will be clear to the skilled artisan, and need not be repeated here. The terms and expressions employed herein are used as terms of description and not of limitation; there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

BIOMARKERS ASSOCIATED WITH ASVD

Through collecting samples from a cohort of individuals with ASVD the present inventors have identified a statistically significant increase in the local concentration of platelet-derived growth factor (PDGF) isoforms "AA" and "AB/BB".

PDGF

Platelet-derived growth factors (PDGFs) not only play an important role in

developmental and physiological processes, but also are directly implicated in human cancer and other proliferative disorders (reviewed in [11]). The human genome consists of four PDGF ligands, PDGF A-D, and two receptors, PDGFRa and PDGFR . Ail PDGF ligands can form functional disulfide-linked homodimers (i.e. AA, BB, CC, and DD) but only PDGF A and B have been shown to form functional heterodimers (i.e. AB). The detection methods used herein do not distinguish between the AB heterodimer and the BB homodimer, hence the "AB/BB" reference above)

The two PDGFRs are structurally related protein tyrosine kinase receptors which also function as homo- and hetero-dimers that differ in their affinities to different PDGF dimers (reviewed in [ 1]). The a subunit of PDGFR has been shown to bind the PDGF A, B and C chains, whereas the β subunit is believed to bind only the B and D chains. PDGF-CC specifically interacts with PDGFR-αα and -αβ, but not with -ββ, and thereby resembles PDGF-AB. PDGF-DD binds to PDGFR-ββ with high affinity, and to PDGFR-αβ to a markedly lower extent and is therefore regarded as PDGFR-ββ specific. PDGF-AA binds only to

PDGFR-αα, while PDGF-BB is the only PDGF that can bind all three receptor combinations with high affinity.

After dimerization, each PDGFR partner phosphorylates the other on specific tyrosine residues located on their cytosolic tails. This autophosphoryiation activates downstream signalling by 1) further boosting the kinase activity of the receptor through the additional phosphorylation of tyrosine residues within the kinase domain, and 2) creating high-affinity docking sites for the Src homology 2 (SH2) domain-containing adapter molecules

(eg, Grb2, Grb7, Crk, Nek, and She) that lack intrinsic enzymatic activity but serve as links to other downstream effectors. The biological responses induced by the different PDGF iigands depend on the relative numbers of the receptor subunits on a given cell type and the specific PDGF dimers present. Activation of PDGF receptors leads to stimulation of cell growth, but also to changes in cell shape and motility; PDGF induces reorganization of the actin filament system and stimulates chemotaxis, i.e., a directed cell movement toward a gradient of PDGF. in vivo, PDGF has important roles during the embryonic development as well as during wound healing.

Overactivity of PDGF has been implicated in several pathological conditions. The sis oncogene of simian sarcoma virus (SSV) is related to the B-chain of PDGF, and SSV transformation involves autocrine stimulation by a PDGF-iike molecule. Similarly,

overproduction of PDGF may be involved in autocrine and paracrine growth stimulation of human tumours. Overactivity of PDGF has, in addition, been implicated in non-malignant conditions characterized by an increased cell proliferation such as fibrotic conditions. PDGF in ASVD and CAD

The importance of PDGF as a mitogen in angiogenesis has led to suggestions that it, amongst an array of other possible molecules, may have a role in atherosclerosis. For example, studies have indicated the localization of PDGF-B to atherosclerotic plaques [15], whilst others have reported that PDGF is released into the coronary circulation of patients with unstable angina [16].

However, the role for PDGF in atherosclerosis, and in particular CAD, is unclear. PDGF-A and PDGF-B are found in both atherosclerotic and normal arterial tissue, suggesting that it has a maintenance (as opposed to pathogenic) function in vascular tissue [17]. In addition, other studies in which the levels of PDGF in the cardiac arteries of patients with CAD has been monitored have not reported any elevation in PDGF levels [18].

Accordingly, up until the work presented herein, there has been no clear evidence showing a local increase in PDGF concentration in the coronary arteries of patients with CAD. The findings of the present inventors are the first reports based on measurements made within the key proximal regions of the cardiac arteries, thereby establishing PDGF as one of the molecules released in this crucial section of artery.

"One or more pre-specified biomarkers"

in view of the above and of the findings of the present inventors, the terms "one or more pre- specified biomarkers" or "PDGF" as used herein is used to mean a molecule selected from the group consisting of PDGF-A, -B, -C, -D, -AA, -AB, -BB, -CC, and -DD. In preferred embodiments the terms "one or more pre-specified biomarkers" or "PDGF" is used to mean PDGF-AA. In other preferred embodiments the term "one or more pre-specified biomarkers" or "PDGF" is used to mean PDGF-AB or -BB, The sequences and database references for the human forms are set out in the "Sequences" section.

Species variants are also encompassed by this invention where the individual is a non- human mammal, as are allelic or other variants of the proteins described in the "Sequences" section below, and any reference to the proteins in that section will be understood to embrace, alleles, homoiogues or other naturally occurring variants.

Thus included within the definition of the biomarkers of the invention are amino acid variants of the naturally occurring sequence as provided in any of SEQ ID NOs: 1-9. Preferably, variant sequences are at least 75% homologous to the wild-type sequence, more preferably at least 80% homologous, even more preferably at least 85% homologous, yet more preferably at least 90% homologous or most preferably at least 95% homologous to at least a portion of the reference sequence supplied (SEQ ID NOs: 1-9). In some embodiments the homology will be as high as 94 to 96 or 98%. Homology in this context means sequence similarity or identity, with identity being preferred. To determine whether a candidate peptide region has the requisite percentage similarity or identity to a reference polypeptide or peptide oligomer, the candidate amino acid sequence and the reference amino acid sequence are first aligned using a standard computer programme such as are commercially available and widely used by those skilled in the art. In a preferred embodiment the NCB! BLAST method is used (http://mvw.ncbi.nlm.nih.gov/BLAST/). Once the two sequences have been aligned, a percent similarity score may be calculated. In all instances, variants of the naturally- occurring sequence, as detailed in SEQ ID NO: 1-9 herein, must be confirmed for their function as biomarker proteins. Specifically, their presence or absence in a particular form or in a particular biological compartment must be indicative of the presence or absence of ASVD in an individual. This routine experimentation can be carried out by using standard methods known in the art in the light of the disclosure herein.

Thus, the present invention provides a method used for assessing the likelihood of a major cardiac event, which method comprises assessing the concentration of PDGF local to a vascular lesion or suspected vascular lesion, wherein the concentration of PDGF

downstream of the vascular lesion that is at least 125%, 150% or 200% of the concentration upstream of the vascular lesion or suspected vascular lesion, or at least 125%, 150% or 200% of the systemic concentration, is associated with a higher risk of a major cardiac event, in some embodiments the vascular lesion is situated within 60mm of the LCx artery ostium, within 80mm of the LAD artery ostium, or within 130mm of the RCA ostium. In some embodiments the PDGF concentration local to the vascular lesion is assessed by comparing the PDGF concentration at a point downstream of the vascular lesion with the PDGF concentration at a point upstream of the vascular lesion, wherein both upstream and downstream points are within the coronary artery (for example, are both downstream of the LCx, LAD, or RCA ostium).

