Abstract
The present application relates to a series of novel peptides and their use in therapy and diagnosis, in particular in relation to atherothrombosis and/or atherosclerosis. Atherothrombosis is characterised by an unpredictable, sudden disruption (rupture or erosion/fissure) of an atherosclerotic plaque, which leads to platelet activation and thrombus formation. Atherothrombosis is the underlying condition that results in events leading to myocardial infarction, ischemic stroke and death. Atherothrombosis is the main cause of death and disability within the population aged 45 or above in industrialized nations (currently 30% of global deaths and 40% of deaths in the UK are caused by Cardio Vascular Disease (CVD); this projection is expected to remain within the next 30 years). The development of atherosclerotic plaques starts during childhood and develops over a range of 50-60 years before the first symptoms of instability occur. Currently, prevention is based on the measurement of risk factors (family history, diabetes, high circulating cholesterol levels, etc). Patients who are at risk undergo early screening and this has been shown to reduce the incidence of complications. The follow up of patients who have suffered acute complications or you have a history of chronic cardiovascular disease is based on a combined approach that does not always reflect the development of the atherosclerotic lesions and the possible appearance of complications. The complex nature and long term development of atherosclerotic cardiovascular diseases demands the development of novel biomarkers for early disease detection, risk assessment and evaluation of response to therapy. Traditional evaluation of patients attending to hospital accident and emergency services with chest pain or other signs or symptoms suggesting acute coronary syndrome is sometimes problematic, time-consuming and most of the time expensive. Moreover, current laboratory investigations designed to assess the status and prognosis of these patients rely on serological markers that provide a very narrow window of action to clinicians and health care professionals to act upon. There is, therefore, a need for improved methods of diagnosing and treating atherothrombosis and/or atherosclerosis. SUMMARY OF THE INVENTION According to one aspect of the present invention, there is provided a peptide or a polypeptide comprising an amino acid sequence selected from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and 51, or a fragment or variant thereof. According to another aspect of the present invention, there is provided a peptide or polypeptide consisting of an amino acid sequence selected from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and 51, or a fragment or variant thereof. Preferably, the fragments or variants thereof comprise an amino acid sequence that has at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% amino acid sequence identity with SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51. Preferably, the fragments thereof comprise at least four, preferably at least five, preferably at least six, preferably at least seven, preferably at least eight, preferably at least nine, preferably at least ten, preferably at least eleven consecutive amino acids from SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51. Fragments may also include truncated peptides that have x amino acids deleted from the N-terminus and/or C-terminus. In such truncations, x may be 1 or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51. Preferably, the fragments or variants thereof are functional fragments or variants thereof. According to another aspect of the present invention, there is provided a nucleic acid sequence which encodes a peptide or a polypeptide of the present invention. Preferably, the nucleic acid sequence is an isolated nucleic acid sequence. Also provided by the present invention is a nucleic acid molecule comprising the nucleic acid sequence of the present invention. Preferably, the nucleic acid molecule further comprises vector nucleic acid sequences. Preferably, the nucleic acid molecule further comprises nucleic acid sequences encoding a heterologous polypeptide. Another aspect of the present invention relates to a host cell which contains the nucleic acid molecule of the present invention. The host cell may be a mammalian host cell or a non-mammalian host cell. Preferably, the nucleic acid sequence is incorporated into a vector, for example a DNA plasmid. As such, in one aspect of the present invention, there is provided a vector, for example a DNA plasmid, comprising a nucleic acid sequence of the present invention. In preferred embodiments, the peptides, polypeptides and nucleic acid sequences of the present invention are atherothrombosis or atherosclerosis specific biomarkers. As such, in one aspect of the present invention, there is provided an atherothrombosis specific biomarker or an atherosclerosis specific biomarker comprising a peptide, polypeptide or nucleic acid sequence of the present invention. Another aspect of the present invention relates to a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention for use in therapy. A further aspect of the present invention relates to a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention for use in therapy. Another aspect of the present invention relates to use of a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention in therapy. A further aspect of the present invention relates to use of a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention in therapy. Preferably, said therapy is selected from the treatment of atherothrombosis and the treatment of atherosclerosis. Another aspect of the present invention relates to a method for treating a patient with a disease, the method comprising administering to a patient a therapeutically effective amount of a peptide or polypeptide of the present invention and/or a therapeutically effective amount of a nucleic acid sequence of the present invention. Another aspect of the present invention relates to a method for treating a patient with a disease, the method comprising administering to a patient a therapeutically effective amount of a combination of two or more peptides or polypeptides of the present invention and/or a therapeutically effective amount of a combination of two or more nucleic acid sequences of the present invention. Preferably, said disease is selected from atherothrombosis and atherosclerosis. Another aspect of the present invention relates to use of a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention in the manufacture of a medicament for the treatment of atherothrombosis or atherosclerosis. A further aspect of the present invention relates to use of a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention in the manufacture of a medicament for the treatment of atherothrombosis or atherosclerosis. Another aspect of the present invention relates to an antibody or fragment thereof that specifically binds to a peptide or polypeptide of the present invention. Preferably, the antibody is conjugated to a detectable marker, for example a fluorescent marker or tag. Preferably, the antibody is a monoclonal antibody. Another aspect of the present invention relates to an antibody or fragment thereof of the present invention for use in therapy. Another aspect of the present invention relates to use of an antibody or fragment thereof of the present invention in therapy. Preferably, said therapy is selected from the treatment of atherothrombosis and the treatment of atherosclerosis. Another aspect of the present invention relates to a method for treating a patient with a disease, the method comprising administering to a patient a therapeutically effective amount of an antibody or fragment thereof of the present invention. Preferably, said disease is selected from atherothrombosis and atherosclerosis. Another aspect of the present invention relates to use of an antibody or fragment thereof of the present invention in the manufacture of a medicament for the treatment of atherothrombosis or atherosclerosis. In preferred embodiments, the methods and compositions of the invention are for treatment of disease at an early stage, for example, before symptoms of the disease appear. In some embodiments, the methods and compositions of the invention are for treatment of disease at a clinical stage. Another aspect of the present invention relates to a method for imaging atherothrombosis or atherosclerosis in a patient, the method comprising administering to a patient an antibody or fragment thereof of the present invention. Further provided is an antibody or fragment thereof of the present invention for use in a method of imaging atherothrombosis or atherosclerosis in a patient. Another aspect of the present invention relates to a composition comprising a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention. A further aspect of the present invention relates to a composition comprising a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention. Also provided is a composition comprising an antibody or fragment thereof of the present invention. Preferably, the composition is a pharmaceutical composition. Preferably, the pharmaceutical composition is a vaccine. Preferably, the composition is for use in therapy, for example in the treatment of atherothrombosis or atherosclerosis. Also provided by the present invention is a vaccine comprising a peptide or polypeptide of the present invention and/or a nucleic acid sequence of the present invention. Another aspect of the present invention relates to a vaccine comprising a combination of two or more peptides or polypeptides of the present invention and/or a combination of two or more nucleic acid sequences of the present invention. According to another aspect of the present invention, there is provided a method for diagnosing atherothrombosis or atherosclerosis in a patient or for identifying a patient at risk of developing atherothrombosis or atherosclerosis, the method comprising: (a) determining an amount of a peptide, polypeptide or nucleic acid sequence of the present invention in a sample obtained from a patient; (b) comparing the amount of the determined peptide, polypeptide or nucleic acid sequence in the sample from the patient to the amount of the peptide, polypeptide or nucleic acid sequence in a normal control; wherein a difference in the amount of the peptide, polypeptide or nucleic acid sequence in the sample from the patient compared to the amount of the peptide, polypeptide or