The present invention relates to assays for biomarkers useful

5 for assessment of cardiovascular events, including but not limited to Acute Myocardial Infarction (AMI), high coronary calcification, and symptomatic non AMI. In particular, according to the present invention, biomarkers relating to degradation fragments of Titin are found to be useful.

0 Titin, also known as connectin, is a sarcomeric protein

expressed in cardiac and skeletal muscle. It is the largest known mammal protein, having a size that can reach up to SVOOkDa ¹ . Its main function and one that is is well described is to act as a long molecular spring by restoring passive tension during

5 myocardial stretch ^2-4 . Titin has two isoforms that are co- expressed in the sarcomere, the N2A which is the larger of the two and is found in both skeletal and myocardial muscle, and the N2B isoform which is smaller, stiffer and is solely found in cardiac muscle ^1,5~7 . Due to the different stiffness properties of the

0 isoforms of Titin it has been proposed that the adaptive or

maladaptive ratio alteration between the two isoforms, in synergy with their corresponding kinase region, during pathologic events could be responsible for affecting the myocardial contractile properties ^{3, 5} ' ^8~12 . Isoform modifications and ratio alterations

5 have been first described in animal models while clinical studies have also reported isoform shifts during dilated cardiomyopathy (DCM) , aortic stenosis (AS) , diastolic heart failure (DHF) and ischemic heart disease (IHD) ^{11, 13~16} . The main limitation of Titin relevant studies lies on the available methods for detecting and

0 quantifying Titin isoform levels, which are either based on

methods that are not sensitive enough such as immunoblotting and gel electrophoresis or techniques which can provide higher

sensitivity such as quantitative RT-PCR but can only provide information about the total amount of Titin isoform levels. All

5 of these methods rely on invasive means of tissue collection and identification. Low sensitivity and specificity might contribute to the poor utilisation of these methods as diagnostic or

prognostic biomarkers and restrain the correlation of Titin isoform levels with functional studies. In contrast, we

discovered according to the present invention that detection of specific degradation fragments of Titin has excellent clinical utility in a number of important clinical situations.

Extracellular matrix (ECM) components are degraded by a number of different proteases including matrix metalloproteinases (MMPs) . MMP derived degradation of proteins generates specific cleavage sites/ fragments which in turn produce new epitopes. We have previously described that neo epitopes may have potential utility as biomarkers of unbalanced ECM remodeling in a number of different pathologies and can be measured in an array of

biological fluids such as serum, plasma and urine ^{17- 22} . A key benefit of this approach is that it is a non-invasive method of measuring specific neo epitopes that represent a unique

Λ fingerprint' of the proteolytic cleavage of the protein and directly reflect specific tissue turnover in both physiology and pathology .

Accordingly, the present invention now provides in a first aspect a method of bioassay for the quantification of peptide fragments comprising a neo-epitope formed in by cleavage of a titin protein by a proteinase, said method comprising contacting a sample comprising said peptide fragments with an immunological binding partner having specific binding affinity for a said neo- epitope and determining the level of binding of said immunological binding partner to peptide fragments in said sample.

Titin proteins include all isoforms of titin, including each of those described above. The titin fragments detected may be in samples derived from any mammalian, for instance a rodent

(including particularly mice and rats), dogs, and primates, including monkeys. However, the samples are preferably from a human. Similarly the titin sequences on which the assays are based may come from any of these sources. Generally, the

fragments detected will be formed in nature and will be naturally occurring in said sample. Said immunological binding partner may have specific binding affinity for peptide fragments comprising a C-terminal neo-epitope of said titin protein or for peptide fragments comprising an N- terminal neo-epitope of said titin protein.