"protein associated with the one or more pre-specified biomarker(s)"

in view of the above and of the findings of the present inventors, the term "protein associated with the one or more pre-specified biomarker(s)" encompasses the PDGF receptor

(PDGFR). "PDGFR" as used herein is used to mean PDGFR-a, -β, -aa, -αβ, and -ββ. The sequences and database references for the human forms are set out in the "Sequences" section. Species variants are also encompassed by this invention where the individual is a non- human mammal, as are allelic or other variants of the proteins described in the "Sequences" section below, and any reference to the proteins in that section will be understood to embrace, alleles, homologues or other naturally occurring variants. Thus included within the definition of the "proteins associated with the one or more pre- specified biomarker(s)" and "PDGFR" are amino acid variants of the naturally occurring sequence as provided in any of SEQ ID NOs: 10-13. Preferably, variant sequences are at least 75% homologous to the wild-type sequence, more preferably at least 80%

homologous, even more preferably at least 85% homologous, yet more preferably at least 90% homologous or most preferably at least 95% homologous to at least a portion of the reference sequence supplied (SEQ ID NOs: 10-13). in some embodiments the homology will be as high as 94 to 98 or 98%. Homology in this context means sequence similarity or identity, with identity being preferred. To determine whether a candidate peptide region has the requisite percentage similarity or identity to a reference polypeptide or peptide oligomer, the candidate amino acid sequence and the reference amino acid sequence are first aligned using a standard computer programme such as are commercially available and widely used by those skilled in the art. In a preferred embodiment the NCBi BLAST method is used (http://www.ncbi. nlm.nih.gov/BLAST/). Once the two sequences have been aligned, a percent similarity score may be calculated, in ail instances, variants of the naturaliy- occurring sequence, as detailed in SEQ ID NO: 10-13 herein, must be confirmed for their function as biomarker proteins. Specifically, their presence or absence in a particular form or in a particular biological compartment must be indicative of the presence or absence of ASVD in an individual. This routine experimentation can be carried out by using standard methods known in the art in the light of the disclosure herein. Treatments of ASVD

Thus, as used herein, the terms "treatments of ASVD" and/or "drugs for treating ASVD" encompass PDGF antagonists such as JI-101 , Orantinib (also known as TSU-68; SU6668), Vatalanib, Pazopanib/ Votrient, Motesanib / AMG 708, Trapidil (triazolopyrimidine), Axitinib (AG013736) Inlyta, AMG 706 (motesanib), BIBF 1 120 (nintedanib), Sorafenib / Nexavar, X- 82, Sunitinib / Sutent / SU1 1248, Crenolanib / CP-868,596; ARO 002, niiofinib, Imatinib / Gleevec / Glivec, Olaratumab (IMC-3G3, see [19]), MEDI-575, Fovista E10030, Vinpocetine, BIBF-1120, Tandutinib, Piperlongumine, and Eupatolide as treatments of <4SVD.

Also encompassed by the terms "treatments of ASVD" and/or "drugs for treating ASVD" is an antibody or other binding moiety which specifically binds to PDGF, In preferred embodiments the binding of the antibody or other binding moiety reduces or inhibits the ability of the PDGF to activate the PDGFR; in some embodiments the antibody or other binding moiety reduces or inhibits the ability of PDGF to bind to and/or activate PDGFR (i.e. if increases the

PDGF/PDGFR disassociation constant by, for example, at least a factor of 2, 5, 10, 100, 1000 or 10000). in some embodiments a protein associated with PDGF is the target per se of the drug. For example, the protein associated with PDGF may be a receptor (i.e. PDGFR) which PDGF binds to elicit its biological effect (the cross-phosphorylation of PDGFR, and activation of its downstream signalling cascade). Thus, the drug may comprise an antibody or other binding moiety which specifically binds to, for example, PDGFR. in preferred embodiments the binding of the antibody or other binding moiety reduces or inhibits the ability of PDGFR to be bound and/or activated by PDGF. In some embodiments the antibody or other binding moiety reduces or inhibits the ability of PDGFR to be bound and/or activated by PDGF (i.e. it increases the PDGFR/PDGF disassociation constant by, for example, at least a factor of 2, 5, 10, 100, 1000 or 10000). in one embodiment the "drug for treating ASVD" is an antibody as defined in EP2049571 B1 (herein termed " EDI-575"). For example, the "drug for treating ASVD" may be an antibody as defined by the claims as granted of EP2049571 B1 , such as antibody which comprises a heavy chain polypeptide comprising the sequence of SEQ ID NO.: 10 as defined in EP2049571 B1 and a light chain polypeptide comprising the sequence of SEQ ID NO.: 12 as defined in EP2049571 B1.

In one embodiment the "drug for treating ASVD" is an antibody as defined in EP2505205A1 (herein termed "IMC-3G3"). For example, the "drug for treating ASVD" may be an antibody as defined by the claims as published of EP2505205A1 , such as antibody which comprises a heavy chain variable region comprising the sequence of SEQ ID NO.: 8 as defined in EP2505205A1 and a light chain variable region comprising the sequence of SEQ ID NO.: 16 as defined in EP2505205A1. In some embodiments the drug for treating ASVD is the antibody known as LY3012207 / Olaratumab (see [19])

The present invention also provides a method of selecting individuals for treatment with MEDI-575 which method comprises assessing a local concentration of one or more pre- specified biomarkers

The present invention also provides a method of selecting or stratifying individuals for a clinical trial with MEDi-575 which method comprises assessing a local concentration of one or more pre-specified biomarkers

The present invention also provides a method of selecting MEDi-575 treatment for an individual, which method comprises assessing a local concentration of one or more pre- specified biomarkers

wherein the treatment is selected if the individual has a measure exceeding the threshold level, and wherein the treatment inhibits or reduces one or more aspect of ASVD, The present invention also provides a method of selecting individuals for treatment with IMC- 3G3 which method comprises assessing a local concentration of one or more pre-specified biomarkers

wherein individuals having a measure exceeding the threshold level are selected for treatment, and wherein the treatment inhibits or reduces one or more aspect of ASVD. The present invention also provides a method of selecting or stratifying individuals for a clinical trial with a I C-3G3 which method comprises assessing a local concentration of one or more pre-specified biomarkers

The present invention also provides a method of selecting I C-3G3 treatment for an individual, which method comprises assessing a local concentration of one or more pre- specified biomarkers

wherein the treatment is selected if the individual has a measure exceeding the threshold level, and wherein the treatment inhibits or reduces one or more aspect of ASVD,

As ASVD progresses from fatty streaks on vessel walls, through intermediate lesions, atheroma, and fibroatheroma to complicated lesions and vulnerable plaques, the profile of biomarkers released by the lesion / plaque is expected to change, as is the type of treatment which is required / most effective.