nucleic acid sequence in the normal control is associated with the presence of atherothrombosis or atherosclerosis or is associated with a risk of developing atherothrombosis or atherosclerosis. According to another aspect of the present invention, there is provided a method for diagnosing atherothrombosis or atherosclerosis in a patient or for identifying a patient at risk of developing atherothrombosis or atherosclerosis, the method comprising: (a) determining an amount of a combination of two or more peptides, polypeptides and/or nucleic acid sequences of the present invention in a sample obtained from a patient; (b) comparing the amount of the determined peptides, polypeptides and/or nucleic acid sequences in the sample from the patient to the amount of the peptides, polypeptides and/or nucleic acid sequences in a normal control; wherein a difference in the amount of the peptides, polypeptides and/or nucleic acid sequences in the sample from the patient compared to the amount of the peptides, polypeptides and/or nucleic acid sequences in the normal control is associated with the presence of atherothrombosis or atherosclerosis or is associated with a risk of developing atherothrombosis or atherosclerosis. According to another aspect of the present invention, there is provided a method for monitoring the progression of atherothrombosis or atherosclerosis in a patient, the method comprising: (a) determining an amount of a peptide, polypeptide or nucleic acid sequence of the present invention in a sample obtained from a patient; (b) comparing the amount of the determined peptide, polypeptide or nucleic acid sequence in the sample from the patient to the amount of the peptide, polypeptide or nucleic acid sequence in a normal control; and (c) repeating steps (a) and (b) at two or more time intervals, wherein an increase in the amount of the peptide, polypeptide or nucleic acid sequence from the patient over time is associated with an increase in the progression of atherothrombosis or atherosclerosis and a decrease in the amount of the peptide, polypeptide or nucleic acid sequence from the patient over time is associated with a decrease in the progression of atherothrombosis or atherosclerosis. According to another aspect of the present invention, there is provided a method for monitoring the progression of atherothrombosis or atherosclerosis in a patient, the method comprising: (a) determining an amount of a combination of two or more peptides, polypeptides and/or nucleic acid sequences of the present invention in a sample obtained from a patient; (b) comparing the amount of the determined peptides, polypeptides and/or nucleic acid sequences in the sample from the patient to the amount of the peptides, polypeptides and/or nucleic acid sequences in a normal control; and (c) repeating steps (a) and (b) at two or more time intervals, wherein an increase in the amount of the peptides, polypeptides and/or nucleic acid sequences from the patient over time is associated with an increase in the progression of atherothrombosis or atherosclerosis and a decrease in the amount of the peptides, polypeptides and/or nucleic acid sequences from the patient over time is associated with a decrease in the progression of atherothrombosis or atherosclerosis. Accordingly, the methods of the present invention can be used to detect the onset, progression, stabilisation, amelioration and/or remission of atherothrombosis or atherosclerosis. Preferably, the control may be from the same patient from a previous sample, to thus monitor onset or progression. However, it is also preferred that the control may be normalised for a population, particularly a healthy or normal population, where there is no atherothrombosis or atherosclerosis. In other words, the control may consist of the level of a peptide, polypeptide or nucleic acid sequence of the present invention found in a normal control sample from a normal subject. Accordingly, in one example of the present invention, there is provided a method of diagnosing or monitoring the progression of atherothrombosis or atherosclerosis, comprising detecting and/or quantifying a peptide, polypeptide or nucleic acid sequence of the present invention or a combination of two or more peptides, polypeptides and/or nucleic acid sequences of the present invention in a biological fluid obtained from a patient. As discussed above, it is preferred that at least two detection and/or quantification steps are provided, spaced apart temporally. Preferably, the steps are spaced apart by a few days, weeks, years or months, to determine whether the levels of the peptides, polypeptides or nucleic acid sequences have changed, thus indicating whether there has been a change in the progression of atherothrombosis or atherosclerosis, enabling comparisons to be made between a level of the peptides, polypeptides or nucleic acid sequences in samples taken on two or more occasions, as an increase in the level of the peptides, polypeptides or nucleic acid sequences over time is indicative of the onset or progression of atherothrombosis or atherosclerosis, whereas a decrease in the level of the peptides, polypeptides or nucleic acid sequences may indicate amelioration and/or remission of atherothrombosis or atherosclerosis. Preferably, the