Said immunological binding partner may have specific binding affinity for a peptide fragment which comprises a neo-epitope formed by cleavage of a titin protein by a protease giving any one of the following partial sequences of titin:

1351 9 ' i GEYVCDCGTD ^Λ 13528 SEQ ^{10 N0 : 1}

13532 ' NVTVEARLIKi U 3542 SEQ ID NO: 2

13543 ' iVEKPLYGVEV ^Λ 1553 SEQ ^{10 N0 : 3} Said immunological binding partner may have specific binding affinity for either of the following sequences at the N terminal of a peptide:

1351 9 ' i GEYVCD SEQ ID NO: 4

13543 ' iVEKPLY SEQ ID NO: 5

or with the following sequence at the C-terminal of a peptide: EARLIKi ' 13542 SEQ ID NO: 6

Said immunological binding partner may preferably have specific binding affinity for the following sequence at the C terminal of a peptide: NVTVEARLIKi ^Λ 13542 .

Preferably, said immunological binding partner is a

monoclonal antibody or a fragment of a monoclonal antibody having specific binding affinity.

Said method is preferably conducted as a competition immunoassay in which said immunological binding partner and a competition agent are incubated in the presence of said sample and the competition agent competes with the peptide fragments in the sample to bind to the immunological binding partner. Optionally, said competition agent is a synthetic peptide or is a purified native peptide formed by cleavage of the protein from which said epitope comes so as to reveal said neo-epitope. The sample is preferably a sample of urine, serum, blood, plasma, or saliva. Included in the invention are methods in which the sample is a patient derived sample, said method further comprising comparing the determined level of said binding of said peptide fragments with values characteristic of (a) comparable healthy individuals and/or (b) a pathological cardiac condition.

In a further aspect, the invention includes an immunological binding partner against a C-terminal or N-terminal neo-epitope formed by proteinase cleavage of a titin protein.

The immunological binding partner may be specifically immunoreactive with the N-terminal of either of the amino acid sequences :

13519' i GEYVCD

13543' iVEKPLY or with the C-terminal of the amino acid sequence:

EARLIKi U3542

The immunological binding partner may be a monoclonal antibody or a binding fragment thereof.

The invention includes in a further aspect a cell line producing a monoclonal antibody described above.

In a further aspect, the invention provides a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of a titin protein by a protease in any one of the partial sequences of said proteins set out above. The peptide may be conjugated as a hapten to a carrier for producing an immune response to said peptide, or immobilised to a solid surface or conjugated to a detectable marker for use in an immunoassay.

In a further aspect, the invention provides an isolated nucleic acid molecule coding for a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of a said protein by a protease in any one of the partial sequences of said proteins set out above.

In a still further aspect, the invention provides a vector comprising a nucleic acid sequence comprising an expression signal and a coding sequence which codes for the expression of a peptide comprising a C-terminal or N-terminal neo-epitope formed by cleavage of a said protein by a protease in any one of the partial sequences of said proteins set out above.

In a still further aspect, the invention provides a host cell transformed with a vector as described above and expressing a said peptide .

In a still further aspect, the invention provides an

immunoassay kit comprising an immunological binding partner as described above, and a competition agent which binds said

immunological binding partner, and optionally one or more of a wash reagent, a buffer, a stopping reagent, an enzyme label, an enzyme label substrate, calibration standards, an anti-mouse antibody and instructions for conducting an assay using said kit.

During digestion of human tissue with an array of exogenous metalloproteases , a large number of proteolyzed peptide products were identified using mass spectrometry ²³ . Among these, a Titin specific fragment 12670' NVTVEARLIK 12679' was identified to be MMP12 cleavage specific. Proteomic analysis revealed that the sequence is unique for the N2B Titin isoform and homologous to human and mouse. Even though at least in murine models MMP12 has been implicated in cardiovascular events such as atherogenesis, the only MMP previously described to have an effect on Titin degradation in relevant bibliography is MMP2 located in the Z-disk region of cardiac sarcomere, contributing to Titin degradation in ischemic and reperfusion related events ^{24, 25} . Due to our mass spectrometry derived finding, we hypothesized that MMP12 may be a more active participant in related pathologic events than

previously described and we set out with the hypothesis that the MMP12 specific fragment of the N2B isoform of Titin identified with mass spectrometry could be potentially useful for monitoring pathologic cardiovascular events.