Accordingly, the present invention also provides a method of timing the application of treatment of an individual with IMC-3G3, which method comprises assessing a local concentration of one or more pre-specified biomarkers

Accordingly, the present invention also provides a method of timing the application of treatment of an individual with a MEDI-575, which method comprises assessing a local concentration of one or more pre-specified biomarkers

PDGF-A

Platelet-derived growth factor subunit A

also known as PDGF-1 , Platelet-derived growth factor A chain, Platelet- derived growth factor alpha polypeptide

P04085

SEQ lD NO:1 ->

MRTLACLLLLGCGYLAHVLAEEAEIPREVIERLARSQIHSIRDLQRLLEIDSVG SEDSLDTSLRAHGVHATKHVPEKRPLPIRRKRSIEEAVPAVCKTRTVIYEIPR SQVDPTSANFLIWPPCVEVKRCTGCCNTSSVKCQPSRVHHRSVKVAKVEY

VRKKPKLKEVQVRLEEHLECACATTSLNPDYREEDTGRPRESGKKRKRKRL KPT (long isoform) SEQ ID NO:2 -

MRTLACLLLLGCGYLAHVLAEEAEIPREVIERLARSQIHSIRDLQRLLEIDSVG SEDSLDTSLRAHGVHATKHVPEKRPLPIRRKRSIEEAVPAVCKTRTVIYEIPR SQVDPTSANFLIWPPCVEVKRCTGCCNTSSVKCQPSRVHHRSVKVAKVEY VRKKPKLKEVQVRLEEHLECACATTSLNPDYREEDTDVR (short isoform) rUUr-D

Name: Platelet-derived growth factor subunit B

also known as PDGF-2, SIS

Uniprot ref: P01127

Sequence:

SEQ ID NO:3 ->

NRCWALFLSLCCYLRLVSAEGDPIPEELYE LSDHSIRSFDDLQRLLHGDP

GEEDGAELDLN TRSHSGGELESLARGRRSLGSLTIAEPAMIAECKTRTEV

FEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQV RKIEIVRKKPIFKKATVTLEDHLACKCETVAAARPVTRSPGGSQEQRAKTPQ TRVTIRTVRVRRPPKGKHRKFKHTHDKTALKETLGA (long isoform)

SEQ ID NO:4 ->

MFIMGLGDPIPEELYEMLSDHSIRSFDDLQRLLHGDPGEEDGAELDLNMTR SHSGGELESLARGRRSLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFL VWPPCVEVQRCSGCCIMNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATV TLEDHLACKCETVAAARPVTRSPGGSQEQRAKTPQTRVTIRTVRVRRPPKG KHRKFKHTHDKTALKETLGA (short isoform)

PDGF-C

Name: Platelet-derived growth factor subunit C

also known as Faliotein, Spinal cord-derived growth factor (SCDGF), VEGF-E Uniprot ref: PQ9NRA1

Sequence:

SEQ ID NO:5 ->

MSLFGLLLLTSALAGQRQGTQAESNLSSKFQFSSNKEQNGVQDPQHERIIT VSTNGSIHSPRFPHTYPRNTVLVWRLVAVEENVWIQLTFDERFGLEDPEDDI CKYDFVEVEEPSDGTILGRWCGSGTVPGKQISKGNQIRIRFVSDEYFPSEP GFCIHYNIVMPQFTEAVSPSVLPPSALPLDLLNNAITAFSTLEDLIRYLEPERW QLDLEDLYRPTWQLLGKAFVFGRKSRVVDLNLLTEEVRLYSCTPRNFSVSIR EELKRTDTIFWPGCLLVKRCGGNCACCLHNCNECQCVPSKVTKKYHEVLQL RPKTGVRGLHKSLTDVALEHHEECDCVCRGSTGG (isoform 1)

SEQ ID NO:6

SLFGLLLLTSALAGQRQGTQAESNLSSKFQFSSNKEQNGVQDPQHERIIT VSTNGSIHSPRFPHTYPRNTVLVWRLVAVEENVWIQLTFDERFGLEDPEDDI CKYDFVEVEEPSDGTILGRWCGSGTVPGKQISKGNQIRIRFVSDEYFPSEP GFCIHYNIVMPQFTEAVSPSVLPPSALPLDLLNNAITAFSTLEDLIRYLEPERW

QLDLEDLYRPTWQLLGKAFVFGRKSRWDLNLLTEEVLQLRPKTGVRGLHK SLTDVALEHHEECDCVCRGSTGG (Isoform 2)

SEQ ID NO:7 ->

SLFGLLLLTSALAGQRQGTQAESNLSSKFQFSSNKEQNGVQDPQHERIIT VSTNGSIHSPRFPHTYPRNTVLVWRLVAVEENVWIQLTFDERFGLEDPEDDI

CKYDFVEVEEPSDGTILGRWCGSGTVPGKQISKGNQIRIRFVSDEYFPSEPS NRGGKiiQLHTS (Isoform 3)

PDGF-D

Name: Platelet-derived growth factor subunit D

also known as Iris-expressed growth factor, Spinal cord-derived growth factor

B (SCDGF-B)

Uniprot ref: Q9GZP0

Sequence: SEQ ID NO:8 ->

MHRLIFVYTLICANFCSCRDTSATPQSASIKALRNANLRRDESNHLTDLYRR

DETIQVKGNGYVQSPRFPNSYPRNLLLTWRLHSQENTR!QLVFDNQFGLEE

AEND!CRYDFVEVEDISETSTI!RGRWCGHKEVPPRJKSRTNQIKITFKSDDYF

VA PGFKIYYSLLEDFQPAAASETNWESVTSSISGVSYNSPSVTDPTLIADAL

DKKIAEFDTVEDLLKYFNPESWQEDLEN YLDTPRYRGRSYHDRKSKVDLD

RLNDDAKRYSCTPRNYSVN!REELKLANVVFFPRCLLVQRCGGNCGCGTVN

WRSCTCNSGKTVKKYHEVLQFEPGHIKRRGRAKTMALVD!QLDHHERCDCI

CSSRPPR (isoform 1)

SEQ ID NO:9

MHRLIFVYTLICANFCSCRDTSATPQSASIKALRNANLRRDDLYRRDETIQVK

GNGYVQSPRFPNSYPRNLLLTWRLHSQENTRIQLVFDNQFGLEEAENDICR YDFVEVEDiSETSTilRGRWCGHKEVPPRiKSRTNQIKITFKSDDYFVAKPGF KIYYSLLEDFQPAAASETNWESVTSS!SGVSYNSPSVTDPTLiADALDKKIAE FDTVEDLLKYFNPESWQEDLEN YLDTPRYRGRSYHDRKSKVDLDRLNDD AKRYSCTPRNYSVNIREELKLANWFFPRCLLVQRCGGNCGCGTVNWRSC TCNSGKTVKKYHEVLQFEPGHIKRRGRAKTMALVDIQLDHHERCDCICSS RPPR (Isoform 2)

PDGFR-g

Name: Platelet-derived growth factor receptor alpha (a)

Uniprot ref: P16234

Sequence:

SEQ !D NO:10 -

MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENEKWQLNSSFSLRCFGES EVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNH TQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPET PVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQT!PFNVYALK ATSELDLEMEALKTVYKSGETIWTCAVFNNEVVDLQWTYPGEVKGKGITML EEIKVPSIKLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGF IEIKPTFSQLEAVNLHEVKHFWEVRAYPPPRISWLKNNLTLIENLTEITTDVE KIQEI RYRSKLKLI RAKEEDSGHYTI VAQN EDAVKSYTFELLTQVPSSI LDLVD DHHGSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNNETSWTILANNVSNIITE IHSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRELKLVAPTLRSELTVA AAVLVLLVIVilSLIVLWIWKQKPRYEIRWRVIESISPDGHEYiYVDPMQLPYD SRWEFPRDGLVLGRVLGSGAFGKWEGTAYGLSRSQPVMKVAVKMLKPTA RSSEKQAL SELKI THLGPHLNiVNLLGACTKSGPIYHTEYCFYGDLVNYLH

KNRDSFLSHHPEKPKKELDIFGLNPADESTRSYVILSFENNGDYMDMKQAD

TTQYVPMLERKEVSKYSDIQRSLYDRPASYKKKS LDSEVKNLLSDDNSEG

LTLLDLLSFTYQVARG EFLASKNCVHRDLAARNVLLAQGKIVK!CDFGLA

RDIMHDSNYVSKGSTFLPVKWMAPESIFDNLYTTLSDVWSYGILLWEIFSLG

GTPYPGMMVDSTFYNKIKSGYRMAKPDHATSEVYEIMVKCW SEPEKRPS

FYHLSEIVENLLPGQYKKSYEK!HLDFLKSDHPAVAR RVDSDNAYIGVTYK

NEEDKLKDWEGGLDEQRLSADSGYIIPLPDIDPVPEEEDLGKRNRHSSQTS

EESAIETGSSSSTFIKREDET!EDIDM DD!GIDSSDLVEDSFL (Isoform 1)

SEG ID NO:11 ->

MGTSHPAFLVLGCLLTGLSL!LCQLSLPS!LPNENEKWQLNSSFSLRCFGES EVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNH TQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPET

PVTLHNSEGVVPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALK GTCHSFLL (!soform 2)

SEQ !D NG:12 - GTSHPAFLVLGCLLTGLSL!LCQLSLPS!LPNENEKWQLNSSFSLRCFGES

EVSWQYPMSEEESSDVEIRNEENNSGLFVTVLEVSSASAAHTGLYTCYYNH

TQTEENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDDSAIIPCRTTDPET

PVTLHNSEGWPASYDSRQGFNGTFTVGPYICEATVKGKKFQTIPFNVYALK

ATSELDLEMEALKTVYKSGETIVVTCAVFNNEVVDLQVVTYPGEVKGKGIT L

EE!KVPS!KLVYTLTVPEATVKDSGDYECAARQATREVKEMKKVTISVHEKGF lEIKPTFSQLEAVNLHEVKHFWEVRAYPPPRISWLKNNLTLIENLTEITTDVE

KIQEIRYRSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFELLTQVPSSILDLVD

DHHGSTGGQTVRCTAEGTPLPDIEWMICKDIKKCNNETSWriLANNVSNIITE

IHSRDRSTVEGRVTFAKVEETIAVRCLAKNLLGAENRELKLVAPTLRSELTVA

AAVLVLLVIVIISLIVLWIWKQKPRYEIRWRVIESISPDGHEYIYVDPMQLPYD

SRWEFPRDGLVLGRVLGSGAFGKVVEGTAYGLSRSQPVMKVAVKMLKPTA

RSSEKQALMSELKIMTHLGPHLNIVNLLGACTKSGPIYIITEYCFYGDLVNYLH

KNRDSFLSHHPEKPKKELD!FGLNPADESTRSSGQGCLSSGTLQELSVDLQ

ARGPC (Isoform 3)

PDGFR-3

Name: Platelet-derived growth factor receptor beta (β)

Uniprot ref: P09619 SEQ !D NG:13 - RLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLT

CSGSAPWWER SQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHN

DSRGLETDERKRLYIFVPDPTVGFLPNDAEELFiFLTEITEITIPCRVTDPQLVV

TLHEKKGDVALPVPYDHQRGFSGIFEDRSYICKTTIGDREVDSDAYYVYRLQ

VSS!NVSVNAVQTWRQGENITLMCIViGNEVVNFEWTYPRKESGRLVEPVT

DFLLD PYH!RSILHIPSAELEDSGTYTCNVTESVNDHQDEKA!N!TVVESGY

VRLLGEVGTLQFAELHRSRTLQWFEAYPPPTVLWFKDNRTLGDSSAGEIAL

STRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVR

VLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNS

SEEESQLETNVTYWEEEQEFEWSTLRLQHVDRPLSVRCTLRNAVGQDTQ

EVI WPHSLPFKVWISAI LALVVLTI ISLI I L! M LWQKKPRYEI RWKVI ESVSSDG

HEYIYVDPMQLPYDSTWELPRDQLVLGRTLGSGAFGQWEATAHGLSHSQ

ATMKVAVK LKSTARSSEKQAL SELKI SHLGPHLNWNLLGACTKGGPi

YHTEYCRYGDLVDYLHRNKHTFLQHHSDKRRPPSAELYSNALPVGLPLPSH

VSLTGESDGGYMDMSKDESVDYVPMLDMKGDVKYADIESSNYMAPYDNY

VPSAPERTCRATLINESPVLSYMDLVGFSYQVANGMEFLASKNCVHRDLAA

RNVL!CEGKLVK!CDFGLARD! RDSNYISKGSTFLPLKWMAPES!FNSLYTT

LSDVWSFGILLWEIFTLGGTPYPELPMNEQFYNAIKRGYRMAQPAHASDEIY

EI Q CWEE FEIRPPFSQLVLLLERLLGEGYKKKYQQVDEEFLRSDHPAiL

RSQARLPGFHGLRSPLDTSSVLYTAVQPNEGDNDYIIPLPDPKPEVADEGPL

EGSPSLASSTLNEVNTSST!SCDSPLEPQDEPEPEPQLELQVEPEPELEQLP

DSGCPAPRAEAEDSFL

[SEQ ID NO:10 of EP2049571B1]

¾1» ¥&X G is L«« ¥§ Qlu Sly Sly Sly ¥al Lys o Sly Sly

1 IS

>s Ala &1¾ S® Sly S^> ® Thr P « Ssr

25 $0 l?yr .&&ft ^s XI® Argr <S1« Ai S y I»ys Sl l&v l8 fsip v¾i

45

S*r fys Il« S« S*3i iSly S® lis lie Tye ¾r Ala A&p Ss ¥&i so 5s m

&c tog

6S S 80 sis $ z sn $er Xs®« A¾g S » As ¾h &Xa V l Siy 3¾¾ Cys

8S SO SS

JU.& A g Sis Sly A ^ 11* Ala Ala to Sly &fe Asp Val Sly Sis*

105 119

§ y h£ h ¥al T¾x ^&l S¾

[SEQ ID NO:12 of EP2049571B1]

As II® Q t\ t h Sin Sa¾* Fro S®r Le S& a S«

X S 10

Arg Val S« II» t!te C s tog

30

T g> ¾S1» QXn &ya ro Sly I*ys la Fro &ys l^u tea Xl¾ 35 45

Ala Sax v&l Sly ¾y val o S®« tof Sax? Sly

55 66

S«s*r Sly Sear Sly ϊ he &&p

65 70 75 fjg- Cys S « GX« hs? ¾?y« S<¾« Asa Ss?<$

85 $0 95

Sly Sla Sty Th to¾ si¾

too X^Q5 [SEQ ID NO:8 of EP2505205A1] -»

QLQ¾l»eL¥»^

[SEQ ID NO:16 of EP2505205A1]

I!¥O ¾TLS:»

CIFAlf SCISC!SC!W

58 Figures

Figure 1 Release of biomarkers from a plaque within an artery and their collection by a catheter. Four samples are taken simultaneously from within a blood vessel. The collected samples are then extracted from the device and are tested for a variety of different biomarkers that may be released within the vessel.