difference in the level of the peptides, polypeptides or nucleic acid sequences is statistically significant, determined by using a "t-test" providing confidence intervals of preferably at least about 80%, preferably at least about 85%, preferably at least about 90%, preferably at least about 95%, preferably at least about 99%, preferably at least about 99.5%, preferably at least about 99.95%, preferably at least about 99.99%. The peptides, polypeptides, nucleic acid sequences and methods of the invention are particularly useful in detecting early stage atherothrombosis or atherosclerosis and are more sensitive than known methods for detecting early stage atherothrombosis or atherosclerosis. Thus, the peptides, polypeptides, nucleic acid sequences and methods of the invention are particularly useful for confirming atherothrombosis or atherosclerosis when a patient has tested negative for atherothrombosis or atherosclerosis using conventional methods. It is preferred that the peptides, polypeptides or nucleic acid sequences of the present invention are indicative of the presence of atherothrombosis or atherosclerosis or the risk of developing atherothrombosis or atherosclerosis when present at a level of at least about 1.5-fold, preferably at least about 2-fold, preferably at least about 5-fold, preferably at least about 10-fold, preferably at least about 20-fold, preferably at least about 50-fold, preferably at least about 100-fold, preferably at least about 200-fold, preferably at least about 300-fold, preferably at least about 400-fold, preferably at least about 500-fold, preferably at least about 600-fold that of a normal control. Also provided by the present invention is a method for monitoring the efficacy of a treatment for atherothrombosis or atherosclerosis, comprising detecting and/or quantifying the presence of a peptide, polypeptide or nucleic acid sequence of the present invention or a combination of two or more peptides, polypeptides and/or nucleic acid sequences of the present invention in a biological sample obtained from a patient. Preferably, in the methods of the present invention, detection and/or quantification of the peptides, polypeptides or nucleic acid sequences is by one or more of MALDI-TOF, SELDI, via interaction with a ligand or ligands, 1-D or 2-D gel-based analysis systems, Liquid Chromatography, combined liquid chromatography and Mass spectrometry techniques including ICAT(R) or iTRAQ(R), thin-layer chromatography, NMR spectroscopy, sandwich immunoassays, enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RAI), enzyme immunoassays (EIA), lateral flow/immunochromatographic strip tests, Western Blotting, immunoprecipitation, particle-based immunoassays including using gold, silver, or latex particles, magnetic particles or Q-dots, and immunohistochemistry on tissue sections. Preferably, detection and/or quantification of the peptides, polypeptides or nucleic acid sequences is performed on a microtitre plate, strip format, array or on a chip. Preferably, detection and/or quantification of the peptides, polypeptides or nucleic acid sequences is by an ELISA comprising antibodies specific for the peptides, polypeptides or nucleic acid sequences, preferably linked to a reporter. Preferably, detection and/or quantification of the peptides, polypeptides or nucleic acid sequences is by a biosensor. Preferably, the sample comprises biological fluid or tissue obtained from the patient. Preferably, the biological fluid comprises urine. It is also preferred that the biological fluid is substantially or completely free of whole/intact cells. Preferably the biological fluid is free of platelets and cell debris (such as that produced upon the lysis of cells). Preferably the biological fluid is free of both prokaryotic and eukaryotic cells. Such samples can be obtained by any number of means known in the art, such as will be apparent to the skilled person. For instance, a patient can be asked to provide a urine sample during a routine consultation. Cell free or substantially cell free samples can be obtained by subjecting the sample to various techniques known to those of skill in the art which include, but are not limited to, centrifugation and filtration. Another aspect of the present invention relates to use of a peptide, polypeptide or nucleic acid sequence of the present invention or a combination of two or more peptides, polypeptides or nucleic acid sequences of the present invention, detectable in a body fluid or tissue, as a biomarker for atherothrombosis or atherosclerosis. Preferably, said use is in a method selected from the group consisting of: clinical screening, methods of prognosis assessment, monitoring the results of therapy, method to identify patients most likely to respond to a particular therapeutic treatment, and drug screening and development. According to another aspect of the present invention, there is provided a kit for use in the methods or uses described above, wherein the kit comprises a ligand capable of binding or specifically recognising a peptide, polypeptide or nucleic acid sequence of the present invention, detectable in a body fluid or tissue and reporter means. Preferably, the kit is an array or chip. Preferably, the kit comprises a microtitre plate, test strip, array or chip.