According to the present invention, the following peptide

sequences and cleavage sites are useful;

Protease Cleavage sites marked i MMP1213519' GEYVCDCGTD ^Λ 13528

MMP1213532' NVTVEARLIKi U3542

MMP1213543' χVEKPLYGVEV ^Λ 1553 The present invention will be further described and

illustrated by the following examples in which reference is made to the accompanying drawings, in which:

Figure 1 shows the reactivity of a monoclonal antibody of the invention to target peptide as measured in Example 1 below;

Figure 2 shows the reactivity of the monoclonal antibody to human serum and urine samples as determined in Example 2; and Figure 3 shows measurements of titin fragment content in human serum from control patients and patients suffering from cardiac diseases as obtained in Example 3.

Example 1

Development of monoclonal antibodies to Titin fragments

All reagents used for experiments were standard high-quality chemicals from Merck (Whitehouse Station, NJ, USA) and Sigma Aldrich (St. Louis, MO, USA) . The synthetic peptides used for monoclonal antibody production were purchased from the Chinese Peptide Company, Beijing, China.

Selection of peptide for immunization

The sequence of the peptide selected for the assay was chosen on the basis of mass spectrometry performed on human tissue ²³ .

Peptide fragments were identified using Uniprot (accession number C0JYZ2) . The first 10 amino acids of each free end of the sequences identified were regarded as a target sequences. All relevant sequences were analyzed for homology and then blasted for homology using the CHECK XX NPS@: network protein sequence analysis ²⁸ . The sequence NVTVEARLIK located between amino acid position 12670' and 12679' (Titin) was selected as immunogen . The sequence was identified by Uniprot and PBIL network protein sequence analysis as being unique to human and mouse Titin. The selected sequence was also found to be present in 6 out of 8 Titin isoforms produced by alternative splicing. These were, isoforms 3 (small cardiac N2-B) , 7 (cardiac novex-2) and 8 (cardiac novex- 1) that are known to present in cardiac muscle (Uniprot accession numbers Q8WZ42-3, Q8WZ42-7 and Q8WZ42-8) and isoforms 2, 4 and 5 (Uniprot accession numbers Q8WZ42-2, Q8WZ42-4 and Q8WZ42-5) ²⁶ ' ²⁷ .

Immunization procedure

Six 4-6 week old Balb/C mice were immunized subcutaneously in the abdomen with 200 emulsified antigen (50 g per

immunization) , using Freund' s incomplete adjuvant (KLH-CGG- NVTVEARLIK SEQ ID NO: 7) . Immunizations were performed at two- week intervals until stable titer levels were obtained. At each bleeding, the serum antibody titer was measured and the mice with the highest antibody titer and best reactivity towards serum and urine were selected for fusion. The selected mice were boosted intravenously with 50 g immunogen in 100 0.9% sodium chloride solution three days before surgical removal of the spleen for cell fusion .

Fusion and antibody screening

The fusion procedure has been described elsewhere ²⁹ .

Briefly, mouse spleen cells were fused with SP2/0 myeloma fusion partner cells. The hybridoma cells were cloned using a limiting dilution method and transferred into 96-well microtiter plates for further growth. Standard limited dilution was used to promote monoclonal growth. Supernatants were screened using an indirect ELI SA, while the biotinylated peptide Biotin-CGG-NVTVEARLIK was used as a catcher peptide on streptavidin-coated microtitre plates . Characterization of clones Native reactivity and peptide binding of the monoclonal antibodies in human serum, plasma and urine was evaluated using a preliminary ELISA with a 10 ng/mL biotinylated peptide coater on a streptavidin-coated microtitre plate and the supernatant from the growing monoclonal hybridoma. Clone specificity was tested against a free peptide (NVTVEARLIK) and a non-sense peptide.