The principle of the system is that if a statistically significant increase in biomarker concentration (a "gradient"; see Figure 2) is seen along the artery in the direction of flow, then this is evidence that that particular biomarker is released within the artery and hence potentially has a role in coronary artery disease.

Figure 1A = full sheathing, Figure 1 B = short sheathing.

An example of a theoretical "true gradient". NP = Normalisation port, PP = proximal port, MP = mid port, DP = Distal Port and VS = Venous sample.

Figure 2A = full sheathing, Figure 2B = short sheathing.

A graph displaying the venous concentration of PDGF-AA plotted against individual patient in the 'First-in-rnan clinical trial'. Individuals having a gradient of PDGF-AA in their coronary artery are indicated by an arrow.

A graph displaying the venous concentration of PDGF-AA plotted against individual patient in the 'First-in-man clinical trial'. Individuals having a gradient of PDGF-AA in their coronary artery are indicated by an arrow.

Examples

Example 1 : First-in-IVIan c nical trial

Data collection

Samples were collected from the coronary artery of each of 30 individuals using the muitiport catheter disclosed in WO2009/090390 (see Figure 1). The device was used in two different modes, "Full sheathing" (where four samples were collected, one from each of the four ports; Figure 1A) and "Short sheathing" (where three samples were collected, one from each of the three most distal ports; Figure 1 B). Short sheathing was used in relatively tortuous arteries where the full length of the sampling region could not be inserted within the coronary artery, therefore the sampling zone of the device cannot be fully unsheathed and hence the most upstream port does not collect a sample.

For full sheathing, 10 samples were analysed for each patient, the samples are organised as follows:

· Two samples from the Normalisation port

Two samples from the Proximal port

Two samples from the Mid port

Two samples from the Distal port

Two samples from the Peripheral sample.

For short sheathing, 8 samples were analysed for each patient, the samples are organised as follows:

Two samples from the Proximal port

Two samples from the Mid port

· Two samples from the Distal port

Two samples from the Peripheral sample.

The samples were then analysed using a Luminex platform a number of kits, including:

The adhesion panel kit from R&D Systems (4-piex polystyrene bead kit) · The Cytokine panel kit (41 -plex magnetic bead kit)

Ail kits were used according to the manufacturers' instructions and all samples were run in duplicate. The following dilutions were used for the plasma

The adhesion panel kit - 1 in 30

The 41-plex Cytokine - neat plasma with an overnight incubation step Four out of the thirty individual samples could not be analysed using the selected mathematical model: three of these individuals had only three samples collected instead of four (due to obstruction of one of the assessing ports by the artery wall) and one of the samples failed during the 41-piex multiple cytokine assay process.

Data Analysis: detection of local release

Three or four samples (depending on sheathing) were simultaneously collected from within the coronary artery. The collected samples were then extracted from the device and tested for a variety of different biomarkers.

The concentration of specific biomarkers can be plotted as shown in Figures 2A and 2B and the relative levels of biomarker concentrations along the artery can be compared to each other and to a sample collected from the circulation outside the coronary artery.

The principle of the system is that if a statistically significant increase in biomarker concentration (a "gradient") is seen along the artery in the direction of flow, then this is evidence that that particular biomarker is released within the artery and hence potentially has a role in coronary artery disease.

The peripheral sample was either a venous sample collected from the cubital vein of the patient (patients 1 to 19) or an arterial sample (patients 20 to 30) collected from an artery peripheral to the coronary artery (i.e. the introducer sheath in either the radial artery (wrist) or the femoral artery (groin)),

For short sheathing, i.e. in those tortuous arteries where the full 1 1.5 cm sampling portion could not be inserted, only three samples were collected by the device, in these samples the NP port does not collect a sample and the PP sample is therefore used as the reference sample against which a distal increase in concentration can be measured.

Data Analysis: local biomarker release

Data from the individuals was analysed with a view to identifying local release of biomarkers from plaques as evidenced by the presence of biomarker concentration gradients in the blood flow immediately downstream of the plaque.

The analytical technique used was to develop a mathematical definition of what a "true" gradient would look like (the hypothesis - i.e. data suggesting local release) and what a "false" gradient would look like (the null hypothesis - i.e. data suggesting that any variation between the samples was random and not due to local release). It could then be determined if the evidence that indicated "true gradients" (i.e. local release) was statistically more significant than data that indicated the opposite (i.e. a "false gradient").

The mathematical definition relied on the basic principle of the technology that only increases indicate local release and any increase should be consistent, i.e. the concentration should not decrease significantly along the artery. A significant decrease in concentration along the artery is incompatible with the principle of the device and is evidence of random variation.

For "full sheathing" a "true" gradient, there had to be a net significant increase along the length of the artery. A false gradient was defined as the opposite. Significance was defined as two times the Root Mean Standard Error (RMSE) as calculated from the sample replicates. The relative frequencies of the true and false gradients could then be compared to determine the strength of the evidence for local release through the detection of true positive gradients. If there was no local release and all variation was random then the incidence of true and false gradients should be the same. A significant increase was defined as where:

DP > PS + z*SE, and

NP > PS - z*SE, and

PP > NP - z*SE, and

MP > NP - z*SE, and

DP > NP + z*SE, and

NOT: PP > NP + z*SE and MP < NP + _Z*SE

A significant decrease was defined where:

DP < PS - z*SE, and

NP< PS + z*SE, and

PP < NP + z*SE, and

MP < NP + z*SE, and

DP < NP ~- z*SE, and

NOT: PP < NP - z*SE and MP > NP - z^*SE

The relative frequencies of the increases and decreases were then compared to determine the strength of the evidence of local release through the detection of true positive gradients, A similar approach was taken with the samples collected by the short sheathing procedures, but it was modified to allow for the fact that only three samples were collected by the device and the "Proximal Port" sample was used as the reference sample as no sample was collected from the Normalisation Port.

A significant increase was defined for short sheathing where:

DP > PS + z*SE, and

PP > PS - z*SE, and

MP > PP - z*SE, and

DP > PP + z*SE

A significant decrease was defined for short sheathing where:

DP < PS - z*SE, and

PP < PS + z*SE, and

MP < PP + z*SE, and

DP < PP - z*SE

RESULTS

Data quality

Four out of the thirty patient samples could not be analysed using the selected mathematical model. Three of these patients (5, 8 and 12) had only three samples collected instead of four. This was due to obstruction of one of the sampling ports by the artery wall preventing blood collection. One of the samples from Patient 11 failed during the 41-piex multiple cytokine assay process and so patient 11 "s cytokine data was not analysed. Ail other patient samples were successfully analysed.

Preliminary results

initial results indicated that the molecules PDGF-AB/BB and PDGF-AA showed significant positive gradients. (NB. PDGF comes in several different isoforms, as the assay cannot differentiate between the PDGF AB and PDGF BB form; hence, the result is defined as "PDGF AB/BB" by the manufacturer.) Intra-coronary artery gradients

For each of the PDGF molecules the percentage of patients with intra-coronary gradients of +/- 2*RMSE was measured (Table 1). Biomarker Sheathing of Patients Patients that showed Patients that collection in group significant Positive showed significant catheter biomarker elevations Negative biomarker

(at Z = 2) elevations (at Z = 2)

PDGF Full 11 45% 0

AB/BB (patient# 10, 15, 22,

23, 25)

Short 15 0

53%

(patient# 6, 7, 13, 16,

20, 27, 28 and 30)

PDGF AA Full 11 0

18%

(patient* 22, 25)

Short 15 0

40%

(patient* 6, 7, 13, 20,

27 and 28)

Table 1 : Intra-coronary artery PDGF AA and/or PDGF AB/BB gradient s

For PDGF AB/BB 50% of patients (5 of 1 1 full sheathings and 8 of 15 short sheathings) had positive gradients. No patients showed any negative gradients.

For PDGF-AA, 31 % of patients (2 of 1 1 full sheathings and 6 of 15 short sheathings) had positive gradients. This data supports the hypothesis that the increased intra-coronary artery concentration is due to local biomarker release within the coronary artery.

Analysis of differences between single locations in the coronary artery and peripheral samples

Biomarkers that showed significant intra-coronary gradients (whether in normal or short sheathing collection modes) were then analysed to see if the individual coronary samples were different to the samples collected outside of the coronary artery.

For this analysis firstly the most upstream sample (i.e. the sample that was used for the reference for the intra-coronary gradients) was compared to the sample collected from a location peripheral to the coronary artery (Table 2). Biomarker Sheathing of patients % of patients with % of patients with collection in group upstream biomarker upstream biomarker catheter greater than smaller than

peripheral fat Z = 4} peripheral (at Z = 4)

PDGF Full 11 82% 0

AB/BB Short 15 67% 0

PDGF AA Full 11 82% 0

Short 15 73% 0

Table 2: Differences in PDGF AA and/or PDGF AB/BB concentrations between the most upstream sample taken from within the coronary artery (i.e. nearest the ostium) and a peripheral sample.

Secondly, the sample collected most distally within the coronary artery (i.e. that collected by the Distal Port - 7) was compared to the sample collected from a location peripheral to the coronary artery (Table 3).

Table 3: Differences in PDGF AA and/or PDGF AB/BB concentrations between the most downstream sample taken from within the coronary artery (i.e. furthest from the ostium) and a peripheral sample.

Through this analysis it was determined if levels of the biomarker upstream and downstream of the coronary artery were higher than those found in vessels peripheral to the coronary system.

The results show that the coronary artery has higher concentrations of PDGF AB/BB and PDGF AA than that shown by peripheral samples for a significant number of CVD patients. This concentration is shown both just within the opening of the coronary artery (i.e. near the ostium) and also at a point 7 to 12cm distal to the opening of the coronary artery. The proportion of individuals who had coronary artery levels of PDGF (AA, AB, or BB) which were significantly higher than a peripheral sample (see Tables 2 and 3) was notably higher than the proportion of individuals who had intra-coronary artery gradients of PDGF (AA, AB, or BB - see Table 1).

The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross- reference.

References

1 , Zaman, A.G., et aL, The role of plaque rupture and thrombosis in coronary artery disease. Atherosclerosis, 2000. 149(2): p. 251-66.

2. ach, F., U. Schonbeck, and P. Libby, CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis, 1998. 137 Suppl: p. S89-95.

3. Kinlay, S., et ai., Inflammation, the endothelium, and the acute coronary syndromes. J Cardiovasc Pharmacol, 1998. 32 Suppl 3: p. S62-6.

4, Blankenberg, S., S. Barbaux, and L. Tiret, Adhesion molecules and atherosclerosis. Atherosclerosis, 2003. 170(2): p. 191-203. 5. Ikonomidis, I., et ai., Inflammatory and non-invasive vascular markers: the muitimarker approach for risk stratification in coronary artery disease. Atherosclerosis, 2008. 199(1): p. 3-1 1.

6. Doliery, C ., et a!., Neutrophil elastase in human atherosclerotic plaques:

production by macrophages. Circulation, 2003. 107(22): p. 2829-36.

7. Miwa, K., A. Igawa, and H. Inoue, Soluble E-seiectin, ICAM-1 and VCAM-1 levels in systemic and coronary circulation in individuals with variant angina. Cardiovasc Res, 1997. 36(1): p. 37-44.

8. Fryburg, D.A. and M.T. Vassiieva, Atherosclerosis drug development in jeopardy: the need for predictive biomarkers of treatment response. Sci Trans! Med, 201 . 3(72): p. 72.

9. Ridker, P.M., et al., Established and emerging plasma biomarkers in the prediction of first atherothrombotic events. Circulation, 2004. 109(25 Suppl 1 ): p. IV6-19.

10. Gerszten, R.E. and T.J. Wang, The search for new cardiovascular biomarkers.

Nature, 2008. 451 (7181): p. 949-52. 11. Heldin, C.-H., and B. Westermark. 1999. Mechanism of Action and In Vivo Role of Platelet-Derived Growth Factor. Physiol. Rev. 79: 1283-1316. 12. Pavan K. Cheruvu, Frequency and Distribution of Thin-Cap Fibroatheroma and Ruptured plaques in Human Coronary Arteries - A Pathologic Study, J Am Coli Cardiol, 2007. 50(10): p. 940-9. 3. John C. Wang, Coronary Artery Spatial Distribution of Acute Myocardial Infarction Occlusions, Circulation, 2004. 110(3): p. 278-84.

14. Stone et al, A Prospective Natural-History Study of Coronary Atherosclerosis, N Engl J Med 201 1 ;364:226-35

15. Ross et al. , Localization of PDGF-B protein in macrophages in all phases of atherogenesis., Science. 1990 May 25;248(4958): 1009-12. 16. Ogawa et al. , Plasma platelet-derived growth factor levels in coronary circulation in unstable angina pectoris., Am J Cardiol, 1992 Feb 15;69(5):453-6.

17. Barrett et al., Plateiet-deriyed growth factor gene expression in human

atherosclerotic plaques and normal artery wail., Proc. Natl. Acad. Sci. USA

Vol. 85, pp. 2810-2814, April 1988.

18. Schirmer et al., Local Cytokine Concentrations and Oxygen Pressure Are Related to Maturation of the Collateral Circulation in Humans., Journal of the American College of Cardiology, Vol. 53, No. 23, pp.2141-2147, 2009

19. Loizos et al. , Targeting the platelet derived growth factor receptor a with a

neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: implications as a potential therapeutic target., Mol Cancer Ther., 2005; 4: pp.369-379.