Isotyping of the monoclonal antibodies was performed using the Clonotyping System-HRP kit, cat.5300-05 (Southern Biotech,

Birmingham, AL, USA) . The selected clones were purified using Protein G columns according to the manufacturer's instructions and dialysed (GE Healthcare Life Science, Little Chalfont,

Buckinghamshire, UK) .

In Figure 1 the reactivity of a monoclonal antibody against the target sequence NVTVEARLIK (circles, dashed line) is compared to the de-selection (and C-terminally elongated) sequence

NVTVEARLIKV (square, solid line) . The data clearly demonstrate a strong reactivity to the target sequence NVTVEARLIK with no detectable binding to the elongated peptide. These data suggest a strong binding affinity with the sequence having a free C-terminal lysine residue, i.e. the neo-epitope specificity of the monoclonal antibody is confirmed.

Example 2

MMP12 Titin assay protocol

The following competitive ELISA protocol was optimised for use with the MMP12 Titin monoclonal antibody. The selected monoclonal antibodies were labelled with horseradish peroxidase (HRP) using the Lightning-Link Horseradish Peroxidase (HRP) antibody labelling kit according to the manufacturer's

instructions ( Innovabioscience , Babraham, Cambridge, UK) . A 96- well streptavidin plate (Roche diagnostics, Basel, Switzerland) was coated with 6.8ng of the biotinylated synthetic peptide, Biotin-CGG-NVTVEARLIK, dissolved in assay buffer 25mM Tris BTB and incubated for 30 minutes at 4°C. 20 L of the peptide calibrator or sample were added to appropriate wells, followed by 100 L of 280ng conjugated monoclonal antibody and incubated for 1 hour at 4°C. Finally, 100 tetramethyl benzinidine (TMB) (Kem-En-Tec cat.4380H, Taastrup, Denmark) was added, and the plate was incubated for 15 minutes at 20°C in the dark. All the above incubation steps included shaking at 300 rpm. After each

incubation step the plate was washed five times in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2) . The TMB reaction was stopped by adding 100 of stopping solution (1% HC1) and measured at 450 nm with 650 nm as the reference. A calibration curve was plotted using a 4-parametric mathematical fit model with a starting concentration of 200 ng for the standard peptide following a 2- fold dilution.

In Figure 2 the reactivity to titin fragments present in human serum is demonstrated. A human serum sample obtained from an individual with AMI was diluted 2-fold and incubated in the MMP12 Titin Elisa described above. The similarity of the curve obtained with human serum and the standard curve suggest that the affinity of the monoclonal antibody is similar against the synthetic peptide NVTVEARLIK and the fragments present in human serum.

Example 3

Assessment of serum samples from subjects with cardiovascular events in the MMP12 Titin ELISA

Serum from subjects respectively diagnosed with Acute

Myocardial Infarction (AMI), high coronary calcification, and symptomatic non AMI were tested in the MMP12 Titin ELISA and compared to healthy controls (Figure 3) .

A statistically significant increase of the marker was measured for all three patient groups. Mean level of controls was measured to be 116.6ng/ml. MMP12 Titin levels were elevated in all patient groups examined, AMI patients mean value 137.9ng/ml (P<0.05), Patients with high coronary calcification 140ng/ml (P<0.05) and Ischemic heart disease 150.6ng/ml (P<0.05) (Figure 3) .

Biochemical markers consisting of protein fragments from ECM degradation may be informative of disease pathology and

progression, which in turn may be useful for diagnostic and prognostic purposes. These markers could potentially detect changes resulting from intervention strategies and serve as surrogate markers of drug efficacy ³⁰ .

In this specification, unless expressly otherwise indicated, the word 'or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator 'exclusive or' which requires that only one of the conditions is met. The word 'comprising' is used in the sense of 'including' rather than in to mean 'consisting of . All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

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