Claims

1. A method used for assessing the likelihood of a major cardiac event, which method comprises assessing the concentration of PDGF local to a vascular lesion or suspected vascular lesion,

wherein the concentration of PDGF downstream of the vascular lesion that is at least 125% of the concentration upstream of the vascular lesion or suspected vascular lesion, or at least 125% of the systemic concentration, is associated with a higher risk of a major cardiac event

2. The method of claim 1 wherein the vascular lesion is situated within 60mm of the LCx artery ostium, within 80mm of the LAD artery ostium, or within 130mm of the RCA ostium.

3. The method of claim 2 wherein the PDGF concentration local to the vascular lesion is assessed by comparing the PDGF concentration at a point downstream of the vascular lesion with the PDGF concentration at a point upstream of the vascular lesion,

wherein both upstream and downstream points are within the coronary artery.

4. A method of selecting individuals for treatment with,

(1) an antibody which specifically binds a protein selected form the group consisting of: IL-1 (Uniprot ref: P01583, P01584), IL-6 (Uniprot ref: P05231), IL-7 (Uniprot ref: P13232), Monocyte chemo attractant protein-1 (MCP-1 ; Uniprot ref: Q6UZ82), TNF-a (Uniprot ref: P01375), IL-18 (Uniprot ref: Q14116), IL-10 (Uniprot ref: P22301), CRP (Uniprot ref:

P02741), serum amyloid A (SAA; Uniprot ref: PODJiS, P0DJI9, P35542), ICAM (interceliular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9U F0), VCAM (vascular cell adhesion molecule; Uniprot ref: P19320), E-selectin (Uniprot ref: P16581), E- selectin (Uniprot ref: P 6581), von Willebrand factor (vWF; Uniprot ref: P04275),

myeloperoxidase (MPO; Uniprot ref: P05164), secretory phospholipase A2 (sPLA2; Uniprot ref:), lipoprotein-associated phospholipase A2 (Lp-PLA2; Uniprot ref: P 14555), Vascular endothelial growth factor (VEGF; Uniprot ref: P15692, P49765, P49767, 043915), placental growth factor (PIGF; Uniprot ref: P49763), hepatocyte growth factor (HGF; Uniprot ref:

P14210), matrix metalloproteinases (MMPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29965), P-seiectin (Uniprot ref: P16109), pregnancy-associated plasma peptide A (PAPP-A), Neutrophil elastase (Uniprot ref:P08246), Tissue Factor (Uniprot ref: P13726), Protein-bound-lnsulin-like growth factor (Uniprot ref: P05019, P01344), Neopterin, Choline, Heat Shock Proteins (Uniprot ref: Q00813, Q03933, Q9ULV5); or (2) a compound selected from the group consisting of: Advicor (Lovastatin with Niacin), Aitocor (Lovastatin), Altoprev (Lovastatin), Atoriip (Atorvastatin), Bayco! (Cerivastatin), Caduet (Atorvastatin with Amlodipine), Canef

(Fluvastatin), Crestor (Rosuvastatin), Inegy (Simvastatin with Ezetimibe), Lescol

(Fluvastatin), Lipex (Simvastatin), Lipitor (Atorvastatin), Lipobay (Cerivastatin), Lipostat (Pravastatin), Lipvas (Atorvastatin), Livaio (Pitavastatin), evacor (Lovastatin), Pitava (Pitavastatin), Pravachol (Pravastatin), Se!ektine (Pravastatin), Simcard (Simvastatin), Simcor (Simvastatin with Niacin), Simiup (Simvastatin), Sortis (Atorvastatin), Torvacard (Atorvastatin), Torvast (Atorvastatin), Totalip (Atorvastatin), Tulip (Atorvastatin), Vytorin (Simvastatin with Ezetimibe), Zocor (Simvastatin), Juvisync (sitagliptin/simvastatin), Liptruze (ezetimibe/atorvastatin), evastatin, Anacetrapib, Evacetrapib, Torcetrapib, Daicetrapib, A G 145 (Amgen), SAR236553/REGN727 Sanofi/Regeneron Pharmaceuticals, RN316 (Pfizer), RG7852 (Anti-PCSK9, IV1PSK3169A), iomitapide (juxtapid, Aegerion

Pharmaceuticals), Mipomersen sodium (Kynamro, !sis Pharmaceuticals/Genzyme), SPC- 4955, REGN-728, PF-05335810, LY-3015014, BMS-962476, ALN-PCS, TA-8995, DRL- 21995, LY-3015014, TAP31 1 , CJ-30Q39, ZYT-1 , KT6-971 , RG-7652, GSK-1292263, CER- 001 , GFT-505, SLx-4090, B S-823778, RVX-208, VIA-3196 / MGL-3196, SPC-5001 , AMG- 145, K-877, alirocumab (REGN-727 / SAR-236553), daicetrapib, JTT-302, DRL-17822, THVC:CBD (GW42003 + GW42004), RN-316 / PF-04950615, MBX-8025 / RWJ-800025, ETO10Q2, ZYH-7, LGT-209, mipomersen sodium, Iomitapide, pioglitazone, anacetrapib, evacetrapib, Ji- 01 , Orantinib (also known as TSU-68; SU6868), Vatalanib, Pazopanib/ Votrient, IVIotesanib / AMG 708, Trapidil (triazolopyrimidine), Axitinib (AG013736) Inlyta, AMG 708 (motesanib), BIBF 1 20 (nintedanib), Sorafenib / Nexavar, X-82, Sunitinib / Sutent / SU11248, Crenolanib / CP-868,596; ARO 002, niiofinib, Imatinib / Gleevec / Glivec, Olaratumab (IMC-3G3, see [19]), MEDi-575, Fovista E10030, Vinpocetine, BIBF-1120, Tandutinib, Piperlongumine, and Eupatolide,

which method comprises assessing a local concentration of one or more pre- specified biomarkers local to a vascular lesion or suspected vascular lesion,

wherein individuals having a PDGF concentration downstream of the vascular lesion or suspected vascular lesion that is at least 125% of the concentration upstream of the vascular lesion or suspected vascular lesion are selected for treatment,

and wherein treatment reduces the risk of a major cardiac event.

5. The method of any one of claims 1 to 4, wherein PDGF is a PDGF-AA dimer, a PDGF-AB dimer, or a PDGF-BB dimer.

8 The method of any one of claims 1 to 5 wherein the concentration of PDGF downstream of the vascular lesion that is at least 150% of the concentration upstream of the vascular lesion, or at least 150% of the systemic concentration, is required for association with increased risk of plaque rupture, or selection for treatment.

7. The method of any one of claims 1 to 8 wherein the major cardiac event is the rupture of a vulnerable plaque.

8. The method of any one of the previous claims wherein the concentration is assessed by non-invasively imaging an individual to whom a PDGF specific binding-moiety has been administered, which binding-moiety optionally coupled do a detectable label.

9. The method of claim 8 wherein the individual has been administered with an anti- PDGF antibody coupled to a radioisotope. 0. A method for assessing the risk of ASVD in an individual, which method comprises assessing a local concentration of PDGF,

wherein a measure exceeding the threshold level is associated with a higher likelihood of ASVD.

11. A method of assessing the efficacy of a drug in treating ASVD in an individual, which method comprises assessing the effect of the drug on the concentration of PDGF, or the effect of the drug on the biological activity of a pathway associated with PDGF.

12. A method of assessing the increased risk of ASVD in an individual caused by a drug, which method comprises assessing the concentration gradient of PDGF local to a vascular lesion or suspected vascular lesion, or the effect of the drug on the biological activity of a pathway associated with PDGF.

13. The method of either one of claims 11 or 12 wherein the pathway is vascular endothelial proliferation or vascular neogenesis.

14. The method of any one of claims 1 1 to 13 wherein the efficacy assessment comprises the steps of:

(a) assessing the concentration of PDGF local to a vascular lesion or suspected vascular lesion;

(b) administering the drug; and then

(c) reassessing the concentration of PDGF local to a vascular lesion or suspected vascular lesion, wherein observed changes in the PDGF gradient are correlated with the efficacy or increased risk of the drug.

15. Use of an increased PDGF concentration local to a vascular lesion or suspected vascular lesion as a diagnostic or prognostic marker of ASVD.

16. Use according to claim 15, wherein an increased PDGF concentration local to a vascular lesion or suspected vascular lesion is used to diagnose a vulnerable plaque.

17. A method of assessing risk of thrombosis or coagulation within a section of a blood vessel, the method comprising collecting a sample of blood from the section of the blood vessel and measuring the dotting ability of the blood. 8. The method of claim 17, wherein the clotting ability of the blood is measured by measuring the level of platelet activation in the sample. 9. A method of treating ASVD comprising administering to an individual in need of treatment a therapeutically effective amount of a drug which lowers the concentration of PDGF local to vascular lesion or suspected vascular lesion, or inhibits the biological activity of a pathway associated with PDGF.

20. Use of a drug which lowers the concentration of PDGF local to a vascular lesion or suspected vascular lesion, or inhibits the biological activity of a pathway associated with PDGF, in the manufacture of a medicament for the treatment of ASVD.

21. A drug which lowers the concentration of PDGF local to a vascular lesion or suspected vascular lesion, for use in the treatment of ASVD,

22. The method of claim 19, the use of claim 20, or the drug of claim 21 , wherein the drug is selected from:

(1) an antibody which specifically binds a protein selected form the group consisting of:

IL-1 (Uniprot ref: P01583, P01584), IL-6 (Uniprot ref: P05231 ), IL-7 (Uniprot ref: P13232), Monocyte chemo attractant protein-1 (MCP- ; Uniprot ref: Q6UZ82), TNF-a (Uniprot ref: P01375), IL-18 (Uniprot ref: Q14116), IL-10 (Uniprot ref: P22301), CRP (Uniprot ref:

P02741), serum amyloid A (SAA; Uniprot ref: P0DJI8, P0DJI9, P35542), ICAM (intercellular adhesion molecule; Uniprot ref: P05362, P13598, P32942, Q14773, Q9U F0), VCAM (vascular cell adhesion molecule; Uniprot ref: P19320), E-selectin (Uniprot ref: P16581 ), E- selectin (Uniprot ref: P16581), von Willebrand factor (vWF; Uniprot ref: P04275), myeloperoxidase (MPO; Uniprot ref: P05164), secretory phospholipase A2 (sPLA2; Uniprot ref:), lipoprotein-associated phospholipase A2 (Lp-PLA2; Uniprot ref: P14555), Vascular endothelial growth factor (VEGF; Uniprot ref: P15692, P49785, P49787, 043915), placental growth factor (PIGF; Uniprot ref: P49763), hepatocyte growth factor (HGF; Uniprot ref: P14210), matrix metailoproteinases (MMPs), MMP-1 (Uniprot ref: P03956), -2 (Uniprot ref: P08253), and -9 (Uniprot ref: P14780), pregnancy-associated plasma peptide A (PAPP-A), sCD40L (Uniprot ref: P29985), P-selectin (Uniprot ref: P18109), pregnancy-associated plasma peptide A (PAPP-A), Neutrophil eiastase (Uniprot ref:P08246), Tissue Factor (Uniprot ref: P13726), Protein-bound-lnsulin-like growth factor (Uniprot ref: P05019, P01344), Neopterin, Choline, Heat Shock Proteins (Uniprot ref: Q00813, Q03933, Q9ULV5); or (2) a compound selected from the group consisting of:

Advicor (Lovastatin with Niacin), Altocor (Lovastatin), Altoprev (Lovastatin), Atorlip

(Atorvastafin), Baycoi (Cerivastafin), Caduef (Atorvastatin with Amiodipine), Canef

(Fluvastatin), Lipex (Simvastatin), Lipitor (Atorvastatin), Lipobay (Cerivastatin), Lipostat (Pravastatin), Lipvas (Atorvastatin), Livaio (Pravastatin), Mevacor (Lovastatin), Pitava (Pitavastatin), Pravachol (Pravastatin), Selektine (Pravastatin), Simcard (Simvastatin), Simcor (Simvastatin with Niacin), Simlup (Simvastatin), Sortis (Atorvastatin), Torvacard (Atorvastatin), Torvast (Atorvastatin), Totalip (Atorvastatin), Tulip (Atorvastatin), Vytorin (Simvastatin with Ezetimibe), Zocor (Simvastatin), Juvisync (sitagliptin/simvastatin), Liptruze (ezetimibe/atorvastatin), Mevastatin, Anacetrapib, Evacetrapib, Torcetrapib, Dalcetrapib, A G 145 (Amgen), SAR236553/REGN727 Sanofi/Regeneron Pharmaceuticals, RN316 (Pfizer), RG7652 (Anti-PCSK9, MPSK3169A), lomitapide (Juxtapid, Aegerion

Pharmaceuticals), IVlipomersen sodium (Kynamro, isis Pharmaceuticals/Genzyme), SPC- 4955, REGN-728, PF-05335810, LY-3015014, BMS-962478, ALN-PCS, TA-8995, DRL- 21995, LY-3015014, TAP31 1 , CJ-30039, ZYT-1 , KT6-971 , RG-7652, GSK-1292263, CER- 001 , GFT-505, SLx-4090, BMS-823778, RVX-208, VIA-3196 / MGL-3196, SPC-5001 , AMG- 145, K-877, alirocumab (REGN-727 / SAR-236553), dalcetrapib, JTT-302, DRL-17822, THVC:CBD (GW42003 + GW42004), RN-316 / PF-04950615, BX-8025 / RWJ-800025, ETC-1002, ZYH-7, LGT-209, mipomersen sodium, lomitapide, pioglitazone, anacetrapib, evacetrapib, JI-101 , Orantinib (also known as TSU-68; SU6868), Vafalanib, Pazopanib/ Votrient, Motesanib / AMG 706, Trapidil (triazolopyrimidine), Axitinib (AG013736) Inlyta, AMG 706 (motesanib), BIBF 120 (nintedanib), Sorafenib / Nexavar, X-82, Sunitinib / Sutent / SU 1248, Crenolanib / CP-868,596; ARO 002, niiotinib, Imatinib / Gieevec / Glivec, Oiaratumab (IMC-3G3, see [19]), MED!-575, Fovista E10030, Vinpocetine, BIBF-1120, Tandutinib, Piperiongumine, and Eupatoiide.